Microbes For Sustainable Insect Pest Management An Eco Friendly Approach Volume 1 1st Ed 2019 978 3 030 23044 9 978 3 030 23045 6 - Compress

Sustainability in Plant and Crop Protection
Md. Aslam Khan

Wasim Ahmad Editors
Microbes for
Sustainable
lnsect Pest
Management
An Eco-friendly Approach - Volume 1
Series Editor
Aurelio Ciancio, Sezione di Bari, Consiglio Nazionale delle Ricerche Istituto per
la Protezione delle Piante, Bari, Italy
More information about this series at http://www.springer.com/series/13031
Md. Aslam Khan • Wasim Ahmad
Editors
Microbes for Sustainable

Insect Pest Management
An Eco-friendly Approach - Volume 1
Editors
Md. Aslam Khan Wasim Ahmad
Department of Biology, Faculty of Science Department of Zoology, Section
Jazan University of Nematology
Jazan, Saudi Arabia Aligarh Muslim University
Aligarh, Uttar Pradesh, India
ISSN 2567-9805 ISSN 2567-9821 (electronic)

ISBN 978-3-030-23044-9 ISBN 978-3-030-23045-6 (eBook)
https://doi.org/10.1007/978-3-030-23045-6
© Springer Nature Switzerland AG 2019

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Foreword
Insect pests are responsible for major losses to agricultural products globally.
Synthetic chemical insecticides have been the primary control agent of insect pests
for decades. Hazards and side effects associated with the extensive and indiscrimi-
nate use of pesticides are well documented and are of great concern. Resistance to
synthetic insecticides is also a critical problem in several parts of the world.
Development of integrated pest management strategies with little or no reliance
on chemical pesticides has become an important goal for modern agriculture.
Biological control is a component of IPM strategies that minimize insecticide spray
applications and move towards ecofriendly systems of pest management. Due to
high specificity, effectiveness, and safety to nontargeted organisms, biological con-
trol agents are considered suitable alternatives to the use of chemical pesticides. In
recent years, biological control using microorganisms like bacteria, fungi, viruses,
and nematodes has emerged as a valuable tool. In the present scenario, it is praise-
worthy to have a book comprising potentials of different entomopathogens in the
sustainable management of insect pests.
The current volume should prove a very timely action in this direction. The dif-
ferent chapters in this book provide valuable information in this regard. The editors
of this volume together with the authors of the individual chapters have made a
remarkable contribution in collating the up-to-date information on sustainable and
ecofriendly management of pernicious insect pests.
v
vi Foreword
This information could be useful for researchers, educators, students, and indus-
try persons for understanding and developing ecofriendly and sustainable pest man-
agement strategies. This book comprehensively addresses various methods related
to sustainable management of insect pests through the expertise of the leading
authors worldwide. Finally, this book in the series Sustainability in Plant and Crop
Protection is highly innovative in covering both basic information and effective
management of insect pests.
Director, Kailash Chandra

Zoological Survey of India
Kolkata, India
Preface
It is generally acknowledged that agricultural yields must be increased in the com-

ing years in order to feed the projected growth of the global human population. An
enormous amount of resources is spent each year, worldwide, to manage and con-
trol insect pests. A great proportion of the agricultural yields is, however, still lost to
insect damage, predominantly in developing countries. Therefore, the need to keep
insect pests away from destroying food crops has become even more urgent.
The negative impacts of synthetic organic pesticides in crop pest management
programs caused tremendous damages to the environment, including the develop-
ment of resistance among target pests, and other detrimental effects on nontarget
organisms, underlying the need for alternative and eco-friendly control methods.
Biological control-based approaches provide a more environment-friendly and
acceptable alternative to traditional chemical control measures, owing to high host
specificity and safety for the environment and mankind.
Microorganisms have been regularly isolated from natural sources around the
world for pest management purposes. Some of them, known as entomopathogens,
may safeguard crops and plants by causing epidemic diseases of insect populations.
They have been widely tested and proved to be very effective against pernicious
pests. Almost all the major insect groups are susceptible to entomopathogens. In
certain developing and developed countries, a number of species and isolates have
been registered for field application on various vegetables, fruits, and other crops of
agricultural, horticultural, and forest importance. It is therefore imperative to study
the entomopathogens and their potential role in achieving a sustainable pest man-
agement goal.
This volume comprises 14 chapters in an attempt to bring available information
on safe use of entomopathogens. The soil constitutes an important reservoir for
harvesting various types of beneficial organisms. Chapters dealing with soil-borne
entomopathogens and their phylogeny provide a review and most updated informa-
tion of their isolation and molecular identification. Fungal pathogen applications
play a key role in biological control programs. In other chapters, thermotolerance
and oxidative stress are examined and explored. A number of entomopathogenic
vii
viii Preface
bacteria are able to kill their host quickly. The information provided upon cytotoxic
factors for insect hemocytes constitutes an important contribution. Nematodes as
biological control agents are safer alternatives for the environment, as shown by the
information provided on their direct and indirect effects on nontarget organisms.
Being highly specific, virulent, and safer to nontarget species, viruses are poten-
tial candidates for use as biological insecticides. A separate chapter on the role of
granuloviruses in IPM contributes a wealth of information on this topic. Biopesticides
in combination with synthetic insecticides are effective, economic, and eco-friendly.
Understanding their interactions will certainly promote their use, as shown in
reviews of synergistic and antagonistic interactions of microbial and chemical pes-
ticides, provided in further chapters.
We hope that this volume will be helpful to students, teachers, researchers, and
industry technicians. We are highly grateful to all the contributors for providing
their expertise in the form of stimulating contributions. Thanks are due to the Head,
Biology Department, and Dean, Faculty of Science, Jazan University, Jazan, for
their moral support. We are grateful to Dr. Aurelio Ciancio, CNR, Bari, Italy, for
including this volume in the Springer series “Sustainability in Plant and Crop
Protection.” We extend our thanks to Springer International team for their generous
cooperation at every stage of the book production.
Jazan, Saudi Arabia Md. Aslam Khan

Aligarh, India Wasim Ahmad
Contents
1 Synthetic Chemical Insecticides: Environmental and Agro

Contaminants........................................................................................... 1
Md. Aslam Khan and Wasim Ahmad
2 Soil-Borne Entomopathogenic Bacteria and Fungi.............................. 23
Tan Li Peng, Samsuddin Ahmad Syazwan, and Seng Hua Lee
3 Molecular Phylogeny of Entomopathogens........................................... 43
Mudasir Gani, Taskeena Hassan, Pawan Saini,
Rakesh Kumar Gupta, and Kamlesh Bali
4 Potential of Entomopathogenic Bacteria and Fungi............................. 115
Lav Sharma, Nitin Bohra, Rupesh Kumar Singh,
and Guilhermina Marques
5 Ascomycota and Integrated Pest Management..................................... 151
Tariq Ahmad, Ajaz Rasool, Shaziya Gull, Dietrich Stephan,
and Shabnum Nabi
6 Thermotolerance of Fungal Conidia...................................................... 185
Flávia R. S. Paixão, Éverton K. K. Fernandes, and Nicolás Pedrini
7 Oxidative Stress in Entomopathogenic Fungi and Its Potential
Role on Mycoinsecticide Enhancement.................................................. 197
Carla Huarte-Bonnet, M. Constanza Mannino, and Nicolás Pedrini
8 Effects of Cytotoxic Factors Produced by Entomopathogenic
Bacteria on Insect Haemocytes............................................................... 207
Carlos Ribeiro and Amélia Vaz
9 Effects of Entomopathogenic Nematodes and Symbiotic
Bacteria on Non-target Arthropods........................................................ 247
Ramandeep Kaur Sandhi and Gadi V. P. Reddy
ix
x Contents
10 Granuloviruses in Insect Pest Management.......................................... 275

Pankaj Sood, Amit Choudhary, and Chandra Shekhar Prabhakar
11 Interactions of Entomopathogens with Other Pest
Management Options............................................................................... 299
Surendra K. Dara
12 Toxicological Prospects on Joint Action of Microbial
Insecticides and Chemical Pesticides..................................................... 317
A. R. N. S. Subbanna, J. Stanley, V. Venkateswarlu,
V. Chinna Babu Naik, and M. S. Khan
13 Entomopathogen and Synthetic Chemical Insecticide:
Synergist and Antagonist......................................................................... 341
Arash Zibaee
14 Current State of Fungal Antagonists with Special
Emphasis on Indian Scenario................................................................. 365
Purnima Das, Lakshmi Kanta Hazarika, Surajit Kalita,
and Somnath Roy
Index ................................................................................................................. 387

Chapter 1
Synthetic Chemical Insecticides:
Environmental and Agro Contaminants
Md. Aslam Khan and Wasim Ahmad
Abstract Synthetic pesticides are indispensable in intensive agricultural produc-

tions. For decades these compounds served as backbone in insect pest management.
Due to persistence and pervasiveness of millions of tonnes of synthetic chemical
pesticides applied, almost every ecosystem has received a negative impact. In the
present chapter an effort has been made to highlight the environmental contamina-
tions caused by synthetic chemical pesticides, their adverse effects on human health
and other non target organisms, the development of resistance in target insect pests,
along with the degradation of synthetic pesticides.
Keywords Synthetic pesticides · Environment · Contaminant · Non target

organism · Human health
1.1 Introduction
Insects, plants, bacteria, fungi and other organisms occur naturally in the environment
but in some situations they can have environmental, health and economic impacts,
and become pests that must be controlled. For many generations, natural pest control
in agricultural practices relied heavily on crop rotation or mixed crop planting (Dayan
et al. 2009). In view of the world rapid human population growth and limited crop-
lands, it is needed to apply all available measures to increase crop production in order
to ensure food security (Zhang 2009) and optimize provision of food at low cost.
In this view, a worldwide agricultural movement that originated in Mexico in
1944, with the primary goal of boosting grain yields, was named as “Green revolu-
M. A. Khan (*)
Department of Biology, Faculty of Science, Jazan University, Jazan, Saudi Arabia
e-mail: [email protected]
W. Ahmad
Department of Zoology, Section of Nematology, Aligarh Muslim University,
© Springer Nature Switzerland AG 2019 1

M. A. Khan, W. Ahmad (eds.), Microbes for Sustainable Insect Pest
Management, Sustainability in Plant and Crop Protection,
https://doi.org/10.1007/978-3-030-23045-6_1
2 M. A. Khan and W. Ahmad
tion”. Based on higher yields new cultivars were selected but most of those varieties
were not widely resistant to pests and diseases. Therefore the pest control issue was
mostly addressed by the use of synthetic chemical pesticides, as an integral part of
the green revolution. More pesticide inputs were, however, needed for the green
revolution, more than in the traditional agricultural systems. Following its success
in Mexico, the green revolution concepts spread all over the world.
As high temperature and humidity are highly conductive to rapid multiplication
of pests, crop losses remain more severe especially in tropical countries. Insect
pests, plant pathogens and weeds cause an estimated 14%, 13% and 13% of loss,
respectively (Pimentel 2009). Without pesticides application a loss of 78% fruits,
54% vegetables, and 32% cereal crops has been reported, due to pest injury (Cai
2008). Therefore, pesticides appeared indispensable in agricultural productions as it
was conceived that they were not a technical luxury rather a necessity for the well
being of mankind. According to the Environmental Protection Agency (EPA 2009)
a pesticide is any substance or mixture of substances intended for preventing,
destroying, repelling, or mitigating any pest like insects, mites, nematodes, weeds
and rats etc. Major categories of pesticides include insecticides, herbicides and fun-
gicides/bactericides, but several other types of biocides, such as nematicides and
rodenticides etc., are also included.
Due to longer residual action and a wide toxicity spectrum synthetic chemical
insecticides became more popular. Dichloro-diphenyl-trichloroethane (DDT) was
the first important synthetic organic insecticide, synthesized by German scientist
Ziedler in 1873 (Othmer 1996). Its insecticidal effect was discovered by Swiss
chemist Paul Muller in 1939. DDT hailed as miracle in its early days because of a
broad-spectrum activity, persistence, insolubility, low cost and ease to apply (Keneth
1992). For decades these compounds served as backbone in insect pest management
and farmers relied heavily upon the conventional groups of synthetic insecticides
such as organochlorines (DDT, BHC), cyclodienes (aldrin, dieldrin, endosulfan),
organophosphates (monocrotophos, quinalphos, chlorpyriphos, profenophos,
dimethoate, phosalone, metasystox, acephate, phorate, methyl parathion), carba-
mates (carbosulfan, carbaryl, thiodicarb, methomyl), pyrethroids (cypermethrin,
deltamethrin, fenvalerate, λ-cyhalothrin), and formamidines (chlordimeform and
amitraz) (Kranthi 2007).
Many of the available pesticides are often a mixture of several chemicals ingredi-
ents mixed together in desired proportions, suspended in appropriate carriers or dilu-
ent materials. Different forms of pesticides include emulsifiable concentrate, wettable
powder, granule, bait, dust and fumigant. A comprehensive review on identity, physi-
cal and chemical properties of pesticides was produced by Tano (2011). Application
of million tonnes and hundred types of synthetic pesticides, however, reduced crop
losses but due to their persistence and pervasiveness, almost every ecosystem on earth
has receive one or more negative impacts (Reuter and Neumeister 2015), with long-
term affect on society. It is now clear that adverse effects of pesticides are wide and
varied. The public has expressed today many concern about the potential health and
environmental impacts of these pesticides. This chapter collates the adverse effects of
synthetic chemical insecticides from the growing body of related literature.
1 Synthetic Chemical Insecticides: Environmental and Agro Contaminants 3
1.2 Background and History
Before 1870s natural pesticides, for instance sulfur in ancient Greece, were used to
control pests. From 1870 to 1945 natural materials and inorganic compounds were
mainly used as pesticides. Since 1945, man-made organic synthetic compounds ter-
minated the era of inorganic and natural pesticides (Zhang et al. 2011). After that
time most pesticides have been synthesized by man, and named as chemical pesti-
cides. Based on chemical classification, insecticides are broadly grouped as: organo-
chlorines, organophosphorous, carbamates and pyrethroids.
For insect pests control most spectacular episodes began in 1945–1946 with the
commercial introduction of organochlorines such as DDT, followed by organophos-
phate and carbamate introduced in the ‘60s (Nicholson 2007). Organochlorines, the
first synthetic organic pesticides to be used in agriculture and in public health, are
contact poisons that apparently act as nervous system disruptors leading to convul-
sions and paralysis of the insect and its eventual death. They have a long-term resid-
ual effect in the environment as they are resistant to most chemical and microbial
degradations. Organophosphorous insecticides are cholinesterase inhibitors in tar-
get pests. As a result, nervous impulses fail to move across the synapses, causing a
rapid twitching of voluntary muscles and hence paralysis and death. Unlike organo-
chlorines, organophosphorous insecticides are easily decomposed in the environ-
ment by various chemical and biological reactions. Carbamates are organic
pesticides derived from carbamic acid. They show high insect toxicity as cholines-
terase inhibitors. The cholinesterase inhibition of carbamates differs from that of
organophosphorous because it is species-specific and reversible (Drum 1980).
Pyrethroids are synthetic analogues of the naturally occurring pyrethrins. They are
known for the fast knocking down effect active against insect pests, with low mam-
malian toxicity and easy biodegradation. As they are particularly susceptible to pho-
tolysis, their uses as agricultural insecticides is relatively impractical.
In each country, regulatory insect risk assessment related to agrochemicals use
and registration follows specific guidelines such as the European Council Directive
91/414 in Europe, and the Federal Insecticide Fungicide and Rodenticide Act in the
United States. In India the pesticides import, manufacture, sale, transport and use
are regulated under a comprehensive statute, the Insecticides act of 1968.
The worldwide consumption of pesticides is around 2–3 million tonnes per year
(United States Environment Protection Agency 2011), of which 45% is consumed
in Europe, 25% in the United States, and 30% in the rest of the world (De et al.
2014). The usage of pesticides in India is only 0.5 kg ha−1, while in Korea and Japan
it is 6.6 and 12.0 kg ha−1, respectively (Abhilash and Singh 2009). Annual pesticide
consumption in different countries is represented in Table 1.1. Total expenditure on
pesticides is about US$ 40 billion per year (Popp et al. 2013). Ssynthetic insecti-
cides entail several types of costs, including internal costs due to the purchase and
application, and various other additional hidden costs due to the impact of treat-
ments on the environment and human health (Bourguet and Guillemaud 2016).
Zhang et al. (2011) reviewed the consumption, pollution and developmental trend
Table 1.1 Annual pesticide consumption by different Asian countries (Abhilash and Singh 2009)
Country Ton a.i. Ton product US $ value (000)
Bangladesh 3635 22100 75000
Cambodia 42 198 226
China 258000 1000000 5670000
DPR Korea 3000 12000 60000
India 41020 164080 820400
Rep. of Korea 26610 100000 842638
Lao PDR 10 40 200
Malaysia 51065 204260 85020
Myanmar 758 3030 15095
Nepal 145 580 2100
Pakistan 32500 129589 172300
Philippines 7934 31735 158675
Sri Lanka 1696 6329 49000
Thailand 49108 132509 253537
Vietnam 24473 50000 159000
of pesticide varieties and reported that the overall consumption structure has under-
gone significant changes since 1960s. The proportion of herbicides in pesticide con-
sumption increased rapidly, and the consumption of insecticides and fungicides/
bactericides declined. Dayan et al. (2009) also reported that herbicides account for
more than half of the volume of all agricultural pesticides applied in the developed
world (Fig. 1.1). Pesticide production in India is dominated by insecticides and
fungicides, followed by herbicides and rodenticides (Fig. 1.2).
The major markets for pesticides are the USA, Western Europe and Japan
(Dinham 2005). Europe is the largest pesticide consumer in the world. United
States, Brazil, Germany, France, China and Japan are the largest pesticide produc-
ers, consumers or traders in the world (Zhang et al. 2011). India ranks twelfth in the
world for the use of pesticides. The Indian pesticides industry started in 1952 with
the BHC technical plant, near Kolkata. Thereafter Hindustan insecticides Ltd. set up
two units to manufacture DDT. Union Carbide then set up a plant in 1969 named as
Union Carbide India Ltd., at Bhopal city. The industry produced various pesticides
for the Indian market till the 1984 Bhopal disaster. Now the Indian pesticide indus-
try comprises of more than 125 basic producers of large and medium scale (Abhilash
and Singh 2009).
Although the Indian average pesticide consumption is far lower than many other
developed economies, the problem posed by the pesticide residues is very severe
(Abhilash and Singh 2009). Inappropriate application of pesticides affects the whole
ecosystem by entering the residues in food chains and polluting soil, air, ground and
surface water (Agnihotri 1999; UN/DESA 2002). For this reason Rachel Carson
(1962) in her book “The Silent Spring” described this era as that of the “rain of
chemicals”.
Fig. 1.1 Comparison of pesticide use in India and worldwide. (Adapted from Abhilash and Singh
2009)
Fig. 1.2 Share of pesticide groups in total pesticide production (technical grade), in India.
(Adapted from Subash et al. 2017)
Monocrotophos, phorate, phosphamidon, methyl parathion and dimethoate are

some of highly hazardous pesticides that are continually and indiscriminately used
in India. As a consequence, the country is now battling against the residual effects
of extensively used pesticides (Rekha and Prasad 2006; Agoramoorthy 2008). The
Supreme Court of India appointed an expert committee to examine all aspects of the
ban on endosulfan and the disposal of the existing quantity of this pesticide. On
May 2011 the Supreme Court of India passed an interim order to ban the produc-
tion, distribution and use of endosulfan in the country. Moreover, the High Court of
Karnataka state directed the State government machinery to provide medical cover
to all persons affected by the use of endosulfan, in certain coastal districts.
As early as 1972, DDT, one of the most noxious pesticides ever used, and related
organochlorinated insecticides, were banned in the United States, by the
Environmental Protection Agency, and then in most other countries because of its
potential harmful effects on the environment, wildlife and humans. During the late
twentieth century, pesticide consumption in the United States declined by 35%
without reducing crop production (SDNX 2005). In Europe, as in the United States,
older pesticides are being re-assessed one-by-one, to ensure that they meet the new
regulatory standards (Damalas and Eleftherohorinos 2011). Overall pesticide con-
sumption in Europe declined in last decades. Since 2001 to 2008 in Europe 704
pesticides were banned, 26% of which were insecticides, 23% herbicides and 17%
fungicides (Karabelas et al. 2009). China has banned the application of high-residual
DDT, HCH and other organochlorined pesticides since 1983. Since 2007, the highly
poisonous organophosphorus pesticides parathionmethyl, parathion, methamido-
phos, and phosphamidon have been banned for using and selling in China.
Recent reports indicate that the use of persistent organic pollutants (POPs) is
declining. Wang et al. (2008) noted that since the use of organochlorined pesticides
(OCPs) has been banned long, the residual OCPs are declining but in some regions
they are still abundant. Covacia et al. (2005) also suggested a slight decline in the
concentrations of DDT from Belgium sediments, due to the restrictions in their usage.
1.3 Environmental Contaminants
Pesticides hold a unique position among environmental contaminants due to their

high biological activity and toxicity. Indiscriminate, inadequate, and improper use
of these synthetic organic inputs in crop pest management programs around the
world caused tremendous damage to the environment, development of resistance in
target pests, pest resurgence, detrimental effects on non-target organisms, and
impact on human health (Casida and Quistad 1998; Shen and Zhang 2000; Niyaki
2010; Al-Zaidi et al. 2011). According to the Food and Agriculture Organization
(FAO) inventory (FAO/UNEP/OECD/SIB 2001), more than 500,000 tonnes of
unused and obsolete pesticides are threatening the environment and public health, in
many countries.
A report published in proceedings of the international academy of ecology and
environmental sciences by Zhang et al. (2011) shows that only 1% of the sprayed
pesticides are effective, the remaining 99% being released to non-target soils, water
bodies and atmosphere, and finally absorbed by almost every organism. Some syn-
thetic pesticides are extremely persistent in the environment because of their resis-
tance to natural breakdown processes and are routinely found in the environment
today. POPs and related degraded products flow into the atmosphere, soils and riv-
ers, resulting in the accumulation of toxic substances, thus threatening the environ-
ment. High-residual pesticides such as DDT have been detected in the Greenland
ice sheet and the bodies of Antarctic penguins, as the result of atmospheric circula-
tion, ocean currents and biological enrichment along food chains.
Environmental costs are those resulting both from pesticide damage to animals,
plants, algae and microorganisms and from induced pest resistance. These costs
may be incurred by farmers or by the society as a whole. In agricultural areas in
which pesticides are used, these substances drift in the air, pollute the soil and
waterways, and are sometimes systemically absorbed by non-target plant species.
Pesticide pollution to the local environment also affects the lives of birds, wildlife,
domestic animals, fish and livestock.
The environmental pollution caused by pesticides in Asia, Africa, Latin America,
the Middle East and Eastern Europe is now serious. Even in earlier years in India the
residuals of DDT, lindane and dieldrin have been much beyond the safety threshold
for fish, eggs and vegetables (Wu 1986). Willet et al. (1998) reported that although
environmental levels of some organochlorines have fallen over time, many can still
be found as contaminants in soils, river or coastal marine sediments, reaching as far
as the deep oceans and poles. The treatise dealing with environmental and pollution
science by Pepper et al. (2006) is worthy to be mentioned here.
Pesticide pollution of environmental waters is a pervasive problem with wide-
spread ecological consequences. During applications, pesticides drift away in the air
and seep into the soil (Pimentel 1995; Gil and Sinfort 2005). Once in the soil, some
soluble compounds may be washed out in runoff water and during soil erosion,
resulting in leaching into rivers and lakes (Chopra et al. 2011). Hence the pesticide
concentration of water bodies can reach the magnitude of several dozen mg/L.
Excess concentration of pesticides into runoff water should be removed to pro-
tect water resources or to achieve drinking water quality. Under Council Directive
98/83/EC in Europe, the legally permitted limit for an individual pesticide in drink-
ing water is 0.1 μg/L, whilst the total of all pesticides must not exceed 0.5 μg/L
(Boobis et al. 2008). Wastewaters from agricultural or industrial activities contain
high levels of pesticide contamination. Agriculture, which accounts for 70% of
water abstractions worldwide, plays a major role in water pollution. Farms dis-
charge large quantities of agrochemicals, organic matter, drug residues, sediments
and saline drainage into water bodies. The resultant water pollution poses demon-
strated risks to aquatic ecosystems, human health and productive activities (UNEP
2016). Chiron et al. (2000) reported that among the different approaches to pesticide
elimination from wastewater, photochemical and ozonation methods appear to be
especially suitable for industrial applications, whereas ozonation is more easily
controlled and easier to adapt to industrial applications than photochemical.
1.4 Impact on Human Health
Despite strict regulations on the registration and use of pesticides, there are major
concerns about their direct impact on human health. There is a growing body of
evidence showing that health hazards of pesticides are serious. People are inevitably
exposed to a cocktail of pesticides through environmental contamination or occupa-
tional use. There are no groups in the human population that are completely unex-
posed to pesticides, and most diseases are multi-causal giving considerable
complexity to public health assessments (Meyer-Baron et al. 2015).
Many pesticides can damage human health (Damalas and Eleftherohorinos
2011). Indiscriminate use and improper handling of synthetic pesticides in agricul-
ture have caused acute (high doses over short periods) and chronic (lower doses
over longer periods of time) health problems in human. Pesticide poisonings are
also divided into productive poisonings, generated in the process of agricultural
production, and living poisonings, i.e., suicide, ingestion, and food intake with
high-residuals etc. (Liu et al. 2008). Exposure to pesticides can occur via a number
of pathways like food, drinking water, residential, occupational (Fig. 1.3), and dif-
ferent routes like oral, inhalation and dermal.
Million cases of pesticide poisonings have been documented every year around
the world. In the USA, the Environmental Protection Agency reports an estimated
300,000 human pesticide poisonings as a part of the cost of their application
(Pimentel and Burgess 2012). Worldwide, the number of severe pesticide
Fig. 1.3 Different pathways of pesticides exposure

poisonings is much higher, as Richter (2002) reports 26 million human pesticide

poisonings with 220,000 deaths occuring each year, the majority in developing
countries. While these countries use only around 25% of the world pesticides, they
experience 99% of deaths linked to pesticides (UNICEF 2018). In developing coun-
tries agricultural pesticides are among the most commonly used substances for self-
poisoning. Gunnell et al. (2007) conservatively estimated that there are 258,234
(plausible range 233,997 to 325,907) deaths from pesticide self-poisoning world-
wide each year, accounting for 30% (range 27–37%) of suicides, globally.
Pesticides have long been proposed as a possible cause of human health, but
information on their health impacts is quite limited in many developing countries.
However, the true extent of the problem is hard to determine for a variety of reasons.
Health-care professionals in rural areas often fail to correctly diagnose poisoning, as
many of the related symptoms are quite general in nature or mimic other common
health problems (FAO 2001). The majority of the pesticide poisonings and deaths in
the developing countries are the result of farmers’ poor handling practices and less
awareness of the relative toxicity of the product they are using. Often they use pes-
ticides at rates more intensive than those recommended by the product labels, dis-
pose residual pesticides into canals or ditches, re-apply them to the same crops, or
spray crops that were not identified for initial use (Huan and Thiet 2000).
Agricultural workers are responsible for mixing and loading pesticide prepara-
tions, spraying, sowing pesticide-treated seeds, harvesting sprayed crops, and clean-
ing and disposing of pesticide containers. Moreover, they are often occupationally
exposed to pesticides. Workers in the pesticide industry are also likely to experience
occupational exposures. The families living in rural areas in which pesticides are
intensively used may also be indirectly exposed to these chemicals, through off-
target pesticide drift (Lee et al. 2011).
Generally, each exposure pathway is the responsibility of a different department
or agency within national governments or international bodies. Hence, assessment
by each route is generally undertaken independently. The risk assessment of pesti-
cide residues in food is currently performed on a compound-by-compound basis.
However, it is the totality of exposure i.e. through multiple routes and multiple
pathways, that determines the actual risk (WHO 2007). The information provided
by Allsop et al. (2015) in the form of “pesticides and our health: a growing concern”
is worthy to be mentioned here.
Unregulated pesticide use and lack of enforcement mechanisms has resulted in
thousand acute and chronic poisoning cases, with effects of varying severity on
human health, from mild ones to death. Exposure to pesticides has also been the
subject of great concern in view of its possible role in the induction of congenital
malformations. Abell et al. (2000) reported that exposure to pesticides to female
workers in flower greenhouses may have reduced fertility, and that exposure may be
part of the causal chain. In a study by Bosma et al. (2000) in the Netherlands, farm-
ers and gardeners showed a higher risk of developing mild cognitive dysfunctions.
Long-term cognitive effects due to low-level exposure to pesticides in occupational
conditions have been reported by Baldi et al. (2001). Clinical reports have shown
that acute intoxication by organophosphates may be responsible for chronic impair-

ment of cognitive functions (Rosenstock et al. 1990).
Acute poisoning, leading to respiratory, gastrointestinal, allergic, and neurologic
disorders, is commonly reported by farmers, and particularly by those carrying out
pesticide applications (Kishi et al. 1995; Hudson et al. 2014). Increasing incidence
of cancer, chronic kidney diseases, suppression of the immune system, among male
and female sterility, endocrine disorders, neurological and behavioral disorders,
especially among children, have been attributed to chronic pesticide poisoning
(Agnihotri 1999). Besides cancers and reproductive effects, nervous system damage
has been reported in terms of peripheral neuropathy and central nervous degenera-
tive disease, with special emphasis on Parkinson’s disease (Checkoway and Nelson
1999). In children organophosphates have been linked to aplastic anemia, the failure
of the bone marrow to produce blood cells, and leukemia. In particular, children
with asthma may have severe reactions to organophosphates (Zahm et al. 1997).
Pesticide exposure has also been associated with elevated cancer risks and repro-
ductive dysfunctions in agricultural workers (Raschke and Burger 1997; Horrigan
et al. 2002). Potential carcinogenicity of a wide range of insecticides, fungicides
and herbicides has been reviewed by the International Agency for Cancer Research
(IARC) and fifty-six pesticides have been classified as carcinogenic to laboratory
animals. It is estimated that cancer patients resulted from pesticide poisoning
account for nearly 10% of total cancer patients (Gu and Tian 2005). Review on
environmental chemicals and breast cancer by Rodgers et al. (2018) is worthy to be
mentioned here. The United Nations Environmental Program (UNEP) has cau-
tiously taken action to protect human health, the environment and the earth from
further destruction by persistent organic pollutants such as DDT and its principal
metabolite, dichloro-diphenyl-trichloro-ethylene (DDE).
To a large extent DDT, the first major synthetic insecticide, replaced lead arse-
nate, a major carcinogenic pesticide used before the modern era (Ames et al. 1990).
Now DDT has emerged as a potential human carcinogen. Both DDT and DDE are
highly lipid soluble, accumulate in fat-containing foods, and travel through the food
chain (Leber and Benya 1994; Spear 1999). DDT exposure in young women during
the peak period of DDT use in the USA predicts breast cancer later in their life time
(Cohn et al. 2007). In India the DDT content in the human body was ever the highest
in the world (Zhang et al. 2011). Anti-androgenic activity has been reported for
DDE, that bioaccumulates in meat and dairy products (Kelce et al. 1995). Compared
with adults, children may be more vulnerable to particular risks due to exposure to
pesticides and related toxic effects. This vulnerability can be due to a number of
factors, including differences in physiology, behavior, and environmental condi-
tions (Goldman 1995; Reigart and Roberts 2001).
Organochlorine pesticides display diverse endocrine activity in vitro and in ani-
mal models. Cholinesterase-inhibiting pesticides are intended for insects but they
can also be poisonous to humans, in some situations. Human exposure to
cholinesterase-inhibiting pesticides can result from inhalation, ingestion, or eye or
skin contact during the manufacture, mixing, or applications of these chemicals.
Insecticide toxicity test includes study with blood tests for cholinesterase inhibition
(reductions in acetylcholinesterase enzyme [AChE] due to contamination by chemi-

cal insecticides use). To test the acute and chronic pesticide poisoning Dasgupta
et al. (2007) conducted an acetylcholinesterase enzyme blood test for 190 rice farm-
ers in the Mekong Delta (Vietnam) and reported high prevalence of pesticide
poisoning.
Pesticides have been also considered as potential chemical mutagens. Various
agrochemical ingredients possess mutagenic properties inducing mutations, chro-
mosomal alterations or DNA damage. Genetic damage associated with high expo-
sure levels of pesticides in human populations is reported by Bolognesi (2003).
1.5 Resistance Among Insect Pests
Melander (1914) first reported insecticide resistance. Since then the subject has
received ever-growing attention. A population is considered resistant if its response
to an insecticide in detection tests drops significantly, below its normal response
(Georghiou and Mellon 1983). This is particularly the case in warmer climates
where insect infestation pressure is high. For all major insecticide classes, resis-
tance has been reported in one or more key pest species (Georghiou 1986), includ-
ing stored product insects. This resistance has steadily increased over the last
30 years (Rex Consortium 2013). The doses of pesticides applied to many crops are,
therefore, almost certainly higher than used in the past, resulting in a greater impact
on the environment. In 1979, the United Nations Environmental Programme
declared pesticide resistance “one of the world’s most serious environmental prob-
lems.” In 2009 the European Union brought in a new framework directive requiring
that all member states should achieve a level of sustainable use of pesticides
(European Union 2009). In view of all these problems, the availability of many
broad-spectrum chemical pesticides is declining, as a result of the evolution of resis-
tance (Ishtiaq et al. 2012), and legislation (Chandler et al. 2011).
In addition to direct mortality, toxic substances can cause physiological responses,
such as changes in biochemical contents of the exposed insects. Broad-spectrum
chemical insecticides have been the primary control agent for agricultural pests,
with about 40% targeted to the control of lepidopteran insects (Brooke and Hines
1999). In the Indian subcontinent, Australia, China and Africa, cotton bollworm,
Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is arguably the most
important agricultural pest. It has a long history of resistance to almost all the insec-
ticide types used for its control (Gunning et al. 1999; Kranthi et al. 2002; Srinivas
et al. 2004). Nair (1981) reported H. armigera “not a serious pest” of cotton in India.
A few years later the pest was noticed to cause heavy economic losses to cotton and
was found to withstand a sustained insecticide pressure. Subsequently high levels of
resistance to synthetic pyrethroids in H. armigera were confirmed by Dhingra et al.
(1988) and McCaffery et al. (1989). By 1992, H. armigera resistance to insecticides
had emerged as a great challenge to pest management in Asia and Australia. Unlike
other lepidopteran species, H. armigera larvae don’t migrate far from their original
host plant, consequently their populations in agricultural areas are exposed to con-
sistent selective pressures, leading to greater resistance to insecticides (Fitt 1994).
Phokela and Mehrotra (1989) stated that pyrethroid resistance in different strains of
H. armigera appears to be mainly due to the high rate of metabolism in resistant
lines. Aurade et al. (2010) however reported that the presence of a P-glycoprotein
could be one of the reasons for insecticide resistance in this pest.
During the late 1970s, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae),
another major pest of subtropical and tropical agricultural crops, was found to
exhibit high resistance to several conventional insecticides recommended for its
control (Ramakrishnan et al. 1984; Tong et al. 2013). Among sucking pests, aphids
were reported to have developed resistance against organophosphates, carbamates
and pyrethroids (Herron et al. 2001; Ahmad and Akhtar 2013). The whitefly Bemisia
tabaci was found resistant to BHC, endosulfan, diamethoate, phosalone, acephate,
monocrotophos, quinalphos, and carbaryl (Prasad et al. 1993).
Because of health, safety, environmental and economic considerations, only a
very limited number of chemicals are available for application to stored grain. A
serious threat to the continued availability of these materials is the development of
resistance in target pests. Stored-grain pests such as lesser grain borer, Rhyzopertha
dominica (F.), red flour beetle, Tribolium castaneum (Herbst), and rice weevil,
Sitophilus oryzae (L.) developed resistance against the fumigant phosphine, a most
important insect control treatment for stored grain, in many regions (Sartori et al.
1990). The last quarter of the twentieth century has seen the withdrawal of many
compounds formerly used as fumigants. Methyl bromide, the fumigant with the
widest range of applications, is scheduled to be completely phased out in the second
decade of the current century under the directive of the Montreal Protocol, an United
Nations agreement on ozone depleting substances. Phosphine is also under regula-
tory review in several developed countries, with some unpredictability in the out-
come (Bell 2000).
Commercial development of insecticidal genes has focused on the Bacillus
thuringiensis (Bt) toxins (Bravo et al. 2007; Pigott and Ellar 2007). More recently
there have been reports of field resistance to Bt crops in cotton bollworm,
Helicoverpa spp. (Luttrell et al. 1999; Ali et al. 2006; Ali and Luttrell 2007; Carriere
et al. 2010), armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) (Storer
et al. 2010), pink bollworm, Pectinophore gosspiella Saunders (Lepidoptera:
Gelichiidae) (Bagla 2010; Dhurua and Gujar 2011), and western corn rootworm,
Diabrotica virgifera LeConte (Lepidoptera: Chrysomelidae) (Gassmann et al.
2011). The most common mechanism for Bt resistance is the disruption of the Bt
toxin binding to the receptors in the host midgut membrane.
Insects that show resistance to one insecticide generally develop resistance to
other classes of insecticides, a phenomenon often referred to as cross-resistance.
This phenomenon resembles multidrug resistance whereby resistance to one drug is
accompanied by simultaneous resistance to a variety of structurally unrelated com-
pounds (Lanning et al. 1996). The complex patterns of cross resistance between
chemical groups and within groups further complicate the use of this strategy. In
some regions, the situation is precarious with insect populations containing multiple
resistances, leaving no effective protective options available. The intensive use of

insecticides to control peach potato aphid, Myzus persicae Sulzer (Hemiptera:
Aphididae) over many years has led to populations that are now resistant to several
classes of insecticide. Myzus persicae has a remarkable ability to evolve mecha-
nisms that avoid or overcome the toxic effect of insecticides, with at least seven
independent selective processes for resistance (Bass et al. 2014). Biochemical and
molecular mechanisms underlying resistance in M. persicae has been reviewed by
Bass et al. (2014). A comprehensive database (Arthropod Pesticide Resistance
Database) by the Insecticide Resistance Action Committee, Michigan State
University, provides thorough information for insect species resistant to different
pesticides, along with the locations where resistance is reported (www.pesticidere-
sistance.com). In the situation outlined above it can be easily concluded that
insecticide-based pest management desperately needs new chemistries with differ-
ent resistance profiles.
1.6 Effects on Non -Target Organisms
Many studies have documented direct and indirect effects of both high and sublethal
doses of pesticides on several wild vertebrates. As stated above, only 1% of the
sprayed pesticides are effective whereas the remaining 99% are released to non-
target soils, water bodies and atmosphere, and finally absorbed by almost every
organism. A possible decline of amphibian population caused by synthetic pesti-
cides has long been suggested (Carey and Bryant 1995; Lips 1998). Davidson
(2004) reported association between the spatial patterns of declines for five amphib-
ian species in California and cholinesterase-inhibiting pesticides, mostly organo-
phosphates and carbamates. Pesticides have a particularly strong impact on birds
(Mitra et al. 2011), through direct deaths and the reduction or elimination of habitats
and food sources, including decreased levels of cereal grains, weed seeds, arthro-
pods etc. Birds are particularly susceptible to cholinesterase-inhibiting pesticides
mostly because, unlike mammals, they have low levels of anticholinesterase detoxi-
fying enzymes (Walker 1983). The effects of pesticides on earthworms (Yasmin and
D’Souza 2010), microarthropods (Adamski et al. 2009), nematodes (Zhao et al.
2013), fungi (Morjan et al. 2002) and microorganisms (viruses, protozoa and bacte-
ria) (Lo 2010; Imfeld and Vuilleumier 2012) within the soil may also have major
environmental consequences.
1.7 Effects on Beneficial Arthropods
For the past 30 years, the effects of pesticides on beneficial arthropods have been the
subject of an increasing number of studies. Insecticide treatments aiming at pest
control also have damaging effects on many non-target terrestrial arthropods
present in agroecosystems, including the natural enemies of agricultural insect pests

(Croft and Brown 1975). Damage to these species may be greater than initially
thought, because such damage can occur even at sublethal insecticide doses
(Desneux et al. 2007). A sublethal dose or concentration is defined as inducing no
apparent mortality in the experimental population.
Experiments on bee physiology have been done by measuring the activity of
enzymes after or during exposure to pesticides. Na+/K+ ATPase is a transmembrane
enzyme that releases energy necessary for cell metabolism. Bendahou et al. (1999)
reported that organophosphorus and pyrethroid led to a decrease in Na+/K+ ATPase
and AChE activities. Thus, the inhibition of Na+/K+ exchange provoked by insecti-
cides might affect a wide range of cellular functions.
Malformations also occur in natural insect enemies after exposure to pesticides
and may lead to reduction in predator or parasitoid efficiency and fitness.
Deltamethrin, a pyrethroid, causes marked dysfunctions in myocardial bee cells
(Papaefthimiou and Theophilidis 2001). Insect growth regulators (IGRs) that dis-
rupt molting and, more generally, act on endocrine systems (Dhadialla et al. 1998)
are also likely to perturb the development of beneficial arthropods. Malformation of
ovaries in the parasitoid Hyposoter didymator Thunberg (Hymenoptera:
Ichneumonidae) exposed to IGRs was reported by Schneider et al. (2004).
Hymenopteran social pollinators oral exposure with diflubenzuron, an IGR, reduced
brood surface area (Chandel and Gupta 1992), weight gain and suppressed develop-
ment of hypopharyngeal glands (Gupta and Chandel 1995).
Insecticides can also interact with the immune capacity of insects. Depending on
the type of insecticide, they can decrease or increase this capacity. Monocrotophos
and methyl parathion applied at 10% of the LC50 decreased the number of plasmato-
cytes in the hemolymph of the predator Rhynocoris kumarii Ambrose and
Livingstone (Hemiptera: Reduviidae) by 16% and 13%, respectively, whereas endo-
sulfan increased these cells by 15% (George and Ambrose 2004). Therefore, insec-
ticides may have an impact on both the immune capacity of a host and the capacity
of parasitoids to evade the host immune reaction. Studies reporting pesticide impacts
on the developmental period of natural enemies typically differ with the biology of
the experimental subject and also by gender. Zanuncio et al. (2003) reported that
exposure of the pentatomid predator Supputius cincticeps Stål (Heteroptera:
Pentatomidae) to permethrin decreased development time for females, whereas this
time increased for males. Reductions in fecundity associated with pesticides may be
due to both physiological and behavioral effects.
1.8 Pesticides Degradation
Microbial degradation of pesticides applied to soil is the main mechanism which

prevents the accumulation of these chemicals in the environment. Microbial adapta-
tion to pesticide degradation was recognized soon after their introduction into
markets, with the pioneer research on biodegradation of 2,4-D (Audus 1949). In a

recent study, Paul et al. (2013) noticed that Azotobacter chroococcum strains
degraded more than 90% of lindane, an organochlorine, under laboratory condi-
tions. When these degraded compounds were tested against lepidopteran larvae, a
lower mortality was recorded against the very high mortality induced at all concen-
trations by non-degraded lindane.
One key factor associated to increased rates of microbial pesticide degradation in
soil is the history (number) of previous applications of the same pesticide, or of
other compounds, having a similar molecular structure. This phenomenon is known
as “accelerated” or “enhanced degradation” (Racke 1990). The list of pesticides
affected by accelerated degradation is long and constantly growing. In a review,
Arbeli and Fuentes (2007) summarized the accelerated degradation processes, pro-
viding a list of important susceptible pesticides. One way to cope with the problem
of reduced pesticide efficacy due to enhanced microbial degradation would be to
increase the amount and/or frequency of applications, which may further generate
other ecosystem problems. Thus, the increased difficulty of pest control clearly
indicates the need for more sophisticated pest management, as the paradigm of rely-
ing almost exclusively on chemicals for pest control may need to be reconsidered.
Insecticides and nematicides known to undergo accelerated degradation / loss of
efficacy are listed as: Aldicarb, Bendiocarb, Benfuracarb, Cadusafos, Carbaryl,
Carbofuran, Carbosulfan, Chlorfenvinphos, Chlorpropham, Chlorpyrifos,
Cloethocarb, Diazinon, Ethoprophos, Fenamiphos, Fensulfothion, Fonofos,
Furathiocarb, Isazofos, Isofenphos, Malathion, Mephosfolan, Methylparathion,
Oxamyl, Parathion, Phorate, Terbufos, Trimethacarb (Arbeli and Fuentes 2007).
1.9 Conclusion
Adverse effects of pesticides are wide and varied, as they hold a unique position
among environmental contaminants. Due to unregulated use and lack of enforce-
ment mechanisms, health hazards caused by pesticides are serious, especially in
developing countries, and no groups in the human population are completely unex-
posed to pesticides or related residues.
For almost all major insecticide classes, resistance has been reported in one or
more key insect pest species. The complex patterns of cross resistance between
chemical groups and within groups further complicate pest management strategies
with chemicals. Direct and indirect adverse effects of pesticides on several non-
target wild vertebrates are also widely documented. To cope with the problem,
instead of relying almost exclusively on chemicals, eco-friendly alternative meth-
ods for insect pest management must be considered.
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Chapter 2
Soil-Borne Entomopathogenic Bacteria
and Fungi
Tan Li Peng, Samsuddin Ahmad Syazwan, and Seng Hua Lee
Abstract Being rich in microorganisms, the soil is an ideal environment and impor-
tant reservoir for harvesting various types of beneficial microorganisms. Soil-borne
entomopathogenic bacteria and fungi have been regularly isolated around the world
to support crop producer in the never-ending arms race of pest management. Among
these microorganisms, entomopathogenic bacteria and their toxins are the most suc-
cessful microbial insecticides also from the commercial point of view. They grouped
into spore- and non-spore-forming entomopathogens, in which the infection process
starts upon ingestion by the susceptible insect hosts. Fungi, on the other hand,
remain relatively underutilized as natural enemies despite their many advantages
over other biological and chemical products. They mainly classified under the class
of Entomophthoromycetes and Sordariomycetes in the larger Ascomycota division,
which consists around 65,000 described species. In comparison to bacteria, fungi
have a wider host range and are especially suitable for controlling pests with pierc-
ing and sucking mouthparts. Entomopathogenic bacteria and fungi can be released
through inundative application methods and therefore play a critical role in inte-
grated pest management (IPM) against several pests. This chapter provides a selec-
tive review on the different types of soil-borne entomopathogenic bacteria and fungi,
including their distribution, infection mechanisms and host ranges.
T. L. Peng (*)
Department of Paraclinical, Faculty of Veterinary Medicine, Universiti Malaysia Kelantan,
Kota Bharu, Kelantan, Malaysia
S. A. Syazwan
Department of Forest Management, Faculty of Forestry, Universiti Putra Malaysia,
Serdang, Selangor, Malaysia
Mycology & Pathology Branch, Forest Biodiversity Division, Forest Research Institute
Malaysia, Kepong, Selangor, Malaysia
S. H. Lee
Institute of Tropical Forestry and Forest Products, Universiti Putra Malaysia,
Serdang, Selangor, Malaysia

https://doi.org/10.1007/978-3-030-23045-6_2
24 T. L. Peng et al.
Keywords Soil-borne · Entomopathogens · Bacteria · Fungi · Bio-insecticides
2.1 Introduction
Soil, the unconsolidated mineral or organic material on the immediate surface of

Earth, is the natural medium for the growth of land plants. It is also a natural
body comprised of solids, liquid, and gases that harbours a complex, living and
unique community on its own. This soil microbial community plays several
important ecological and physiological functions (Sofo et al. 2014). In agroeco-
systems, the soil microbial communities are essential for plant nutrition and
health (Gattinger et al. 2008).
Soil microbial community in a narrow sense consists of viruses and organisms
such bacteria, fungi, algae and protozoa (Sims 1990), that support a food web
formed by nematodes and other micro-arthropods (Jones et al. 2010). Soil microor-
ganisms in general maintain balances within soil through various roles, acting as
e.g. decomposers, nitrogen binders, pathogens, etc. (Waldrop et al. 2000; Glick
2010). They contribute to ensure soil health by sustaining its structure and composi-
tion, in turn sustaining plants’ growth (Jacoby et al. 2017). Even more interesting,
some of these organisms are able to protect crops and plants by regulating harmful
pests such insects and other arthropods (AGP – FAO 2018).
Microorganisms that are able to cause a disease on an insect pest and, in some
extent on other arthropods, are known as entomopathogens (Tanzini et al. 2001).
They are natural enemies that kill or debilitate pests, keeping their populations
under control (Lacey et al. 2015). Entomopathogens also represent a large compo-
nent of the world’s biodiversity. There are for example about 750–1000 species of
fungi being causal agents of insect diseases (St. Leger and Wang 2010). Although
extensive studies and researches have been performed on entomopathogens, either
as causal agents in a natural condition or in exploitation for biological control
(Davidson 2012), only a small proportion of available species has been studied in
depth for crop protection, and subsequently commercialized (Lacey et al. 2015).
Among the mentioned soil microorganisms, bacteria and soil fungi are the most
abundant (Beed 2011). Therefore, much interests and efforts have been invested in
these two major groups in biological pest control. As for bacteria, also the propor-
tion of fungi known to infect insects and several other arthropods is large.
Entomopathogenic fungi can be taxonomically divided into 6 phyla:
Chytridiomycota, Blastocladiomycota, Kickxellomycotina, Basidiomycota,
Ascomycota and Entomophthoromycota (previously a subphylum under
Zygomycota). Among these 6 phyla, substantial numbers of insect pathogenic spe-
cies are found in Ascomycota and Entomopthoromycota (Mora et al. 2017). The
two most important orders are the Entomophthorales (Entomopthoromycotina:
Entomophthoromycetes) and the Hypocreales (Pezizomycotina: Sordariomycetes).
2 Soil-Borne Entomopathogenic Bacteria and Fungi 25
Detailed life-cycles of entomopathogenic fungus belonging to Entomophthorales

and Hypocreales have been described (Augustyniuk-Kram and Kram 2012). Slight
differences are present between these orders, mostly considering two stages: (i)
proliferation, whereby Hypocreales species proliferate inside the host body in the
form of yeast-like hyphal bodies, multiplying by budding (Prasertphon and Tanada
1968), whereas members of Entomophthorales produce protoplasts (Butt et al.
1996); and (ii) sporulation, whereby Hypocreales produce only asexual spores or
sexual spores after host death (known as hemibiotrophic), whereas Entomophthorales
can produce both asexual and sexual spores before host death (termed biotrophic)
(Roy et al. 2006).
Entomopathogenic fungi are widespread throughout the world, ranging from
Antarctic to the Arctic Circle (Bridge et al. 2005; Eilenberg et al. 2007), reaching
highest abundance at the equator level (Aung et al. 2008)(. Different habitats are
known to be occupied by different group of entomopathogenic fungi, the distribu-
tion of these fungi being very much influenced by both biotic and abiotic factors.
For instance, Hypocreales dominate soil layers, whereas Entomophthorales are
mostly found at the arboreal level (Vandenberg and Soper 1978; Sosnowska et al.
2004). These differences could be mainly due to the presence of hosts at different
habitat niches and to abiotic factors that affect the transmission of fungi.
Various aspects collectively contribute to the “success” of an entomopathogenic
fungus. An advantage, however, does not necessarily imply that a specific fungus is
the best candidate for biological control. Hypocreales, for example, have a vast
spectrum of potential victims but may not be suitable when beneficial insects (i.e.,
parasitoids) occur, that might also be infected by these fungi (Augustyniuk-Kram
and Kram 2012). The relatively host-specific and obligate Entomophthorales, on the
other hand, might not be sustainable in the environment after their host has been
being wiped out and a continuous augmentation effort might be needed, is not cost-
effective (Pell et al. 2001).
Regardless of their systematics and even biology, these fungi and bacteria are all
pathogenic, and their effectiveness in infecting their hosts is so significant that they
can become a crucial factor regulating the population of many insects. Nevertheless,
the success of their use requires a thorough knowledge about the biology and ecol-
ogy of both fungi and hosts. It also requires recognition of abiotic and biotic factors
that may interfere with their effectiveness. Biological control, rather than entirely
replacing chemical products, is intended to reduce their use through their integra-
tion with other controlling measures to keep pest populations at a suitable, low
damage level.
Soil-borne entomopathogenic bacteria and fungi regularly isolated around the
world for pest management (Bruck 2004). The use of entomopathogenic bacteria
and fungi in pest management systems has a long and rich history. Despite a variety
of obstructions, there are plenty of opportunities and benefits for the use of natural
enemies in insect pest management. In general, both entomopathogenic bacteria and
fungi play a critical role in integrated pest management (IPM) due to environmental
and human safety concerns, development in pests of resistance to insecticides,
increases in pesticides costs, etc. (Gangwar 2017). This chapter provides a selective
review on the different types of soil-borne entomopathogenic bacteria and fungi,

including their distribution, the primary infection mechanisms and their host ranges.
2.2 Soil-Borne Entomopathogenic Bacteria
Most of the discovered entomopathogenic bacteria are soil-borne aoecies such as

Brevibacillus laterosporus (Oliveira et al. 2004; Ruiu et al. 2007), Lysinibacillus
sphaericus (Persinoti et al. 2018), Clostridium bifermentans (Leja et al. 2011),
Serratia entomophila (Villalobos et al. 1997), and numerous other entomopatho-
gens. Bacteria are one of the most diversified microbiota existing in soil (Riesenfeld
et al. 2004). Soil biota refers to organisms living in the ground, interacting within its
environment, and involved in many ecological processes (Ritz et al. 2004).
Entomopathogenic bacteria provided the most common biological control agents
for the management of insect pest populations in plantations. There are two main
groups of entomopathogenic bacteria classified by their abilities to sporulate, either
obligately or facultatively. Meanwhile, the facultative-sporulating group also
divided into two groups known as crystalliferous, with the ability to form crystalline
toxins, or non-crystalliferous species.
More than a century ago, the discovery of entomopathogenic bacteria began with
the first isolation from diseased silkworm larvae, Bombyx mori (Ishiwata 1901). The
curiosity to examine a diseased silkworm and describing it as the sotto disease led
to the first discovery of entomopathogenic bacteria in the world (Ishiwata 1901).
The study continued by describing the morphology of isolated Bacillus spp. from
Mediterranean flour moth, Anagasta kuehniella, named as Bacillus thuringiensis; in
conjunction with the name of the city in which the moth found, Thuringia, in
Germany (Berliner 1915). The discovery of Bacillus thuringiensis, also known as
Bt, kick-started the biological pesticide discovery and subsequent developments,
with applications depending on the technology availability, the industry demand
and the raising awareness towards the impact of pesticides on environment and
climate.
2.2.1 Isolation
Even though almost all of the studied entomopathogenic bacteria are soil-borne spe-
cies, few new species of entomopathogenic bacteria directly discovered from soil
samples. Most findings proceeded either from diseased insects or insects’ cadavers.
Despite this issue, several studies involved the isolation of soil-borne entomopatho-
genic bacteria, from various sampled places and soil conditions. Bacillus thuringi-
ensis, Paenibacillus popilliae and Paenibacillus lentimorbus discovered from
diseased insect larvae (Ishiwata 1901; Berliner 1915; Dutky 1940). Chromobacterium
substugae, capable of killing the Colorado potato beetle larvae, Leptinotarsa
decemlineata was directly obtained from forest soil samples in central Maryland,
USA (Martin et al. 2007).
Several protocols were applied, which resulted in many soil-borne entomopatho-
genic bacteria isolated from soil samples. By using 1 g of soil, different dilution
techniques, and treatments applied before streaking on to media. Based on Ernandes
and Da Rosa experiment, the soil sample was dispersed into 10 mL saline solution,
serial diluted, heated for 12 min at 80 °C and kept on ice for 5 min before streaked
on nutrient agar medium (Ernandes and Da Rosa 2014). Meanwhile, González cul-
tured 1 g of soil in nutrient broth, incubated for 24 h at 30 °C, shook at 150 rpm
before seeding on nutrient agar (González et al. 2013). Several techniques can
also be applied to confirm the isolated bacteria species, including traditional Gram
staining, phenotypic description, determination of respiratory type, colony and cul-
ture morphology and also biochemical tests, that may aid in the validation of the
isolates species.
2.2.2 Mechanism of Action
The insecticidal mechanism of Bacillus thuringiensis is well known by the scientific

community. Over a century of works and studies focusing on this remarkable spe-
cies yielded fruitful informations on each stage infecting the insects host. By notic-
ing the occurrence of a second body in the sporangium from the Steinhaus
publication in 1951, also present in the Berliner previously published the image,
Hanney hypothesized that the body inidcated as a “parasporal crystal” had a major
role in pathogenesis (Berliner 1915; Steinhaus 1951; Hanney 1953). The production
of parasporal crystals, also known as δ-endotoxin (Cry proteins), within the Bt spo-
rangium followed the sporogenesis time course, being approximately completed
within 24 h in typical bacterial media (Federici et al. 1990). Brevibacillus laterospo-
rus also produced similar parasporal crystals which also have an insecticidal activ-
ity on the infected hosts (Zubasheva et al. 2010). Variation in parasporal crystals
produced by different B. laterosporus strains in different hosts were also discov-
ered. Different insecticidal roles of excreted toxins were shown in the infection of
various species of Coleopteran hosts.
The insecticidal crystal is activated once ingested into the insect digestive sys-
tem, harming its gut membranes by a pore-forming mechanism (Pigott and Ellar
2007). The alkaline condition of insect midgut solubilizes the ingested crystal pro-
teins that are processed into the active form, due to reactions with midgut-excreted
proteases (Lee et al. 2003). Subsequently, activated parasporal proteins(δ-
endotoxins) attach to the receptor sites on the gut epithelium causing the formation
of pores (Martin et al. 2007; Chilcott et al. 1983). This process will imbalance
osmotic regulations thus leading to a midgut paralysis and finally cell lysis. Rupture
of insects midgut will produce a solution leakeage within the insects cavity, imbal-
ancing the hemocoel pH level and causing a septicaemia, leading the infected
insects to death.
The specificity of entomopathogenic bacteria towards hosts depends on the pres-

ence of a corresponding cellular receptor of the crystal proteins in the host of inter-
est. The suitable cellular receptors for the δ-endotoxin to bind and activate are vary
and show a high affinity binding site, affecting host specificity and selection of
entomopathogenic bacteria species (Honée and Visser 1993). In conclusion, several
criteria should be taken to ensure the success of entomopathogenic bacteria applica-
tion on insects pests, which are: (1) optimal condition of insects midgut (pH of
midgut juice), (2) presence of suitable cellular binding receptors for the δ-endotoxin
activation on infected gut epithelium, and (3) production of effective proteases
digesting the activated δ-endotoxin.
2.3 Soil-Borne Entomopathogenic Fungi
Back to 1888, the Russian microbiologist Elie Metchnikoff was the first to identify
an entomopathogenic fungus from a wheat cockchafers. Krassilstick then mass-
produced the same fungus and used in the field against the sugar-beet weevil (Vega
et al. 2009). His work after that ignited curiosity around the world for experimenta-
tion with “friendly fungi” against insect pests (Lord 2005), and searching for poten-
tial natural enemies towards insect pests was springing since then, until now. There
are different groups of entomopathogenic fungi in various habitats, among which
soil is the environment where these fungi may be most commonly found (Keller and
Zimmerman 1989).
Entomopathogenic fungi are a group of phylogenetically diverse and heterotro-
phic microorganisms. They are taxonomically classified into the six previously
mentioned phyla (Mora et al. 2017) although some researchers group them into five
phyla namely Chytridiomycota, Mycosporidia, Basidiomycota, Ascomycota and
Entomophthoromycota (Araújo and Hughes 2016). Ascomycota and
Entomophthoromycota are the two phyla with the most important groups of ento-
mopathogenic fungi due to their substantial numbers (Maina et al. 2018) and thus
focused on in this chapter.
The occurrence and distribution of entomopathogenic fungi vary and mainly
depend on the behavioural patterns of their hosts. Although the emphasis will be
given to soil-borne Entomophthorales and Hypocreales, the occurrence of these
fungi in aquatic and arboreal habitats are equally crucial as within these habitats
they contribute to the regulation of arthropod populations. Entomophthorales, pre-
viously under Zygomycetes, are one of the lower fungi predominantly known as
associated with aquatic habitats. This clade shows remarkable adaptations in which
up to four spore types can be produced by the same species to facilitate dispersal
and infection, both aerially and aquatically (Descals and Webster 1984).
Both Entomophthorales and Hypocreales are abundant in forest habitats, includ-
ing untouched humid, tropical forests or man-managed coniferous forests. They are
especially ubiquitous at the understorey and below (Fig. 2.1), where the forest can-
opy buffers these layers from extremes of temperature and humidity, creating a
Fig. 2.1 Diagram of the forest layer showing entomopathogenic fungi evolutionary diversity in
forest habitats (Samson et al. 1988). Although both Entomophthorales and Hypocreales found
under the canopy layer, they dominate and develop succession at different layers, where forest soil
litter and surface are dominated by Hypocreales, whereas understory trees and canopy mostly
show Entomophthorales. (Sosnowska et al. 2004)
stable microclimate conducive to continual fungal activity, even during extreme

seasons (Samson et al. 1988).
2.3.1 Entomophthorales (Entomopthoromycotina:

Entomophthoromycetes)
Entomophthorales (Schröter, 1893) is an order now treated under

Entomophthoromycota, containing soil saprotrophic, opportunistic, broad-
spectrum, or specialized insect pathogenic genera (Humber 2012). There are
approximately 280 species in the phylum Entomophthoromycota, classified within
three classes and six families (Humber 2012; Gryganskyi et al. 2013).
Entomophthorales is the only order in class Entomophthoromycetes and consists of
approximately 17 genera classified under four families (Humber 2012). This order
is notable for the epizootics they induce in populations of many insect orders,
including Hemiptera, Homoptera, Lepidoptera, Coleoptera, Orthoptera and Diptera.
The modes of formation and germination of resting spores and the nature of
vegetative growth and development are used to separate families (Humber 1989).
The classification of genera in Entomophthorales, at present, follows the three sys-
tems proposed by Humber (1989), Keller (1991) and Balazy (1993). They classified
according to: (1) morphology of primary and secondary conidia, conidiophores
and/or conidiogenous cells; (2) number of nuclei per conidium; (3) mode of dis-
charge and formation; (4) pathobiology (e.g., host symptoms). Further down, the
details of the primary conidia (size and shape); the number of nuclei per conidium
and the basis of host insect species in the species identification (Balazy 1993).
Entomophthorales have conidia and resting spores at different stages of life-
cycle, divided into: (1) conidial cycle and (2) resting spore cycle (Fig. 2.2). The
former allows the propagation and spread of the disease, the latter provides to the
Fig. 2.2 General scheme of the Entomophthorales life-cycle. (a): conidiophores within infected
host grow and emerge out from the cadaver; finger shaped conidiophores show one apical spore,
which is then forcibly ejected to drift on winds until they attach to another fly and start the cycle
again. (b): infected host is compelled to find soil to land on; its abdomen becomes brittle and even-
tually falling apart to release the resting spores into the soil; the spores remain dormant throughout
unfavourable conditions (winter) and attach to emerging hosts in favourable conditions (early
spring). (c): host are infected by conidia drifted on winds, or by direct contact with cadavers
(asexual), or by spores when in contact with soil where the spores resided. (d): infection starts with
the conidia, asexual, non-motile fungal spores, attaching to a fly; the primary conidia then pierce
through the exoskeleton with germ tubes; one long germ tube arises from the secondary conidium
to branch out inside the insect hemocoel; hyphae then grow and start consuming the hemolymph
and internal organs. (e): resting spores attached to the host may start an infection in resting spores
cycle, as in the conidial cycle. (Roy et al. 2006)
survival during unfavourable conditions. Both cycles are supposed to start in the
same way, in which the conidia or spores adhere to the host cuticle and form a pen-
etration tube. Unlike conidia, it is unknown at which stage resting spore formation
is induced and if there are changes at the nuclear level. Resting spores are described
as azygospores, however, their mode of formation has never been observed in detail.
The infection mechanism of entomopathogenic fungi is generally the same. For
a successful infection, the entomopathogenic fungus must first able to penetrate the
insect cuticle. In Entomophthorales, the conidia can easily achieve their adhesion
process by having A larger size and extra cell wall layers or mucus, that enable them
to attach firmly to the host cuticle (Eilenberg et al. 1986). Conidia then germinate
and produce germ tubes that penetrate through the pores or the layers of the epicu-
ticle, procuticle and epidermal via mechanical pressure and production of cuticle-
degrading enzymes (Vega et al. 2012). After reaching the hemocoel, the multiplication
takes place either by protoplasts or by hyphae. These structures colonise the abdo-
men or, more commonly, the whole body of the host. Most host species die at this
stage, as a result of a combination of effects that comprise physical damage of tis-
sues, toxicity, cells dehydration due to loss of fluids, and consumption of nutrients.
Many genera in this order contain obligate insect-pathogenic species, character-
istically biotrophic with a narrow host range, capable of producing epizootics in
natural insect populations and common among foliar insects (Pell et al. 2001). For
instance, if conditions are right, one Entomophthorales species can cause epizootics
of flies, with a 70–90% prevalence, killing the majority of flies. This makes mem-
bers of this lineage as good candidates for biological control of multiple fly popula-
tions. Some important genera and their corresponding host range are summarized in
Table 2.1.
2.3.2 Hypocreales (Pezizomycotina: Sordariomycetes)
The order Hypocreales (Lindau, 1897) classified in Sordariomycetes, one of the

largest classes in the Ascomycota with more than 600 genera and 3000 known spe-
cies (Kirk et al. 2008). This order consists of biotrophic fungi: they are mainly plant
and insect pathogens, mycoparasites, and endophytes, as well as saprotrophic spe-
cies (Sung et al. 2008; Zhang et al. 2006). Many important entomopathogenic fungi
that have been developed into commercial mycoinsecticides belong to this order,
including species of genera Lecanicillium, Isaria, Beauveria, Metarhizium,
Pochonia, Cordyceps and others. Most of these species may be cultured artificially,
thus are relatively easy for mass production. Although efforts in developing them as
inundative biological control agents cannot be obliterated, in biocontrol strategies
understanding of the ecology of entomopathogenic fungi are equally essential to
achieve sustainable effects (Rai et al. 2014).
Pezizomycotina is the most numerous, morphologically, and ecologically com-
plex ascomycetes (Schoch et al. 2009). Members of the Hypocreales, without
exemption, are morphologically diverse with complex life cycles often with two
Table 2.1 Most studied Entomophthorales (including main lineages in the family
Entomophthoraceae) and associated hosts
Genus Host References
Batkoa Homoptera (aphids) Humber (2005)
Hemiptera
Coleoptera
Diptera
Lepidoptera
Conidiobolusa Aphids, other Homoptera Humber (2005)
Other insects (but weak
pathogen)
Entomophaga Lepidoptera Humber (2005)
Othoptera
Entomophthora Muscoid flies,other Diptera Humber (2005)
Aphids, other Homoptera

Erynia Aphids, other Homoptera Li and Humber (1984), Steinkraus and
Diptera Kramer (1989), and Capinera (2008)
Lepidoptera
Trichoptera
Eryniopsis Coleoptera (Cantharidae) Prischepa et al. (2011)
Diptera (Nematocera) Steinkraus et al. (2017)
Furia Diptera (Calliphoridae; Samson and Nigg (1992)
Rhagionidae)
Lepidoptera Prischepa et al. (2011)
Macrobiotophthoraa Nematodes Bernard and Arroyo (1990)
Massospora Cicadidae (Magicicada spp.) Cooley et al. (2018)
Meristacruma Nematodes Gryganskyi et al. (2013)

Orthomyces Homoptera (Aleyrodidae) Steinkraus et al. (1998)
Pandora Homoptera Humber (2005)
Sacrophagid flies, other Diptera
Plutella xylostella, other

Lepidoptera
Strongwellsea Umbonia sp. Samson et al. (1988) and Chien and
Acraea sp. Hwang (1997)
Zoophthora Aphids, other Homoptera Humber (2005)
Weevils, other Coeloptera Capinera (2008)
a
Taxa under other families with biocontrol importance: Macrobiotophthora, Conidiobolus =
Ancylistaceae; Meristacrum = Meristacraceae (Humber 2012)
stages, anamorph (asexual stage) and teleomorph (sexual stage), often leading to the
identification of the same species with two different taxonomic names, one per
stage. However, in several cases, the sexual stage of some Hypocreales may never
or only rarely be produced, and anamorphs are used for taxonomic purposes (Taylor
2011; Mora et al. 2017). Besides, sequences from the nuclear small subunit
ribosomal RNA gene (nSSU rDNA) or other genes are also employed to solve the
complexity in classifying this order, to adopt the principle of “one fungus, one
name” (Zhang et al. 2006; Hawksworth 2011).
The Hypocreales once contained more than 70 genera in four major families
(Rossman et al. 1999). Subsequent estimates showed approximately 237 genera and
2647 species, in seven families (Kirk et al. 2008). Among this order, the former
unique family Clavicipitaceae has split into three monophyletic families:
Clavicipitaceae, Cordycipitaceae, and Ophiocordycipitaceae, including the most
well-known entomopathogenic fungi. These three families are generally distin-
guished based on the host infected. For instance, spider pathogens are mostly found
within the Cordycipitaceae, while Clavicipitaceae are common in scale. Pathogens
of ants, termites, or dipterans are often found in the Ophiocordycipitaceae (Sung
et al. 2007). Other insect orders such as Hemiptera, Coleoptera, Lepidoptera,
Thysanoptera, and Orthoptera also comprise most of the targets.
The sexual (teleomorphic) stages of Hypocreales appear to be more host special-
ized, while their asexual (anamorphic) counterparts appear as more generalist.
Among these entomopathogenic fungi, asexual stages often precede the production
of sexual stages (Sung et al. 2007). Many Hypocreales have a well-developed para-
sitic phase that infects the host’s body. Taking the most commercialized species
Beauveria bassiana as an example, it is the anamorph of Cordyceps bassiana (Li
et al. 2001). The infection pathway of this species begins with attachment of conidia
to the insect cuticle, followed by germination and penetration through the cuticle
and proliferation within the host. This fungus is then switching to a saprotrophic
nutrition (hemibiotrophic), by colonizing the cadaver to maintain hyphal growth
and produce new conidia, even after the host’s death (Evans 1988). Beauveria bassi-
ana is well characterized for infecting several insects. It is also recorded with vari-
able strains which differ substantially in their ability to produce pathogenesis.
Cordyceps bassiana, on the other hand, shows a more specialized behaviour
(Bhushan et al. 2016). It is, perhaps, the most studied sexual stage in association
with arthropods. The host range of Cordyceps, in a classical sense, is very broad
and includes several orders such as Hymenoptera, Hemiptera, Orthoptera, Diptera,
Blattodea, Mantodea, Dermaptera, Odonata, Phasmatodea etc. Coleoptera and
Lepidoptera are the major host orders in which nearly 60% of Cordyceps species
are recorded. Cordyceps are not monophyletic, as teleomorph stages occur in all
the three families mentioned above. It is an intriguing genus considering the same
fungal species differ in their ecology, depending on the reproduction stage
(Hesketh et al. 2009).
Entomophthorales and Hypocreales are the two main orders of entomopatho-
genic fungi distributed in wide terrestrial ecosystems. They are playing vital roles in
regulating the insect density, balancing the ecosystem in natural conditions, and
Table 2.2 Differences between Entomophthorales and Hypocreales

Entomophthorales Hypocreales
Narrow host range (Burges 1981; Pell et al. Narrow host range (Teleomorph) to Broad
2001) host range (Anamorph) (Vega et al. 2012)
Dominantly distributed in temperate forests Dominantly distributed in tropical forests
(Evans 1982) (Evans 1982)
Biotrophic (Charnley 2003; Charnley and Biotrophic Hemibiotrophic (biotrophic +
Collins 2007) saprotrophic) (Charnley and Collins 2007)
Penetrate directly using germ tubes (Hajek and Penetrate using appressoria (Shah and Pell
Delalibera 2010) 2003; Sandhu et al. 2012)
Proliferation trough protoplasts (Boomsma et al. Proliferation trough hyphae (Boomsma
2014) et al. 2014)
Toxin production rarely found / not known The toxin produced (Freimoser et al. 2003b;
(Boguś and Scheller 2002; Freimoser et al. Sandhu et al. 2012)
2003a)
Transmission Transmission
Forcibly discharged: asexual conidia Forcibly discharged: sexual ascospores
Passively released: sexual resting spores Passively released: asexual conidia
(Boomsma et al. 2014) (Boomsma et al. 2014)
reducing crop damage in many crops. They share some characteristics in common,
yet showing uniqueness in many other ways (Table 2.2).
2.4 Current Status as Biopesticides
Controlling the insect pest population is always a worldwide priority, with many
industries and areas of study directly or indirectly connected and responding to this
need. The increasing of the insect pest population is often due to poor agricultural
practices, affecting crop yields, with severe impacts on the economy and food secu-
rity issues. Also, awareness towards environmental conservation is rising world-
wide, and the application of highly toxic pesticides forbidden in many countries.
Therefore, biopesticides emerge as the times required. Sporleder and Lacey (2013)
stated that differing from conventional chemical pesticides; biopesticides are bio-
control agents-containing products used to control pest through specific biological
effects. Biopesticide products potentially can be categorized into microbial insecti-
cides, Plant-Incorporated Protectants (PIPs), and biochemical pesticides. Among
these three classes, microbial pesticides contain microorganisms such as bacteria,
fungi, or virus that kill insects and weeds. This section focuses on the application of
soil-borne entomopathogenic bacteria and fungi as biopesticides.
Using entomopathogenic bacteria and fungi as biopesticides offer some advan-
tages such as: (1) they are more environment-friendly compared to chemical pesti-
cide; (2) they are unharmful to mammals and other non-target organisms; and (3)
they allow a prolonged pest control owing to their higher persistence (Singh et al.
2017). Nevertheless, some drawbacks have also identified regarding biopesticides
Table 2.3 Example of commercially available biopesticides derived from entomopathogenic

fungi and bacteria (Chandler et al. 2011; Mishra et al. 2015; Singh et al. 2017)
Microorganism Product name Target
Fungi
Beauveria bassiana Mycotrol® Coding moth, pine caterpillar, European
corn borer
Culicinomyces clavisporus Mycar Mosquito larvae
Coniothyrium minitans Contans WG Sclerotinia spp.
Chondrostereum purpureum Chontrol Cut stumps of hardwood trees and shrubs
Paecilomyces lilacinus MeloCon WG Plant parasitic nematodes in the soil
Aureobasidium pullulans Blossom Protect Fire blight, postharvest diseases
Bacteria
Bacillus thuringiensis var Dipel DF Caterpillars
kurstaki
Bacillus subtilis QST713 Serenade ASO Botrytis spp.
Pasteuria usgae Pasteuria usgae Sting nematode (Belonolaimus
BL1 longicaudatus)
Agrobacterium radiobacter Galltrol – A Crown gall disease
k84
Pantoea agglomerans C9-1 BlightBan C9-1 Fire blight
including: (1) longer time is required to observe their effects on targeted hosts, com-
pared to chemical pesticides; (2) higher costs; (3) different control measures are
needed, due to their high degree of host specificity; (4) a frequent treatments are
necessary to maintain a long-term impact (Singh et al. 2017).
Many biopesticides, mostly based on bacteria and fungi, have already been for-
mulated and commercially manufactured to tend the global market. According to
Kabaluk et al. (2010), the whole biopesticide market accounts for 60% bacterial
biopesticides, followed by fungal (27%), viral (10%) with a remaining 3% as
“other” biopesticides. The most common bacterial insecticides include Bacillus
species, with B. thuringiensis (Bt) as the most widely used (Mishra et al. 2015;
Khan et al. 2016). Table 2.3 lists some examples of commercially available biopes-
ticides derived from entomopathogenic fungi and bacteria.
2.5 Conclusion
Soil is rich in microorganisms and serves as an ideal reservoir for harvesting various
types of beneficial species of entomopathogenic bacteria and fungi. Many biopesti-
cides derived from these microorganisms are used in pest management. Many
cutting-edge innovations and developments in the industry enhanced the effective-
ness and applicability of bacteria and fungi as a biological control agent. They may
complement chemical pesticide applications to build up optimal protocols for insect
pest management. Discovery of most adapted biological control agents such as
entomopathogenic bacteria and fungi is one of the steps required for sustainable
IPM practices. They may reduce the environmental impact of pests by decreasing
the amount of chemical pesticide usage in the plantation industry. This concept
indirectly will reduce carbon footprint and thus helps in combating climate change.
Unfortunately, despite the wide variety of commercialized products, the usage of
biopesticides worldwide is still not encouraging. The lack of awareness among
farmers is one of the main constraints that limits the prevalence of biopesticides.
Application of chemicals is highly price-dependent, as conventional chemical pes-
ticides lower price and longer shelter life appear more attractive. Practically, being
often highly specific, biopesticides are not as effective or generalist as chemical
pesticides and occasionally, they may perform inconsistently in the field, inducing
farmer to lose trust in their efficacy. Therefore, the intervention of government is of
utmost important to achieve a balance between the two approaches, through the
definition of more stringent rules on the use and impact of conventional chemical
pesticides. Research and development of more effective and cheaper biopesticides
is also vital in order to promote and encourage people to adopt safer IPM approaches.
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Chapter 3
Molecular Phylogeny of Entomopathogens
Mudasir Gani, Taskeena Hassan, Pawan Saini, Rakesh Kumar Gupta,

and Kamlesh Bali
Abstract Insects, like other organisms, are susceptible to a variety of diseases

caused by bacteria, viruses, fungi, protozoan and nematodes. Insect pathogens show
a wide variety of interactions with their hosts that facilitate their replication and
transmission, including strategies for evading the host’s defences towards inva-
sion of microorganisms, and for manipulating their hosts physiology and behaviour.
By applying a wide range of molecular techniques and approaches, better under-
standings of these interactions and of the roles played by both host and virulent
genes have been understood.
The control of insect pests with entomopathogens is an unique approach, in that
naturally occurring host-pathogen relations are manipulated to the benefit of man,
protecting agricultural crops and forests or controlling insect vectors of diseases.
The isolation and identification of a pathogen followed by the phylogenetic classifi-
cation of entomopathogens are the basic principles in insect pathology. Full genomic
DNA sequencing techniques are used to assess the genetic diversity and phyloge-
netic analyses of entomopathogens. Alternatively, specific genes of interest can be
targeted for sequencing. Sequences of single gene have been extensively used to
assess phylogenetic relationships of known and novel isolates. However, the lack of
sufficient resolution and disagreement with other gene phylogenies has prompted
investigation of other genes and methods to further explore evolutionary relation-
ships of entomopathogens. Therefore, phylogenies based on combined sequences of
shared genes or the complete genome sequencing have been found to be more
robust, providing more phylogenetic information and increasing robustness of evo-
M. Gani (*) · P. Saini

Central Sericultural Research & Training Institute, Central Silk Board, Ministry of Textiles,
Govt. of India, Kashmir, Jammu and Kashmir, India
T. Hassan
Department of Zoology, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
R. K. Gupta · K. Bali
Division of Entomology, Sher-e-Kashmir University of Agricultural Sciences and
Technology, Jammu, Jammu and Kashmir, India

https://doi.org/10.1007/978-3-030-23045-6_3
44 M. Gani et al.
lutionary hypotheses. With the increasing efficiency and lower cost of whole
genome sequencing, whole-genome studies of entomopathogens will refine knowl-
edge about their evolutionary history and enable direct insight into the biology of
entomopathogenesis.
Keywords Entomopathogen · Evolution · Genomics · Phylogeny · Taxonomy
3.1 Introduction
Insects constitute the most diverse group of living organisms in the world, with
more than one million species. They live almost everywhere, from steamy tropical
jungles to cold polar regions. The microorganisms that are capable of causing dis-
eases in insects are known as Entomopathogens (Greek entoma = insect, pathos =
suffering, gennaein = to produce). The disease causing agents are ubiquitous in
nature and have the capability to invade and reproduce in an insect and spread to
infect other ones.
Several species of naturally occurring noncellular agents (viruses), prokaryotes
(bacteria), eukaryotes (fungi and protists) and multicellular animals (nematodes)
infect a variety of insects and cause diseases (Table 3.1). Insect diseases include: (a)
diseases of productive insects (honey bees, silkworm, lac insect); (b) diseases of pol-
linators (e.g. honey bees, bumble bees, Syrphid flies etc.); (c) diseases of biological
control agents (Coccinellids, predatory stink bugs, praying mantis etc.); (d) diseases
in natural insect pest populations (gypsy moth, codling moth, Alfa alfa looper etc.)
and (e) diseases of edible insects (grub, caterpillar etc). These infectious microorgan-
isms can be separated into four broad categories of opportunistic, potential, facultative
and obligate pathogens based on their capability to infect hosts (Onstad et al. 2006).
One the basis of the route of infection, the entomopathogens are classified as
ingested (viruses, bacteria, protozoa) and penetrating microorganisms (fungi and
nematodes). All of them effectively suppress pests when applied artificially as
microbial pesticides or biopesticides (Saxena 2008) (Table 3.2). The use of micro-
organisms for biological control is commonly referred to as microbial control, an
approach that includes introduction, augmentation (inoculation and inundation) and
conservation (Eilenberg et al. 2001).
The branch of entomology that deals with the study of insect diseases is known
as Insect Pathology. In a broad sense, it refers to observations concerning the cause,
symptomatology and epizootiology of insects diseases and the study of the resulting
structural, chemical and functional body alterations. Insect diseases are generally
recognised by obvious symptoms of infection such as movement and irritability,
discolouration, changes in body size and shape, and physiological disturbance. The
scope of insect pathology encompasses many subdisciplines in entomology. The
basic concepts include the cause of the diseases and the classification and phylog-
eny of entomopathogens. Phylogenies provide a fundamental framework within
which we can fit the knowledge produced on all aspects of biology (Lecointre and
Le Guyader 2006). It also tells us how closely related the involved organisms are to
3
Table 3.1 List of common entomopathogens along with their virulence traits
Pathogen Pathogenic trait
1. Viral diseases
Baculoviruses Usually cessation of feeding a few hours before death. Swoolen intersegmental regions, Larvae
typically turn dark brown or black upon death and are extremely fragile, breaking open and spilling
the liquefied internal contents at the slightest touch.
Entomopoxviruses (EPV) Infected cells, primarily fat body, become hypertrophied and tissues disintegrate. Lepidoptera show
external symptoms of whitening and softening.
Diagnosis include spheroids and spindles (protein inclusions with no virions occluded) in host cell
cytoplasm.
Polydnaviruses (PDV) Injected along with the wasp egg into the body cavity of a lepidopteran host caterpillar and infects
cells of the caterpillar. Infection does not lead to replication of new viruses, rather it affects the
caterpillar's immune system. Without the virus infection, phagocytic hemocytes (blood cells) will
encapsulate and kill the wasp egg but the immune suppression caused by the virus allows for
survival of the wasp egg, leading to hatching and complete development of the immature wasp in
the caterpillar.
Iridoviruses Infection causes cell fusion, nuclear alterations, cell contraction and formation of masses in the
cells. Infected cells remain "larval", even in adult insects. Most infections are systemic, but the fat
body is particularly affected. Infections are usually lethal but low dosages may allow maturation of
the host; however, infected pupa and adults are often abnormal. Adult honey bees cluster and crawl
abnormally. White spots occur under the integument of some lepidopteran larvae (e.g. silk worms).
Common RNA Viruses
Cytoplasmic Polyhedrosis Virus (CPV) or cypoviruses Strong effects include developmental lags, lower weight gain, etc. Heavily infected larvae may fail
to pupate and usually die. Usually the nuclei of infected tissues are normal, but in heavy infections
the cells can become hypertrophied. In fruit flies, abdomens are swollen and discoloured before the
flies die.
Birnaviruses This family of viruses has only been detected in natural field populations of Culicoides (biting
midges). It replicates in the cytoplasm of cells and is somewhat systemic but does not infect fat
body tissues.
(continued)
45
Table 3.1 (continued)
46

Sacbrood virus (SBV) Develops in the cytoplasm of fat body cells in honey bee larvae. The virus kills larvae but is
relatively benign in adults. The internal organs liquify and the integument remins intact assuming
the typical saccular aspect. Brood cells holding infected larvae are recognizable because they are
not capped or are only partially capped. Heads of infected larvae darken and turn upward.
Dicistroviridae, including: Immature queens die and turn black. Paralyses adults. The disease can be transient, or can weaken
Israel acute paralysis virus (IAPV), Kashmir bee virus colonies.
(KBV), Black Queen Cell Virus (BQCV), Acute bee The virus infects the cytoplasm of hindgut cells and neural ganglia. Adults are symptomatic:
paralysis (APV or ABPV), Chronic bee paralysis, bloated, crawl instead of fly, lose their hairs, tremble and eventually die.
Drosophila C virus, Cricket paralysis virus, Aphid
lethal paralysis virus, Viral Flacherie of the silkworm
(Bombyx mori), Triatoma virus of Triatoma infestans, a
hemipteran vector of Chagas disease
2. Bacterial diseases
a. Spore-forming bacteria
Bacillus thuringiensis Bt possesses 170 or more genes on chromosomes and plasmids for delta endotoxin production
(crystal or “Cry” toxins) with specificity to insect gut tissues. Different combinations of these genes
produce toxicity to particular insect orders. The three best known subspecies are:
Bt kurstaki with lepidopteran specific toxicity
Bt israeliensis with dipteran specific toxicity (Aedes spp. are most susceptible)
Bt tenebrionis with coleopteran specific toxicity
Protoxins of Bt δ-endotoxin are released from the crystal inclusions and, in a susceptible host, are
activated. The toxin binds to receptor sites on the brush borders of the midgut columnar cells and
creates pores that interfere with ion transport across the cell membranes. Feeding cessation and
paralysis occur quickly.
M. Gani et al.
3
Bacillus sphaericus B. sphaericus, a strict aerobe, is a pathogen of mosquitoes. Culex spp. are most susceptible,
followed by Anopheles and Aedes. The most virulent species have parasporal crystals and produce
two toxins, both required for pathogenesis. Unlike Bt, the endospore (reproductive spore) and the
crystal are contained in an ‘exosporium’ that binds them together.
B. sphaericus invades the posterior midgut cells and gastric caecae. Some cells swell and vacuolate,
and midgut cells separate from each other. Peristalsis ceases, paralysis occurs, and larvae die within
two days.
Paenibacillus larvae P. larvae is the aetiological agent of American foulbrood in honey bees. A facultative anaerobe, this
species does not produce a toxin crystal. Rods enter the midgut cells by phagocytosis. Those that
survive gain access to the haemolymph where they reproduce.
Adult bees are not affected but may harbor bacteria in the gut and serve to transmit the pathogen to
larvae.
Diagnosis: Larvae typically die late in the development period, turn brown and are of a sticky or
‘ropey’ consistency. Eventually, the carcasses dry into persistent scales at the bottom of brood cells,
maintaining the bacterium in uncleaned hives.
Paenibacillus popilliae – Type A Milky Disease P. popilliae vegetative forms have a ‘footprint’ appearance with both an endospore and a crystal.
The crystal appears to confer some toxicity (related to delta endotoxin of Bt) but toxicity doesn’t
affect the course of the disease.
Ingested spores multiply in the digestive tract where they penetrate midgut cells. The cells
produced in the midgut are arrow-shaped, perhaps aiding penetration into the haemolymph. Spores
are sometimes encapsulated by the haemocytes but can continue to reproduce inside the capsules.
The host dies of septicaemia. All larvae die, but some survive the first phases or waves of
sporulation.
P. lentimorbus - Type B Milky Disease This species has no crystal and is less pathogenic. Infected larvae continue to grow and moult for
some time. Larvae turn milky brown and haemolymph is clotted; circulation becomes blocked.
(continued)
47
48

Brevibacillus laterosporus Toxin produced during the vegetative phase of the bacterial growth cycle. Toxin maintained during
sporulation
Lysinibacillus sphaericus Mode of action likely similar to that of B. thuringiensis
Clostridium bifermentans Mode of action likely similar to that of B. thuringiensis
b. Non-spore forming bacteria
Serratia marcescens and S. liquifasciens Ubiquitous in nature and common in the gut contents of insects but highly proteolytic and virulent
if the haemocoel is breached.
Some strains of S. marcescens are red-pigmented, rendering the dead host with a bright red or
reddish tinge.
Serratia entomophila Cessation of feeding and gut clearance. Amber coloration of the grub. Invasion of the haemocoel
Melissococcus pluton - European foulbrood M. pluton is a seasonal (spring) pathogen of honey bees. The pathogen multiplies in the alimentary
tract and is confined in the peritrophic membrane, a chitin and protein microfiber lining of the gut
of insect (and other organisms).
M. pluton produces a toxin that diffuses across the gut to other tissues.
Photorhabdus sp. Released of symbiotic bacteria in the haemocoel of infected insects by nematodes of the family
Heterorhabditidae. Invasion of the haemocoel by the bacteria, provoking toxaemia and septicaemia
Xenorhabdus sp. Release of symbiotic bacteria in the haemocoel of infected insects by nematodes of the family
Steinernematidae. Invasion of the haemocoel by the bacteria provoking toxaemia and septicaemia
Pseudomonas entomophila Strong perturbation of the midgut epithelium. Food uptake blockage
3. Fungal diseases
Ascomycota (Beauveria bassiana and Metarhizium Smooth skinned larvae sometimes change colour shortly before death. Dead insects appear
anisopliae) mummified and feel “bready” to touch. Host bodies may be covered with powdery conidia - the
colour of conidia varies and is species specific. B. bassiana turns insects snowy white to yellowish;
M. anisopliae whitish to dark green.
Entomophthoralean Fungi (Entomophthora, They produce both conidia on the external surface of the dead host and either sexual, asexual
Entomophaga, Massospora spp.) resting spores (or both) internally. For these genera, disease in living insects is difficult to detect
and it becomes evident just before or at death.
M. Gani et al.
3
4. Microsporidia
Nosema, Vairimorpha, Octosporea spp. Microsporidia are typically orally transmitted or transovarially transmitted from infected female to
the offspring. Heavily infected larvae develop more slowly and may become sluggish in the last
stages of disease. Failure to pupate successfully is frequent for heavily infected immature hosts.
Some fat body microsporidia may completely fill the cells and cause hypertrophy, resulting in a
swollen appearance and pale colour. Infected adults usually have shorter life spans and fecundity
often declines.
5. Protozoan diseases
– Effects tend to be chronic and transmission ranges from oral to transovarial. Many species are
commensal or opportunistic pathogens that only cause pathology when numbers build to very high
levels in the gut (e.g. Trypanosoma, Crithidia)
6. Nematodes
Mermithids Matures in the living host; the host dies upon egress.
a well-known behaviour of mermithid- and nematomorph-infested insects is water seeking.
This behaviour, induced by parasitism, allows emergence of the nematode/ nematomorph in water,
which these species require to survive and lay eggs.
Steinernema and Heterorhabditis nematodes Rhabditid nematodes in genera Steinernema and Heterorhabditus inject mutualistic bacteria
(species specific - typically Xenorhabdus spp. in Steinernema spp. and Photorhabdus spp. in
Heterorhabditis spp.) that kill the host via septicaemia and prevent saprophytes from reproducing.
Tissues disintegrate, broken down by the bacteria. The invading nematodes mature, mate (if
necessary) and produce offspring. When offsprings are in the 3rd stadium (often still protected by
the second instar cuticle), they are released from the host via mouth, anus and other body openings.
These infective juveniles (IJs, also called Dauer larvae) enter the environment to seek or ambush a
new host.
7. Rickettsiosis and Mycoplasma spp.
Wolbachia Effects are host species-specific and notably include: cytoplasmic incompatibility that kills
offspring (often in the egg stage); male killing (males die during development); feminisation
(infected males develop as females); and parthenogenesis.
(continued)
49
50

8. Degenerative diseases & Neoplastic diseases
a. Genetic diseases Lethal factors (mutants or deficiencies) - most genetic mutations are harmful. Malformations can
occur in every body tissue and structure.
Sterility factors - Many kinds of sterility factors are possible including malformation of organs and
inability to produce eggs or sperm. In biological control programs, chemical or irradiation
mutations in males cause sterility in the treated insects. The sterile males are released to compete
with wild males for females, their mating producing no offspring.
Structural alterations - Any imaginable malformations - missing or deformed wings, supernumerary
appendages, deformed body parts.
Tumours - Both malignant and non-malignant tumours occur, although non-malignant are most
common. Tumour types include benign ovarian tumours, melanotic tumours, neoplasms, imaginal
disk (wing formation), and invasive neuroblastomas.
Gynandromorphs - or intersex mutants are usually sterile.
b. Nutritional diseases Nutritional disease can occur in natural insect populations when the host is an outbreak species and
population density increases until the normal food supply is depleted, or when the total food source
is in short supply, independently of the population density (e.g. stores of honey for overwintering
bees dissipate if pollen is in short supply or bees cannot fly in persistent poor weather conditions).
In laboratory colonies, nutritional issues are a perpetual problem, particularly with meridic diets
where all requirements may not be met despite years of nutritional studies. Symptoms include
failure to grow and develop and deformed wings. Starvation mortality is also possible.
M. Gani et al.
3 Molecular Phylogeny of Entomopathogens 51
Table 3.2 Entomopathogens as biopesticides with their trade name and target pest
Tradenames of
Microbial Control Agent Biopesticides Target Pests
Bacteria
Bacillus thuringiensis Agree WG and XenTari Lepidoptera
subsp. aizawai DF
B. thuringiensis subsp. Mosquito Beater WSP Diptera
israelensis
B. thuringiensis subsp. CoStar, DiPel ES, Lepidoptera
kurstaki Monterey B.t., and
Thuricide
B. thuringiensis subsp. Novodor FC Coleoptera
tenebrionis
Paenibacillus popilliae Milky Spore Powder Japanese beetle, Popillia japonica
Fungi
Beauveria bassiana BotaniGard ES, One or more pests of Acarina,
Mycotrol-ESO, Coleoptera, Diptera, Hemiptera,
Myco-Jaal and Hymenptera, Lepidoptera, Orthoptera,
Naturalis-LL Thysanoptera and others
Hirsutella thompsonii ABTEC Hirsutella
Isaria fumosorosea NoFly WP and Pfr-97
WDG
Lecanicillium lecanii Phule Bugicide
L. longisporum Vertalec
Metarhizium anisopliae BioCane, Metarril and
Ory-X
M. brunneum Met52 EC
Paecilomyces lilacinus MeloCon WG Plant-parasitic nematodes
Pochonia chlamydosporia Poch_ar
Nematodes
Heterorhabditis Nemasys and Terranem Several orders of soilborne pests
bacteriophora
Steinernema carpocapsae Ecomask and NemAttack
S. fatiae Entonem, Fungus Gnat
& Rootknot Exterminator
and Scanmask
H. heliothidis and S. Double-Death
carpocapsae
Viruses
Granulovirus (GV) Lepidoptera
Cyadia pomonella GV CYD-X and MADEX
HP
Nucleopolyhedrovirus
(NPV)
Helicoverpa zea NPV Gemstar LC
Spodoptera exigua NPV Spod-X LC
Lymantria dispar NPV Gypchek
52 M. Gani et al.
each other (e.g., all animals are more closely related to each other than they are to
plants) and how we can recognize groups of organisms (e.g., animals form a group).
Pathogens are often grouped on the basis of phenotypic similarity (e.g., hosts,
predilection sites, infection route, microscopy) or similarities concerning the

induced disease (e.g., symptoms, diagnostic procedures). There are many other
important uses of phylogenies (Harvey et al. 1996), including the study of co-phy-
logeny of hosts and pathogens (e.g., understanding the role of hosts in a pathogen
evolution) and biogeography (e.g., understanding the spread of pathogens). Different
pathogens have varying distributions, different patterns of spread and rates of evolu-
tion. These differences result in a broad range of variations at the genetic and geo-
graphic levels. For example, a phylogenetic tree can be produced to reflect the
geographic locations of the samples in order to investigate the spread of a disease,
whereas the so-called molecular clocks can be applied to estimate the age of impor-
tant events in the origin and spread of new pathogens.
Most pathogens are unicellular or multicellular without specialized tissues,
which severely limits the number and range of available characters. Traditionally,
the characters used for phylogenetic and taxonomic analyses have been based
mainly on life cycle features, disease characteristics and ultrastructure. It may be
rather difficult to determine homologies among such characters (i.e., their evolu-
tionary comparability), so that related characters are being compared, although the
data are also regrettably incomplete for most species. Consequently, phylogenies
based solely on these characters are rare and not particularly robust. For this reason,
molecular data have now become the predominant character information source for
phylogenetic studies. As DNA mutates, the sequences change and, as pathogens
spread, they bring these changes with them. Molecular data are being generated
using DNA-DNA hybridization, randomly amplified polymorphic DNA (RAPD),
restriction fragment length polymorphism (RFLP), amplified fragment length poly-
morphism (AFLP), protein and nucleotide sequences. Of these, nucleotide sequences
are by far the most common, used in general for all taxa, not only pathogens. Despite
the growing understanding of the phylogeny of entomopathogens, our knowledge of
their evolutionary processes is still limited. This chapter provides a comprehensive
review on the taxonomy and evolution of entomopathogens and the contribution of
genomics in deciphering their phylogeny.
3.2 Molecular Phylogenetics and its Importance
Molecular phylogenetics is the branch of phylogeny that analyzes genetic, heredi-

tary molecular differences, predominately in DNA sequences, to gain informations
on an organism's evolutionary relationships. Conserved sequences, such as mito-
chondrial DNA, are expected to accumulate mutations over time. Assuming a con-
stant rate of mutation, they provide a sort of molecular clock for dating divergence
events. Molecular phylogeny uses such data to build a "relationship tree" that shows
the probable evolution of various organisms. There are several methods available to
perform a molecular phylogenetic analysis. Phylogenetic analysis of molecular

sequences (the actual predominant type of data) usually consists of three distinct
procedures: (i) sequence alignment, (ii) character coding and (iii) tree building.
A tree from a single molecular sequence represents only the phylogeny of that
specific gene the sequence belongs to, which does not necessarily reflect the phy-
logeny of the whole organism (Doyle 1992). Just how many genes might be required
to reconstruct the entire organismal phylogeny is still an open question (Gatesy
et al. 2007). Reconstructing a phylogenetic history is conceptually straightforward,
although it may take a long time to explicate the most appropriate approach (Hennig
1966). The objective is to infer the ancestors of the contemporary organisms and
their ancestors too, all the way back to the most recent common ancestor of the
group being studied. Ancestors can be inferred because the organisms share unique
characteristics. That is, they have features that they hold in common and that are not
possessed by any other organisms. Phylogenesis scientists therefore feel confident
that these methods also apply to situations where direct knowledge of the past is
absent. Even the Insecta, which is usually considered to be the prime example of a
poorly known group, has ~ 1 million species known thus far, out of an estimated
total of 4.5–30 million.
To date, most pathogen phylogenies have been based on the sequence of a single
gene, usually the nucleotide sequence of the small-subunit (16S or 18S) ribosomal
RNA gene. Indeed, most of the reclassification of bacteria since the late 1970s has
been based principally on this gene (Sapp 2009). Other genes sequenced include
those for host recognition or for dealing with the host immune system, often
sequenced as part of projects producing new drugs or vaccines. These genes are
often unique to each taxonomic group or are subjected to strong selective pressures,
thus resulting not necessarily useful for phylogeny. In particular, bacterial genomes
often show clusters of functionally related genes such as those for antibiotic resis-
tance (Hedges 1972), which can affect phylogenetic analysis. Consequently, the
data are rather fragmentary for many taxonomic groups. A multigene phylogeny is
therefore unlikely to be produced from these current data sets. Several “analysis
pipelines” have appeared recently, mostly aimed at microbiologists, which do
indeed combine several computer programs together to perform a fast, single phy-
logenetic analysis. These include BIBI (Devulder et al. 2003), PhyloGena
(Hanekamp et al. 2007), WASABI (Kauff et al. 2007), AMPHORA (Wu and Eisen
2008), and ASAP (Sarkar et al. 2008). Probably a better approach is provided by
services that allow the mixing and matching of various programs (e.g., Dereeper
et al. 2008; Gouy et al. 2010). Attempts have even been made to provide descrip-
tions of “standard procedures” for phylogenetic analysis (Peplies et al. 2008).
MEGA (molecular evolutionary genetics analysis, https://www.megasoftware.net/)
is an analysis software that is user-friendly and free to download and use. It is
capable of analyzing both distance-based and character-based tree methodologies.
MEGA also contains several options one may choose to utilize, such as heuristic
approaches and bootstrapping. Bootstrapping is an approach that is commonly used
to measure the robustness of a topology in a phylogenetic tree, which demonstrate
54 M. Gani et al.
the percentage at which each clade is supported after numerous replicates. In gen-
eral, a value greater than 70% is considered significant.
Phylogenetics is important because it enriches our understanding of how genes,
genomes, species (and molecular sequences more generally) evolve. The primary
objective of molecular phylogenetic studies is to recover the order of evolutionary
events and to represent them in evolutionary trees that graphically depict relation-
ships among species or genes over time.
3.3 Entomopathogenic Viruses
3.3.1 Origin, Natural History and Geographical Distribution
The term entomopathogenic refers to those microorganisms that are capable of

attacking insects using them as hosts to develop part of their life cycle (Delgado and
Murcia 2011). From an applied point of view, they reduce insect pest populations to
levels that do not cause economic damage to crops (Tanzini et al. 2001) or regulate
disease vectors (Scholte et al. 2004). They are also defined as facultative or obligate
parasites of insects, with a high capacity for sporulation and survival (Delgado and
Murcia 2011). The viruses that attack and kill insects are called “entomopathogenic
viruses” (EV) and are reported from virtually every insect order. The diseases
caused by EV are known since the 16th century. The grasserie disease of silkworm
(Bombyx mori L.; Lepidoptera: Bombycidae) caused by B. mori nucleopolyhedro-
virus (BmNPV) was described since 1524 by Italian bishop Marco Vida da Cremona
in the poem “De Bombyce”. Later on another viral disease, Sacbrood was described
in the honeybee (Apis mellifera L.; Hymenoptera: Apidae) in 1913. Two Italian
scientists, Maestri (1856) and Cornalia (1856), first described the occlusion bodies
(OBs) of the BmNPV and Paillot (1926) described the granulovirus (GVs) for the
first time in the larvae of cabbage butterfly (Pieris brassicae; Lepidoptera:
Pyralidae). Ishimori (1934) identified the cypovirus infection in B. mori by the
abnormal presence of dense OB in the insect midgut. The introduction of electron
microscopy greatly advanced the knowledge of EV, allowing Bergold to observe the
rod-shaped nucleocapsids within the OBs of baculoviruses. The current view on the
evolution of baculoviruses states that they evolved from non-occluded viruses
infecting midgut tissue to occluded viruses infecting midguts (γ- and δ-baculoviruses)
and finally to occluded viruses with the ability to spread the infection to other tis-
sues (α- and β-baculoviruses) (Herniou and Jehle 2007). The most likely scenario is
that baculoviruses have gained over time new features to infect more cell types,
becoming more independent from the host cell machinery.
Steinhaus and his collaborators (1950–1970) tested baculoviruses as biological
control agents in the field by applying a nucleopolyhedrovirus (NPV) to control the
alfalfa caterpillar (Colias eurytheme Boisduval; Lepidoptera: Pieridae). The first
introduction of a baculovirus resulting in successful regulation of a pest insect,
European spruce sawfly Gilpinia hercyniae occurred accidentally in 1930 in Canada

(Bird and Elgee 1957). The first commercial viral insecticide, Elcar for the control
of Heliothis/Helicoverpa complex (Lepidoptera: Noctuidae) was marketed in 1975
by Sandoz Agro, Inc. Some key examples of their use in biocontrol are Anticarsia
gemmatalis (Ag) MNPV used on over two million Ha of soybean in Brazil (Moscardi
1999; Moscardi and Santos 2005) and H. armigera SNPV (HearNPV) used to con-
trol cotton bollworm in China (Zhang et al. 1995). To date, many commercial prod-
ucts based on baculoviruses are available. SPOD-X™, based on Spodoptera exigua
(Se) MNPV is used in Europe and USA to control S. exigua larvae, mainly in green-
houses (Kolodny-Hirsch et al. 1997). Mamestrin™ based on Mamestra brassicae
(Mb) MNPV is used on cabbage, tomatoes and cotton to control cabbage moth (M.
brassicae), American bollworm (H. armigera), diamondback moth (Plutella xylo-
stella), potato tuber moth (Phthorimaea operculella) and grape berry moth
(Paralobesia viteana). GemStar™ based on H. zea (Hz) SNPV provides control of
pests belonging to the genera Helicoverpa on a wide variety of crops, for these pests
are polyphagous. The best commercial example of a GV in biocontrol is Cydia
pomonella (Cp) GV being sold under at least five commercial names,
Carpovirusine™, Madex™, Granupom™, Granusal™ and VirinCyAP. Also forest
insects are being controlled with baculoviruses and several formulations of
Lymantria dispar (Ld) MNPV (Gypchek™, Virin-ENSH™), Orgyia pseudotsugata
(Op) MNPV (TM-BioControl-1™ and Virtuss™) and Gylpinia herciniae NPV
(Abietiv™) are currently available in the market.
Natural population of insects in any ecosystem are regulated in time by the action
of both biotic and abiotic factors. The biotic factors include entomophagous insects
such as parasitoids and predators and entomopathogens such as viruses, bacteria,
fungi and nematodes. Abiotic factors include climate, soil, air, space and light
(Hostetter and Bell 1985). Insect viruses have been studied for many years due to an
intrinsic interest in the general study of invertebrate diseases and, more particularly,
because of their potential as environmentally benign pest management agents
(Evans 1986). The study of EV ecology and of their potential use for pest manage-
ment began with the pioneering work of Steinhaus (1956a). Early in the 20th cen-
tury the disease observed in silkworms was attributed to a virus infection, and in
1947 the visualization of rod-shaped virions, known to be characteristic of the virus
family Baculoviridae, was reported. Out of 12 families, baculoviruses have been
studied in detail because of their potential as pest control agents (Black et al. 1997;
Van Beek and Hughes 1998) and, more recently, for their prominence as expression
vectors for a wide range of heterologous genes (Miller 1988; Choi et al. 1999). The
advantages of baculoviruses for pest control include their restricted host range
(Gröner 1986), non-target effects on useful insects and lack of toxic residues, allow-
ing growers to treat their crops even shortly before harvest, with low probability to
develop stable resistance (Monobrullah 2003).
Large quantities of baculoviruses are released into the environment during natu-
ral epizootics, which are common, widespread, and often important in regulating
insect population levels (Federici 1978). There is evidence that the amount of virus
56 M. Gani et al.
particles which is artificially spread into the environment for insecticidal purposes
is minimal compared with that produced during such epizootics.
The baculoviruses have been reported from orders Lepidoptera, Diptera and
Hymneoptera. Among them the nucleopolyhedroviruses (NPVs) attracted the atten-
tion of pest control scientists looking for an alternative to pesticides, because they
cause a highly infectious disease that kills the host in 5-7 days. These viruses attack
some of the most important Lepidopteran crop pests including species of Helicoverpa
and Spodoptera.
Some related GV species are also highly infectious, e.g. the C. pomonella (apple
codling moth) GV and P. xylostella (diamond back moth) GV. However not all GVs
are as fast acting as NPVs, because morphologically they have a single envelop with
a single nucleocapsid per OB (Winstanley and O’Reilly 1999). In general, the host
range of most NPVs is restricted to one or a few species of the genus or family of
the host from which they were originally isolated. This factor also represents an
important commercial draw back, restricting the use of these products to specific
key pests or closely related pest complexes, such as Helicoverpa species
(Chakraborty et al. 1999). Some of the few exceptions, having a broader host range
are: (i) the Alfalfa looper, Autographa californica MNPV, infecting more than 30
species from about 10 insect families, all within the order Lepidoptera; (ii) the
Celery looper, Anagrapha falcifera NPV, infecting more than 31 species of
Lepidoptera from 10 insect families and (iii) the Cabbage moth, Mamestra brassi-
cae MNPV, which was found to infect 32 out of 66 tested Lepidopteran species
from 4 different families (Doyle et al. 1990; Hostetter and Puttler 1991).
The maintenance of baculoviruses in insect populations requires transmission of
the virus particles from infected to healthy individuals. Transmission in baculovi-
ruses is thought to be primarily horizontal, via susceptible larvae ingesting OBs
persisting in the environment. The OBs may be further distributed by excrements of
infected larvae (Vasconcelos et al. 1996), rain (Kaupp 1981) and predators, such as
birds (Entwistle et al. 1993). Baculoviruses can also be released in the environment
by human activity, for example by application of sprays. Baculoviruses can also be
transmitted vertically from adults to their young (Easwaramoorthy and Jayaraj
1989; Fuxa et al. 2002). Vertical transmission occurs by surface contamination of
eggs (transovum transmission) or virus passing within the egg (transovarian trans-
mission), including transfer of latent infections. A latent virus is a non-replicating
form that can be reactivated to an infective state by some stressors (Fuxa and Richter
1992) such as host crowding, fluctuations in temperature or relative humidity, irra-
diation, dietary changes, chemical stress, parasitization and inoculation by a second
pathogen (Burden et al. 2003; Cooper et al. 2003).
Epizootics of baculoviruses irregularly occur in agricultural, forest and horticul-
tural pests and have the potential to influence host population dynamics (Weiser
1987). Mathematical models have been used to study and gain insights in these
complex systems. The theoretical relationships of host-pathogen dynamics of
insects have been widely explored in mathematical models starting with those of
Anderson and May (1981). More recent models have incorporated modifications
such as variation in transmission parameters (Getz and Pickering 1983), vertical
transmission (Régnière 1984), both density dependence and vertical transmission

(Vezina and Peterman 1985), nonlinear transmission (Hochberg 1991), density
dependence (Bowers et al. 1993; Bonsall et al. 1999), host stage structure (Briggs
and Godfray 1995), heterogeneity in susceptibility (Dwyer et al. 1997), discrete
generations and seasonal host reproduction (Dwyer et al. 2000), and sublethal infec-
tion (Boots and Norman 2000).
The EV are naturally occurring and widely distributed throughout the world.
Genetic variations appear to be an essential component of natural baculovirus popu-
lations. Some variations may be kept in part by the inclusion of different genotypes
in the same infectious particles, but also because they can confer a greater fitness to
a virus populations. This is the basis on which natural selection drives the evolution
of viral lineages adapted to new environmental conditions, such as new host species.
Agrotis segetum NPV (AgseNPV) have been isolated only in Europe (Lipa et al.
1971; Allaway and Payne 1983) as its host, A. segetum is found only in Europe,
while Agrotis ipsilon NPV (AgipNPV) has been identified only in the United States
(Boughton et al. 1999; Prater et al. 2006), although the host, A. ipsilon is found in
both, the United States and Europe. This distribution of hosts and viruses provokes
the hypothesis that both viruses are descendants from a common ancestor, which is
supported by their phylogenies, and then separated into two species, by adapting to
different hosts, A. segetum and A. ipsilon, found in the same ecological niches.
Since AgseNPV performs well in both hosts, AgseNPV may be the NPV naturally
occurring in European populations of both insect species. However, it must be con-
sidered that the European AgipNPV has not been discovered yet. Interestingly it
was reported that AgipNPV is equally effective against both Agrotis spp. while
AgseNPV is about 40 times more effective on A. segetum than on A. ipsilon
(El-Salamouny et al. 2003). It is hard to find an explanation at this point for the
equal AgipNPV performance in both insect species, given the fact that in natural
conditions it never encounters A. segetum larvae. With the genetic information of
both AgseNPV and AgipNPV, and the observation that both viruses replicate in a
cell culture derived from the tissues of A. ipsilon the possibility to study this aspect
further is now open.
Geographical heterogeneity in host-pathogen interactions is thought to play
indeed a major role in evolutionary processes (Cory and Myers 2003). Pathogens
likely adapt to local host populations, likely due to genetic variability in host and
virus (Ebert 1994). Leucoma salicis is a species native for Europe, introduced into
North America at the beginning of 20th century (Langor 1995). Orygia pseudotsu-
gata is a native species for North America, and is believed not to occur in Europe.
Sequence analysis of a few core genes of two viruses isolated from these hosts,
Leucoma salicis NPV (LesaNPV) and Orygia pseudotsugata NPV (OpMNPV),
showed their very close relationship (98% nucleotide identity). It was then hypoth-
esized that these viruses originated from one species that evolved by adaptation to
different hosts and environmental conditions, and hence showed considerable varia-
tion at the genetic level. The Kimura-2 parameter proposed as a criterion for virus
species demarcation was 0.010 for polyhedrin gene sequence of LesaNPV and
OpMNPV, indicating that these two viruses represent actually the same species.
58 M. Gani et al.
Geographical separation might have led to speciation resulting in two viruses, from
which one still is able to infect both insect species (LesaNPV), while the other is not
(OpMNPV). LesaNPV and OpMNPV thus exemplify evolutional phenomena dif-
ferent from those of AgseNPV and AgipNPV.
Many forest insects belonging to Lepidoptera (often from the family Lymantriidae)
and Hymenoptera are known for their population cycles (Myers 1988). Populations
of these species rise and fall over a predictable period of time. Baculoviruses iso-
lated from these species appear to be specialists. Specialization may result from
constant and predictable host availability, in annual cycles. Selection pressure is
then switched to improved infectivity against one insect host. Leucoma salicis and
Orgyia pseudotsugata are examples of forest lepidopterans showing cyclic popula-
tion outbreaks. Intuitively there should be a trade-off associated with a broad host
range. Generalist viruses, which obviously are less specialized, would be expected
to be also less infective against their multiple hosts, than the specialist viruses.
However, generalist viruses, in most susceptible hosts, are instead highly infectious
(Goulson 2003). The use of molecular techniques to distinguish virus genotypes
revealed that the host is usually infected with a mixture of genotypes and that main-
taining this mixture is evolutionary favorable because it enables fast adaptation to
changing host and environment conditions (Simón et al. 2004).
In a given condition, especially in cyclic insect populations with regular out-
breaks in a predictable time frame, a pathogen adapts to its host and some genotypes
are more prevalent than others. A drastic change in genotype ratios can be seen
when the virus is replicating in cell culture, in which the number of defective geno-
types almost immediately increases after just one passage (Heldens et al. 1996,
Pijlman et al. 2001). Defective genotypes are also present in natural baculovirus
populations and are critical for high infectivity (Muñoz et al. 1998). Expanding the
host range will be reflected in a shift in virus genotype ratios and may even lead to
disappearance of some genotypes.
Other mechanisms, such as mutations (Pijlman et al. 2001), homologous recom-
bination (Arends and Jehle 2002), transposon insertions (Jehle et al. 1995) and hori-
zontal gene transfer, including the introduction of host genes (Hughes and Friedman
2003), allow optimal adaptation of baculoviruses to local host and environmental
conditions, which eventually may result in the evolution of new virus species.
Successful (fatal) infection of an insect usually involves a number of stages.
However, the ability/inability of the virus to establish infection may depend only on
a single gene mutation. Croizier et al. (1994) reported that a change in only two
amino acids of the AcMNPV helicase gene extended its host range to B. mori larvae.
Thus we can expect that the susceptibility of the host may often be a matter of
chance of a single mutation.
Spodoptera exigua MNPV (SeMNPV) is believed to be monospecific, infecting
only S. exigua. However, it should be mentioned that host range studies for this
virus are very limited and so far cover only few insect species (Gelernter and
Federici 1986; Smits and Vlak 1988; Simón et al. 2004). It has been confirmed that
five proteins (P74, PIF1, PIF2, PIF-3 and PIF4) associated with the ODV envelope
are indispensable for oral infectivity and that a deletion of any of them eradicates
oral infectivity (Song et al. 2008; Fang et al. 2009). Thus per os (oral) infectivity
factors (PIFs) are believed to be the main determinants of infectivity. The SeMNPV
was non-infective for A. segetum larvae. However, transcription of the delayed early
genes was detected in the midgut cells. This brings into question how specific PIFs
are and suggesting at least that PIFs may be not the only determinants of oral infec-
tion. Apparently, the virus infection can be blocked at any following stage and the
entry to midgut cells does not necessarily result in a successful infection. Some
events in the midgut cells obviously prevent development of secondary infection. If
this occurs at the level of virus gene expression, DNA replication, assembly of new
nucelocapsids (NCs) or re-packaging of NCs into BVs, still remains unknown for
SeMNPV infection in A. segetum.
3.3.2 Taxonomy and Evolution
EV classification, just as for any other type of viruses, follows the indications of the
International Committee on Taxonomy of Viruses (ICTV) (Van Regenmortel et al.
2000). The taxonomic classification of viruses is outlined in the Report of the ICTV
(Fauquet et al. 2005), the last being the 10th report (http://ictv.global/report/), as
well as the ICTV Taxonomy and Index to Virus Classification and the Nomenclature
Taxonomic Lists and Catalogue of Viruses that can be found on the National Centre
for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/
ICTVdb/Ictv/index.htm). Therefore, like other viruses, the criteria being followed
for their classification include, among many others: type of genetic material (i.e.
singe- or double-stranded DNA, singe- or double-stranded RNA, positive or nega-
tive strand), virion morphology and size (i.e. icosahedral, rod-shaped, etc.), pres-
ence of an envelope surrounding the virion, presence of an OB engulfing the virions,
host and host range. All viruses falling into one of these nucleic acid classifications
are further subdivided on the basis of whether the nucleocapsid (protein coat and
enclosed nucleic acid) assumes a rod like or a polygonal (usually icosahedral)
shape. The icosahedral shape viruses are further subdivided into families on the
basis of the number of capsomeres making up the capsids. Finally, all viruses fall
into two classes depending on whether the nucleocapsid is surrounded by a lipopro-
tein envelope. However, the ultimate criterion is the sequencing of the genetic mate-
rial which determines not only the discrimination between viral species, but also the
evolutionary relationship among viruses within the same group.
The diversity of insect viruses is extensive. They are classified into different
families including DNA and RNA viruses (Van Regenmortel et al. 2000) (Table 3.3).
Most of the non-occluded (virions not occluded in a protein matrix) or non-
aggregated viruses are not visible under light microscopy. The occluded viruses —
Granuloviruses (GV), Nucleopolyhedrovirus (NPV), Entomopoxvirus (EPV) and
Cytoplasmic Polyhedrosis virus (CPV) — are the most commonly observed due to
the incorporation of the particles into a protein matrix which is large enough to be
visible under light microscopy.
60 M. Gani et al.
Table 3.3 Groups of entomopathogenic viruses

Host stage
Nucleic Nucleocapsid Occlusion usually
Family acid symmetry body Recorded host orders infected
Baculoviridae dsDNA, Baciliform + Lepidoptera , Larvae,
circular Hymenoptera, Diptera sometimes
pupae and
adults
Reoviridae dsRNA Isometric + Lepidoptera, Diptera, Larvae, pupae,
linear Hymenoptera, adults
Coleoptera
Poxviridae dsDNA, Ovoid + Lepidoptera, Larvae, pupae,
linear Coleoptera, Diptera, adults
Hymenoptera,
Orthoptera
Iridoviridae dsDNA Isometric – Diptera, Coleoptera, larvae
linear Lepidoptera
Parvoviridae ssDNA Isometric – Lepidoptera, Diptera, Larvae, pupae,
Blattoidea, Odonata, adults
Orthoptera
Picornaviridae ssRNA Isometric – Diptera, Hemiptera, larvae
Hymenoptera,
Lepidoptera,
Orthoptera
Ascoviridae dsDNA Rod-ovoid – Lepidoptera Larvae
circular
Polydnaviridae dsDNA Rod, Fusiform – Parasitic Hymenoptera Adults
circular
Rhabdoviridae ssRNA Bullet shaped – Diptera, larvae
Nodaviridae ssRNA Isometric – Lepidoptera, Diptera, Larvae, adults
linear Coleoptera
Birnaviridae dsRNA icosahedral – Diptera adult
linear
Bunyaviridae ssRNA circular – Diptera, Hemiptera adult
Iflaviridae ssRNA Spherical – Lepidoptera, Larvae, adults
Hymenoptera,
Hemiptera, bee
parasitic mites
(Acarina)
Tetraviridae ssRNA Isometric – Lepidoptera Larvae
linear
Dicistroviridae ssRNA Isometric – Diptera, Hemiptera, Larvae, adults
linear Hymenoptera,
Lepidoptera,
Orthoptera
The vast majority of studies are focused on members of Baculoviridae, as a good

number of these viruses are used as biological control agents for the management of
insect pests (Copping and Menn 2000; Lacey et al. 2002; Szewczyk et al. 2008). In
the past the classification of Baculoviridae was based on morphology, and was
divided into the two genera NPVs and GVs (Theilmann 2005). NPVs form large
(0.15-15 μm), polyhedral-shaped OBs called polyhedra, which contain many viri-
ons that can harbour a single nucleocapsid (SNPV) or multiple nucleocapsids
(MNPV). The GVs form smaller (0.3–0.5μm), cylindrical (granule-like) OBs called
granules, which contain a single virion with a single nucleocapsid (Fig. 3.1). Virion
dimensions are in the size range of 30-60 nm × 260-360 nm for GVs, and 40-140 nm
× 250-400 nm for NPVs. The major component of the OBs is a single, viral encoded
protein called polyhedrin in NPV, and granulin for GV. The latter is genetically and
serologically closely related to the NPV polyhedrin. However, differing from NPV,
the host range of GV is narrower and mostly restricted to a single species. NPVs
have been isolated from lepidopteran and non-lepidopteran hosts, whereas GVs
were only found till now in lepidopterans.
A new division was recently proposed on the basis of the comparison of genomic
sequences which indicated that virus phylogeny followed more closely the classifi-
cation of the hosts than the virion morphological traits (Jehle et al. 2006). In this
Fig. 3.1 On the basis of the OB morphology, baculovirus were originally divided in two major
groups: the Nucleopolyhedrovirus (NPVs) and the Granulovirus (GVs). NPVs occlusion bodies
are called polyhedra and their major occlusion protein is called polyhedrin. The GV occlusion
bodies are called granules or capsules and their major protein is granulin. (Source: Haase et al.
2013)
62 M. Gani et al.
classification the family Baculoviridae contains four genera: Alpha baculoviruses

(lepidopteran specific NPVs), Beta baculoviruses (lepidopteran specific GVs),
Gamma baculoviruses (hymenopteran specific NPVs) and Delta baculoviruses
(dipteran-specific NPVs). These four major groups of baculoviruses were identified
already in the ‘80s by N-terminal sequencing of major OB proteins (Rohrmann
et al. 1981). At that time hymenopteran baculoviruses were represented only by
European pine sawfly, Neodiprion sertifer nucleopolyhedrovirus (NeseNPV). The
European crane fly, Tipula paludosa (Tipa) NPV was the dipteran NPV. It has been
shown that concatenated sequences of three conserved genes [polyhedrin (polh),
late expression factor-8 (lef-8) and late expression factor-9 (lef-9)] or a combined
phylogeny of the separate analyses of each of these, yield the same tree topologies
as the analysis of complete genome sequences (Herniou and Jehle 2007). This indi-
cated that the multiple conserved genes approach is strongly advised for identifica-
tion of new baculovirus isolates.
Insect viruses are named in acronyms, according to their host(s) and the viral
group to which they belong to. For example, the entomopoxviruses are named EPV,
the iridoviruses are IV, and the cytoplasmic polyhedrosis viruses (cypoviruses) are
CPV. The Autographa californica multiple nucleopolyhedrovirus is named
AcMNPV. Therefore, all nucleopolyhedroviruses are named NPV, just as the granu-
loviruses are named GV. Still, lepidopteran baculoviruses was divided in NPVs
(group I and II) and GVs by genomic sequence data (Van Oers and Vlak 2007).
When genome sequences became available from NPVs infecting sawfly and mos-
quito species, it was clear that lepidopteran NPVs and GVs were closer to each
other, than to NPVs from dipterans and hymenopteran hosts.
There is a relatively large group of unassigned viruses that infect invertebrates
(Fauquet et al. 2005). These are viruses whose key characteristics do not readily fit
those of existing genera. Many of them are small ribonucleic acid (RNA) viruses
(SRV) from Drosophila or honeybees, while others are well-characterized viruses
such as Oryctes rhinoceros virus and Heliothis virus 1, which were once thought to
be baculoviruses (Fauquet et al. 2005).
The evolution of baculoviruses is governed by the same rules as that of any
organism as can be seen by the changes in genotypes in viral populations. For evolu-
tion to happen there must first a genetic variation on which natural selection or
genetic drift can act. It also depends on genetic variations in the insect hosts popula-
tions. Viruses in this co-evolutionary duet have of course more chances to adapt than
the hosts, due to a shorter replication time, higher offspring numbers and a high
natural heterogeneity. Since baculoviruses can persist outside their host for long
times, also the environment exerts a selective pressure. The first hypothesis for evo-
lution of baculoviruses in relation to their host stated that baculoviruses have
evolved first in one insect order and then colonized other groups (Rohrmann 1986).
The second postulated that the association between baculoviruses and their hosts
dates back to the origin of insects and that they coevolved with their host during
evolutionary time (Federici 1997). Recently, these two hypotheses have been tested
and the results lead to a new hypothesis, postulating that ancestral baculoviruses
horizontally infected hosts of different orders and that a progressive specialization
into different baculovirus lineages later took place (Herniou et al. 2004). Support
for this latter hypothesis proceeds from the fact that the phylogeny of baculoviruses
follows that of different hosts within an order, hence reflects the pattern of insect
families, but does not clearly reflect the evolution of insect orders.
Based on sequencing studies, a criterion for demarcating baculovirus species has
been depicted. The evolutionary distance between a pair of sequences usually is
measured by the number of mutual nucleotide (or amino acid) substitutions. One of
the models used to estimate the evolutionary distance between sequences is the
Kimura 2-parameter. This method corrects for differences in the rates of transition
(conserving the nucleotide ring number, i.e. A → G or C → T and viceversa) and
transversion (changing the nucleotide ring number, i.e. A → T or C and C or T → G
and viceversa). It allows weighing a quality of difference between transition and
transversion (Kimura 1980). The proposed criterion suggests that when the Kimura
2-parameter between single or concatenated genes is larger than 0.050, two viruses
are distant enough to be considered as different species. As a consequence, the pro-
posed rules to discriminate baculovirus species are as follows: two (or more) bacu-
lovirus isolates belong to the same species if the Kimura-2-parameter between
single and/or concatenated polh, lef-8 and lef-9 nucleotide sequence is smaller than
0.015. Two viruses should be considered as different virus species if that distance is
bigger than 0.050. For the pair of viruses with the distance between 0.015 and 0.050
complementary information i.e. biological characteristics such as host range should
be provided for species demarcation (Jehle et al. 2006). Viral species are defined as
“a monophyletic group of viruses whose properties can be distinguished from those
of other species by multiple criteria” (Adams et al. 2013).
3.3.3 Genomics and Phylogeny
Over the last decade, there has been an explosion of genomic data on insect viruses,
primarily because sequencing technologies have improved drastically in accuracy
and speed, and whole-genome sequencing has become an affordable procedure. As
a result, knowledge on the EV genomics has expanded significantly, yielding more
accurate conclusions on the virus-hosts co-evolution (Herniou et al. 2004). The EV
genetic is very intricate and complex, because most virus genomes may be as large
as 300 kilobase pairs (kb). By far, the most studied group at the genomics level is
the Baculoviruses, with 172 genomes belonging to viruses from Lepidoptera,
Hymenoptera and Diptera totally sequenced (Wennmann et al. 2018) (Table 3.4).
Five genomes of CPVs have been sequenced so far, Bombyx mori (BmCPV-1),
Lymantria dispar (LdCPV-1), Dendrolimus punctatus (DpCPV-1), Lymantria dis-
par cypovirus 14 (LdCPV-14), Trichoplusia ni CPV 15 T (nCPV-15) (Mertens and
Bamford 2009).
Length and size of fragments vary according to each CPV species. That is, frag-
ments are separated and visualized by agarose electrophoresis producing particular
banding patterns in the gel, called electropherotypes. The CPVs genomes are com-
64 M. Gani et al.
Table 3.4 Entirely sequenced genomes of baculoviruses

Genome Genebank
Group Virus Name Abbreviation length (kb) accession no.
Lepidoptera NPV Antheraea pernyi AnpeMNPV 126.246 EF207986
Group I MNPV – L2
Antheraea pernyi MNPV AnpeMNPV 126.629 DQ486030
Antheraea pernyi – NPV AnpeNPV 126.593 LC194889
Anticarsia gemmatalis AgMNPV 132.239 DQ813662
MNPV
Anticarsia gemmatalis AgMNVP 132.077 KR815471
MNPV – 43
MNPV – 26
MNPV – 35
Autographa californica AcMNPV 133.894 L22858
MNPV – C6
Autographa californica AcMNVP 133.926 KM609482
MNPV - WP10
Autographa californica AcMNVP 133.966 KM667940
MNPV - E2
Bombyx mori NPV – T3 BmNPV 128.413 L33180
Bombyx mori NPV – India BmNPV 126.879 JQ991010
Bombyx mori NPV – C1 BmNPV 127.901 KF306215
Bombyx mori NPV – Brazil BmNPV 126.861 KJ186100
Bombyx mori BmNPV 126.125 JQ991008
NPV – Zhejiang
Bombyx mori BmNPV 126.843 JQ991011
NPV – Guangxi
Bombyx mori NPV – Cubic BmNPV 127.465 JQ991009
Bombyx mandarina BomaNPV 126.770 FJ882854
NPV – S1
Bombyx mandarina BomaNPV 129.646 JQ071499
NPV – S2
Catopsilia pomma CapoNPV 128.058 KU565883
NPV – 416
Choristoneura fumiferana CfMNPV 129.593 AF512031
MNPV
C. fumiferana DEF MNPV CfDEFMNPV 131.160 AY327402
Choristoneura murinana ChmuNPV 124.688 KF894742
NPV – Darmstadt
C. occidentalis NPV ChocNPV 128.446 KC961303
C. rosaceana NPV ChroNPV 129.052 KC961304
(continued)

Genome Genebank
Condylorrhiza vestigialis CoveNPV 125.767 KJ631623
NPV
Dashchira pudibunda DapuNPV 136.761 KP47440
NPV – ML1
Dendrolimus kikuchii DekiNPV 141.454 JX193905
NPV – YN
Epiphyas postvittana NPV EppoNPV 118.584 AY043265
Hyphantria cunea NPV HycuNPV 132.959 AP009046
Lonomia oblique LoobNPV 120.023 KP763670
MNVP – SP2000
Maruca vitrata NPV MaviNPV 111.953 EF125867
Orgyia pseudotsugata OpMNPV 131.990 U75930
MNPV
Philosamia Cynthia ricini PhcyNPV 125.376 JX404026
NPV
Plutella xylostella PlxyNPV 134.417 DQ457003
NPV – CL3
Rachiplusia ou NPV RoNPV 131.526 AY145471
Thysanoplusia orichalcea ThorNPV 132.978 JX467702
NPV – p2
Lepidoptera NPV Adoxophyes honmai NPV AdhoNPV 113.220 AP006270
Group II
Adoxophyes orana NPV AdorNPV 111.724 EU591746
Agrotis ipsilon AgipNPV 155.122 EU839994
NPV – Illinois
Agrotis segetum AgseNPV-A 147.544 DQ123841
NPV-A – Polish
Agrotis segatum AgseNPV-B 148.981 KM102981
NPV-B – English
Apocheima cinerarium ApciNPV 123.876 FJ914221
NPV
Buzura suppressaria BusuNPV 121.268 KM986882
NPV – Guangxi
Buzura suppressaria NPV BusuNPV 120.420 KF611977
–Hubei
Clanis bilineata ClbiNPV 135.545 DQ504428
NPV – DZ1
Chrysodeixis chalcites ChchNPV 149.624 JX560542
NPV – TF1H
NPV – TF1C
NPV – TF1B
(continued)
66 M. Gani et al.

Genome Genebank
NPV – TF1A
NPV – TF1G
Chrysodeixis chalcites ChchNPV 149.622 AY864330
NPV
Chrysodeixis includens ChinNPV 140.859 KU669291
NPV – 1C
NPV – 1B
NPV – 1D
NPV – 1A
NPV – 1G
Chrysodeixis includens ChinNPV 139181 KU669293
NPV – 1F
Ectropis obliqua NPV – A1 EcobNPV 131.204 DQ837165
Ectropis obliqua EcobNPV 130.145 KC960018
NPV – unioasis
Euproctis pseudoconspersa EupsNPV 141.291 FJ227128
NPV – Hangzhou
Helicoverpa armigera HearSNPV 131.403 AF271059
SNPV – G4
Helicoverpa armigera HearSNPV 130.759 AF303045
SNPV – C1
Helicoverpa armigera HearSNPV 154.196 EU730893
SNPV
Helicoverpa armigera HearSNPV 132.425 AP010907
SNPV NNg1
Helicoverpa zea SNPV HzSNPV 130.869 AF334030
Helicoverpa armigera HearSNPV 130.992 JN584482
SNPV – Australia
Helicoverpa armigera HearSNPV 130.442 KJ909666
SNPV – AC53
Helicoverpa armigera HearSNPV 130.460 KU738896
SNPV – AC53C1
SNPV – AC53C6
SNPV – AC53T5
SNPV – H25EA1
(continued)

Genome Genebank
SNPV – LB3
SNPV – LB6
SNPV SP1B
SNPV –SP1A
SNPV – LB1
Helicoverpa armigera HearSNPV 136.760 KT013224
SNPV – L1
Helicoverpa zea HzSNP 130.890 KJ004000
SNPV – HS18
Helicoverpa zea SNPV – HzSNP 129.694 KM596835
Brasil/South
Heimileuca sp. NPV HespNPV 140.633 KF158713
Lambdina fiscellaria LafiNPV 157.977 KP752043
NPV – GR15
Leucania separata NPV LeseNPV 168.041 AY394490
Lymantria dispar MNPV LdMNPV 161.046 AF081810
Lymantria dispar MNPV LdMNPV 157.270 KU377538
–BNP
Lymantria dispar LdMNPV 162.658 KT626571
MNPV – Japan-3041
Lymantria dispar LdMNPV 163.138 KF695050
MNPV – Korea-2161
MNPV – Spain-3054
Lymantria dispar LdMNPV 164.158 KP027546
MNPV – 27/2
Lymantria dispar LdMNPV 161.727 KU249580
MNPV – Russia-27/0
Lymantria dispar LdMNPV 161.712 KM386655
MNPV – Russia-3029
Lymantria dispar LdMNPV 159.729 KX618634
MNPV – RR01
Lymantria dispar LdMNPV 161.006 KU862282
MNPV – USA-45/0
MNPV – USA-Ab-a624
Lymantria xylina NPV- 5 LyxyNPV 156.344 GQ202541
Mamestra brassicae MbMNVP 154.451 JX138237
MNVP – CHb1
(continued)
68 M. Gani et al.

Genome Genebank
Mamestra brassicae MbMNVP 153.890 KJ871680
MNVP – Cta
Mamestra brassicae MbMNVP 152.710 JQ798165
MNVP – K1
Mamestra configurata MacoNPV-A 155.060 U59461
NPV-A 90/2
Mamestra configurata MacoNPV-A 153.656 AF539999
NPV-A 90/4
Mamestra configurata MacoNPV-B 158.482 AY126275
NPV-B
Operophtera brumata OpbuNPV 119.054 MF614691
NPV – MA
Orgyia leucostigma NPV OrleNPV 156.179 EU309041
CFS-77
Peridroma PespNPV 151.109 KM009991
sp. NPV – GR-167
Perigonia lusca NPV PeluNPV 132.831 KM596836
Pseudoplusia includens PsinNPV 139.132 KJ631622
NPV – IE
Spodoptera exigua multiple SeMNPV 135.395 HG425349
NPV – HT-SeSP2A
NPV – HT-SeG26
NPV – HT-SeG24
NPV – HT-SeG25
NPV – VT-SeAl1
NPV – VT-SeAl2
NPV – VT-SeOx4
Spodoptera exigua multiple SeMNPV 135.611 AF169823
NPV
Spodoptera frugiperda SfMNPV 135.611 AF169823
MNPV
Spodoptera frugiperda SfMNPV 131.330 EF035042
MNVP– 3AP2
Spodoptera frugiperda SfMNPV 134.239 KF891883
MNPV –Colombian
Spodoptera frugiperda SfMNPV 132.565 EU258200
MNPV – 19
(continued)

Genome Genebank
Spodoptera frugiperda SfMNPV 128.034 JF899325
MNPV – G(def)
Spodoptera frugiperda SfMNPV 132.954 HM595733
MNPV – Nicaraguan
Spodoptera littoralis SpliNPV 137.998 JX454574
NPV – AN1956
Spodoptera litura SpltMNPV 139.342 AF325155
MNPV – G2
Spodoptera litura NPV – II SpltNPV II 148.634 EU780426
Sucra jujube NPV - 473 SujuNPV 135.952 KJ676450
Trichoplusia ni SNPV TnSNPV 134.394 DQ017380
Urbanus proteus NPV – UrprNPV 105555 KR011717
Br/South
Lepidoptera GV Adoxophyes orana AdorGV 99.657 AF547984
GV – En
Adoxophyes orana AdorGV 99.507 KM226332
GV – Mi
Agrotis segetum GV AgseGV 131.680 AY522332
Agrotis segetum GV – L1 AgseGV 131.442 KC994902
Agrotis segetum GV - DA AgseGV 131557 KR584663
Choristoneura occidentalis ChocGV 104.710 DQ333351
GV
Clostera anachoreta ClanGV 101.487 HQ116624
GV – HBHN
Clostera anastomosis ClanGV-A 101.818 KC179784
GV-A Henan
Clostera anastomosis ClanGV-B 107.439 KR091910
GV-B
Cnaphalocrocis medinalis CnmeGV 111.246 KU593505
GV - Enping
Cnaphalocrocis medinalis CnmeGV 112.060 KP658210
GV
Cryptophlebia leucotreta CrleGV 110.907 AY229987
GV – CV3
Cydia pomonella GV – M1 CpGV 123.500 U53466
Cydia pomonella GV – S CpGV 123.193 KM217573
Cydia pomonella GV – M CpGV 123.529 KM217575
Cydia pomonella GV – 112 CpGV 124.269 KM217576
Cydia pomonella GV – E2 CpGV 123.858 KM217577
Cydia pomonella GV – I07 CpGV 120.816 KM217574
Diatraea saccharalis GV DisaGV 98.392 KP296186
Epinotia aporema GV EpapGV 119.082 JN408834
Erimmyis ello GV ErelGV 102.759 KJ406702
(continued)
70 M. Gani et al.

Genome Genebank
Helicoverpa armigera GV HearGV 169.794 EU255577
Mocis sp. GV MospGV 134.272 KR011718
Mythina unipuncta GV MyunGV 144.673 KX855660
Phthorimaea operculella PhopGV 119.217 AF499596
GV – Tu
Phthorimaea operculella PhopGV 119.004 KU666536
GV – Sa
Pieris rapae GV – (1) PiraGV 108.592 GQ884143
Pieris rapae GV –E3 PiraGV 108.476 GU111736
Pieris rapae GV – (2) PiraGV 108.658 JX968491
Plodia interpunctella PlinGV 112.536 KX151395
GV – Cambridge
Plutella xylostella PlxyGV 100.999 AF270937
GV – K1
Plutella xylostella GV – T PlxyGV 100.978 KU529794
Plutella xylostella GV – M PlxyGV 100.986 KU529793
Plutella xylostella GV – C PlxyGV 100.980 KU529791
Plutella xylostella GV – K PlxyGV 101.004 KU529792
Plutella xylostella PlxyGV 100.941 KU666537
GV – SA
Pseudaletia unipuncta PsunGV 176.677 EU678671
GV – Hawaii
Spodoptera frugiperda SfGV 140.913 KM371112
GV – VG008
Spodoptera litura GV – K1 SpltGV 124.121 DQ288858
Trichoplusia ni TnGV 175.360 KU752557
GV – LBIV12
Xestia c-nigrum GV XecnGV 178.733 AF162221
Hymenoptera Neodiprion abietis NPV NeabNPV 84.264 NC008252
NPV
Neodiprion lecontei NPV NeleNPV 81.755 NC005906
Neodiprion sertifer NPV NeseNPV 86.462 NC005905
Diptera NPV Culex nigripalpus NPV CuniNPV 108.252 AF403738
Source: Wennmann et al. (2018)
prised in average by 10 dsRNA fragments with a varying length of 0.4–4 kb.

Different electropherotypes from cypovirus were initially identified on the basis of
differences in the migration patterns of their genome segments during gel electro-
phoresis, which was used to identify them. The sum of all these fragments adds up
to a total genome length of 19–25 Kb. So far, 14 different electropherotypes have
been identified (Martens et al. 1989). Due to these studies, it is known that the
smallest fragment contains the coding gene for the CPV’s polyhedrin, which is the
major OB component.
The molecular biology of Entomopoxviruses has been limited by the lack of

genomic information. EPV genomic organization and molecular mechanisms of
replication, pathogenesis and host range are largely unknown. Known EPV genomes
are constituted of a single dsDNA molecule of 130 to 375 kb in length. Such an
extensive genome may have the potential to code for as many as 150 to 300 genes
(Arif 1984). A peculiarity of these genomes is the presence of isometric DNA
sequences at the terminal ends of the molecule, constituted by inverted palindromic
motifs.
Two EPVs genomes have been sequenced so far, from migratory grasshopper,
Melanoplus sanguinipes (MsEPV) (Afonso et al. 1999) and from red hairy caterpil-
lar, Amsacta moorei (AmEPV) (Bawden et al. 2000). The 236-kb MsEPV genome
contains a subset of genes shared between all sequenced poxviruses, and allowed
the concept of a common, universally shared genetic core of poxvirus genes.
Poxvirus core genes included many of those associated with RNA transcription,
post-transcriptional modification, DNA replication and core structural proteins.
The EPV genes are classified according to phases of expression as: (a) early (b)
intermediate and (c) late genes. Early genes are expressed before the genomic DNA
is replicated. Even, some genes are expressed before the DNA is totally released
from the capsid. It is known that proteins expressed at the early events of infection
inhibit the synthesis of macromolecules by the host cell. These early genes code for
non-structural proteins, including enzymes involved in DNA replication, and modi-
fication of DNA and RNA, as well as those implicated in the inactivation of host
defence mechanisms. Additionally, these genes code for transcriptional factors used
by intermediate genes. During the intermediate phase, viral DNA replicates and
intermediate genes code for transcriptional factors used by the late genes. Finally,
late genes are expressed once the viral DNA has been replicated and code mostly for
structural proteins, both for the assembling of the capsid and the formation of spher-
oids. Interestingly, some of these genes also code for transcriptional factors used by
early genes.
Only two entomopathogenic Invertebrate Iridescent Virus (IV) genomes have
been sequenced to date, IIV-6 or Chilo iridescent virus (Jakob et al. 2001) and iri-
descent virus type 3 (IIV-3) or mosquito iridescent virus (Delhon et al. 2006). The
IV genome is packed as a circularly permuted and terminally redundant ds-DNA
molecule of 140–303 kb (Goorha and Murti 1982). IV genomes range in size from
105 to 212 kb and contain 96 to 234 largely non-overlapping open reading frames
(ORFs), a G-C content ranging from 27% to 55% and complex repeat sequences
mostly located between coding regions. Genomes exhibit little to no co-linearity
among genera (Delhon et al. 2006).
The replication process of IV genome is highly complex. Replication starts early
after infection, as DNA replicates in the nucleus, producing similar or shorter cop-
ies. Replication continues in the nucleus but some copies are transported to the
cytoplasm, where replication goes on and recombination occurs among the copies
constructing a complex of concatamers (Ward and Kalmakoff 1991). So far, it is
known that a virus encoding enzyme called DNA integrase-recombinase may be
involved in the recombination of small pieces of DNA as well as in the resolution of
72 M. Gani et al.
the concatamer, just before the DNA is packed within the capsids. Transcription of
the early genes starts with the RNA polymerase II from the host, which has been
modified by one of the viral proteins (Jakob and Darai 2002).
Only three genomes of ascoviruses have been fully sequenced: Trichoplusia ni
ascovirus 2c (TnAV-2c), Spodoptera frugiperda ascovirus1a (SfAV-1a) and
Heliothis virescens ascovirus 3e (HvAV-3e) (Cui et al. 2007).
The study of gene content has the potential to show the extent of variation
between baculovirus genomes whereas the comparison of genomes may provide
valuable insights into their evolution and biology (Herniou et al. 2003). Baculovirus
genomes range in size from approximately 80–180 kb (Theilmann 2005). Currently,
the baculovirus with the largest genome is Xestia c-nigrum NPV (XecnGV) with
178,733 bp (Hayakawa et al. 1999), while the smallest belongs to Neodiprion lecon-
tei NPV (NeleNPV) with 81,755 bp (Lauzon et al. 2004). The recently determined
complete genome of Antheraea proylei nucleopolyhedrovirus (AnprNPV) is
126,930 bp in lenth and encodes 147 ORFs (Shantibala et al. 2018). The number of
predicted ORFs found in sequenced baculoviruses encoding 50 or more amino acids
range from approximately around 89–181 ORFs. The average G + C content is quite
variable in baculoviruses, ranging from around 30 to 60%.
A distinctive feature of most sequenced baculoviruses is the presence of repeat
regions or homologous regions (hrs) dispersed throughout the genome, ranging
from approximately 3 to 17 hr/repeat regions. In NPVs, most hrs contain 30 bp
palindromes within direct repeats and are similar to other NPV hrs, whereas GV
repeat regions are more variable and often lack palindromes (Wormleaton et al.
2003). Hymenopteran baculovirus genomes contain repeated regions that do not
conform to the typical structure of hrs in lepidopteran NPVs. Comparative analysis
provided insight into their evolutionary history in that differences and similarities in
amino acid sequences and gene order aided in the division of baculoviruses into
groups, sharing gene characteristics and overall genome relatedness.
More closely related viruses share a higher degree of gene co-linearity. Hu et al.
(1998) developed a method called “gene parity plots” that compared the positions
of homologous genes in different genomes and is used to show conservation between
baculovirus genomes. Another method for comparing and classifying genomes is
the use of phylogenetic trees (Herniou et al. 2003).
Early attempts to infer relationships between baculoviruses and to study their
evolution were approached by phylogenetic analyses of the amino acid and nucleo-
tide sequences of the polh and granulin (gran) gene, which constitute the OB matrix
of NPVs and GVs, respectively. The phylogenetic analyses based on polyhedrin
gene sequences further subdivided lepidopteran NPVs into group I and group II
NPVs (Zanotto et al. 1993). This division moreover appeared to correlate with the
utilization of two different budded virus (BV) envelope fusion proteins. Group I
NPVs contain the major envelope glycoprotein GP64, which mediates membrane
fusion (Blissard and Wenz 1992). Group II NPVs as well as GVs lack GP64 protein,
but contain a functional homolog designated as F protein (Westenberg et al. 2002).
The major OB proteins polyhedrin and granulin are made in large amounts and
for this reason large quantities can be obtained and sequenced from the N-terminus
(Rohrmann et al. 1981). Moreover, the genes coding for polyhedrin and granulin
are highly conserved and thus easily identified in new baculovirus isolates, by DNA
hybridization or more commonly by PCR analysis with degenerate primers (Gani
et al. 2017). Single gene phylogenies, however, may occasionally lead to misinter-
pretation as they not always reflect authentic relations between viruses. This was for
example the case for the AcMNPV polh gene, which appeared to have a mosaic
structure resulting most probably from recombination events (Lange et al. 2004).
While biological data as well as sequence information for other AcMNPV genes
clearly show that AcMNPV belongs to group I NPVs, its polh gene is most closely
related to the homologue in Trichoplusia ni SNPV, a group II NPV. Nevertheless,
polh gene phylogenies as well as phylogenies of other conserved genes usually
reflect baculovirus relationships and evolution. In fact, polh was until recently the
only choice for analysis of large numbers of baculoviruses due to the limited
sequences available for other genes.
The use of a concatenation of shared genes has been shown to produce more reli-
able trees (Herniou et al. 2003). This is done by producing concatamers of the core
proteins found in all baculoviruses, aligning their amino acid sequences and gener-
ating trees that reflect different taxonomic divisions. Gene order and phylogeny
provides essential information on the evolution and relatedness of baculoviruses.
Comparative analysis of all completely sequenced baculoviruses revealed a set of
core genes conserved in all genomes that have essential roles in the baculovirus life
cycle (Garcia-Maruniak et al. 2004). There are 38 conserved core genes found in all
baculoviruses (Wennmann et al. 2018). Most of them have a known function within
the genome, either required for RNA transcription, DNA replication or as structural
and auxiliary proteins. Identification of genes that are essential or that stimulate
DNA replication in baculoviruses has provided a basis for elucidating the process
by which they replicate their genomes (Kool et al. 1995). Baculovirus DNA repli-
cates in the nucleus and they carry their own complement of genes encoding DNA
replication proteins. Four of these genes are found in all sequenced baculoviruses to
date: DNA polymerase, DNA helicase (p143), lef-1 and lef-2 (Herniou et al. 2003).
A list of additional genes involved in DNA replication found in lepidopteran bacu-
loviruses also includes: lef-3, ie-1 and me53 (Herniou et al. 2003; Lange and Jehle
2003). An additional gene, dbp (DNA binding protein), is found in lepidopteran and
dipteran baculoviruses (Lauzon et al. 2004).
Four major groups of baculovirus genotypes were identified by sequencing the
N-terminus of purified OB proteins (Rohrmann 1986). (i) the dipteran group, whose
OB proteins did not show any relatedness to those of other baculoviruses, (ii) the
hymenopteran group, with limited relatedness to polyhedrins of the lepidopteran
viruses, (iii) the nucleopolyhedroviruses and (iv) the granuloviruses. These main
clusters were later corroborated using DNA sequence analyses of other genes (lef-8,
ac22-pif2) (Herniou et al. 2004) and by genome sequence comparisons, which
revealed that a NPV isolated from the mosquito Culex nigripalpus (CuniNPV) did
not have a homologue of the polyhedrin gene, but used a different gene to encode its
OB protein (Afonso et al. 2001). Whether any of the single gene trees indeed repre-
74 M. Gani et al.
sented the phylogeny of the baculoviruses had not been evaluated until whole
genome sequence comparisons were performed.
Herniou et al. (2001) was the first study to use the information derived from
whole genomes to infer a baculovirus phylogeny and compare the trees based on
complete genomes to those based on single genes. The inference of phylogeny
based on complete genomes followed three complementary approaches using: (i)
the concatenated sequences of the core genes present in all baculoviruses, (ii) gene
order and (iii) the gene content of the genomes. These approaches had separately
been successfully applied to the phylogenetic analyzes of herpesviruses (Montague
and Hutchison 2000). Applying these three methods on 9 and 13 complete genomes,
the phylogeny of baculoviruses was clarified, permitting the comparison of single
gene trees (Herniou et al. 2003). Of all the core genes, comparisons of only seven
genes (ac22/pif-2, ac81, ac119, ac142, ac145, lef-8 and lef-9) produced the same
phylogenetic tree comparisons based upon complete genomes (Herniou et al. 2001).
Despite the limitations of single gene trees, their use in phylogenetic analysis is
still needed when less characterized viruses are being investigated. In order to over-
come the limitation of single gene trees, a small number of phylogenetically infor-
mative genes may be used. Herniou et al. (2004) developed degenerate PCR primers
that are suited to amplify highly conserved gene fragments within pif-2 (ac22) and
lef-8 (ac50), which can be directly subjected to DNA sequencing and molecular
phylogenetics (Herniou et al. 2004). By using this approach the separation of the
baculovirus tree into four main groups as described above was validated for an
unprecedented number of viruses.
Lange et al. (2004) also succeeded in developing degenerate primers for con-
served sequences within lef-8, lef-9 and polh/gran of Lepidoptera-specific NPVs
and GVs (Lange et al. 2004). Using this method, a comprehensive phylogenetic tree
of 117 baculoviruses was inferred by Jehle et al. (2006). Baculovirus phylogeny
based on the maximum-likelihood (ML) method [using RAxML (randomized
accelerated maximum likelihood)] software was recently inferred by Wennmann
et al. (2018), using concatenated 38 core-gene amino acid sequences from 172
entirely sequenced baculovirus genomes. It was found that the 38 core-gene data
provided a more accurate method to define distinctions between baculovirus species
than the original 3-gene concept.
A baculovirus phylogenetic tree using 37 core genes from 81 baculovirus com-
plete genomes was constructed by Nguyen et al. (2018) (Fig. 3.2). On the basis of
complete genome analyses (Herniou et al. 2001; Herniou et al. 2003; Lauzon et al.
2004) and gene phylogenies (Herniou et al. 2004; Lange et al. 2004; Jehle et al.
2006), the following conclusions can be derived:
(i) Baculoviruses comprise at least four distinct clades, which should be consid-
ered as different genera. These are (a) lepidopteran-specific NPVs, (b)
lepidopteran-specific GVs, (c) hymenopteran-specific NPVs and (d) dipteran-
specific NPVs;
(ii) the lepidopteran-specific NPVs fall into two most likely monophyletic clades,
the so-called group I and group II NPVs, utilizing different envelope fusion
proteins for cell-to-cell spread;
Fig. 3.2 Molecular phylogenetic analysis by Maximum Likelihood method. The tree was con-
structed using 37 core genes from 81 baculovirus complete genomes. Bootstrap value resulted
from 1000 replications is shown in each node. Red arrow – HytaNPV. (Source: Nguyen et al. 2018)
76 M. Gani et al.
(iii) within group I NPVs, two monophyletic clades (Ia and Ib) have been defined
(Jehle et al. 2006). Clade Ia includes Autographa californica NPV (AcMNPV)
and variants of this virus isolated from other insects, as well as Rachiplusia ou
MNPV, Bombyx mori NPV, and Thysanoplusia orichalcea NPV. These viruses
infect members of the families Noctuidae, Bombycidae, Pyralidae and
Plutellidae. Clade Ib comprises hosts from the Lepidoptera families Arctiidae,
Geometridae, Lymantriidae, Notodontidae, Nymphalidae, Saturniidae, and
Tortricidae. There are further group I NPVs, which do not belong to clade Ia
and Ib;
(iv) group II NPV relationships are characterized by long branches and weak reso-
lution at the basal nodes, which suggests that this group is more ancient than
group I NPVs (Jehle et al. 2006). However, their host range is apparently
restricted to fewer lepidopteran families, as group II NPVs have mostly been
isolated from higher Macrolepidoptera families, such as Noctuidae,
Lymantriidae, Lasiocampidae and Geometridae. Thus far, the only
Microlepidoptera-specific group II NPVs have been identified from Adoxophyes
honmai (Tortricidae) and Wiseana cervinata (Hepialidae). Improved virus
sampling might show an increase in the number of lepidopteran host families
infected by group II NPVs;
(v) GVs have so far been identified from 9 Lepidoptera families including
Noctuidae, Arctiidae, Plutellidae, Nodontidae, Pieridae, Tortricidae,
Bombycidae Sphingidae, and Gelechiidae. Similar to group II NPVs basal
resolution is weak and the branches are generally long, suggesting an ancient
radiation of these viruses. Midgut infecting GVs (Harrsinia billions GV) and
slow killing GVs (Adoxophyes orana GV, Xestia c-nigrum GV and Trichoplusia
ni GV) are scattered within the vast majority of fast GVs causing systemic
infection (Federici 1997; Wormleaton and Winstanley 2001). This clearly indi-
cates that these traits are not phylogenetically informative and therefore not
suitable for a natural classification of GVs. GVs isolated from Tortricidae
appear to have a monophyletic origin irrespective of their pathogenesis and
replication speed (Lange et al. 2004; Jehle et al. 2006).
3.4 Entomopathogenic Bacteria (EB)
Bacteria may be generally defined as unicellular and ubiquitous microorganisms

possessing a single chromosome deprived of a nucleus membrane and having ribo-
somes of the 70S type. These organisms proliferate through binary fission, a process
resulting in daughter cells that are essentially identical copies of the mother cell.
Bacteria constitute a large domain of prokaryotic microorganisms. Typically a few
micrometres in length, they have varying shapes which include spherical, rod-like
and spiral cells. Bacteria were among the first life forms to appear on Earth and
inhabit all environments, including soil, oceans and land waters, acidic hot springs,
radioactive waste and the deep portions of the Earth's crust. They also live in sym-
biotic and parasitic relationships with or within plants and animals.
The ancestors of modern bacteria were unicellular microorganisms, the first
forms of life to appear on Earth, about four billion years ago. Although bacterial
fossils exist, such as stromatolites, their lack of distinctive morphology prevents
them from being used to examine their evolutionary history or to date the time of
origin of a given species. The most recent common ancestor of bacteria and archaea
(the other branch of Prokaryotes) was probably a hyperthermophile that lived about
2.5–3.2 billion years ago (Di Giulio 2003; Battistuzzi et al. 2004).
As with other living organisms, insects are intimately associated with bacteria at
all stages of their lives. EB infecting insects have sizes ranging from less than 1 μm
to several μm in length. Insects inhabit diverse niches and interact with various
bacteria to form relationships that range from mutualistic symbiosis to pathogene-
sis. Depending on the relationship, symbiotic associations can be divided into com-
mensalism, mutualism and parasitism (Moya et al. 2008). The mechanisms leading
to these interactions are presumed to have ancient origins and to have developed
throughout a long co-evolutionary process (Vilcinskas 2010). Pathogenic interac-
tions require a high degree of specialisation and intimate contact with the host. In
this respect, many of the molecular mechanisms used by bacterial pathogens and
mutualists are similar. Furthermore, the same microorganism can behave differ-
ently, depending on the fitness of the host and the environmental circumstances,
turning from beneficial to detrimental, thus complicating the arbitrary definition of
bacterial behaviour. Therefore, the concepts of mutualism and pathogenesis are not
clearly differentiated, but rather a matter of balance between the bacteria and the
insect host in terms of fitness, reproductive success, feeding and influence of other
symbionts.
Given that insects and bacteria are some of the most numerous organisms on our
planet, their interactions hold significance in many areas. Most bacteria currently
are spore forming members of the bacterial family Bacillaceae and Enterobacteraceae.
In Bacillaceae the genus Bacillus is the most important. The insect pathogenic
Bacilli occur in healthy and diseased insects and can also be isolated from many
other habitats including insect frays, soil, plants, granaries and aquatic environ-
ments. Over 90 species of naturally occurring insect-specific (entomopathogenic)
bacteria have been isolated from insects, plants and soil, but only a few have been
studied intensively. Much attention has been given to Bacillus thuringiensis (Bt)
(Firmicutes: Bacillaceae) a species that has been developed as a microbial insecti-
cide and safe alternative for the management of insect pests (Lacey et al. 2001;
Lacey and Kaya 2007). Indeed, Bt originally discovered in 1901 by Ishiwata and
later rediscovered and isolated by Berliner in 1915 in Germany from the diseased
larvae of the Mediterranean flour moth, Ephestia kuhniella has been the most stud-
ied and broadly used in microbial control. Screening programs have identified thou-
sands of different strains of Bt all of which have a limited host range but together
span a wide range of insect orders (Lepidoptera, Diptera, Coleoptera, Hymenoptera,
78 M. Gani et al.
Homoptera, Orthoptera and Mallophaga) and even other organisms such as nema-
todes, mites and protozoa (Feitelson et al. 1992). Manonmani and Balaraman (2001)
isolated several strains of B. thuringiensis from various sources, i.e. soil, water,
larvae and roots of aquatic weeds representing diverse habitats. These strains were
examined for their flagellar antigenicity, mosquito larvicidal activity and protein
composition. Among these, one strain belonging to the serotype B. thuringiensis
thompsoni (H-12) was found to be highly toxic to different species of mosquitoes.
Nine different indigenous B. thuringiensis isolates were recovered from the soil of
cotton fields in different Egyptian governorates namely, Minofiya, Sharkiya,
Gharbiya, Kalyoubiya, Dakahliya and Kafr El-Sheikh (Saker et al. 2012). The dis-
covery that Bt spore-associated toxins are extremely virulent and can persist in the
environment with high potency, prompted the development of bacterial spray for-
mulations (Wilcox et al. 1986) and transgenic plants expressing certain Bt toxins
(Fischhoff et al. 1987). Different Bt varieties produce a crystal protein that is toxic
to specific groups of insects. Sprays of sporulated B. thuringiensis have a long his-
tory of safe use for pest control in agriculture (Paul et al. 2017). Sprays of B.
thuringiensis subsp. israelensis (Bti) and of Bacillus sphaericus (Bsp) have been
used to control disease-carrying mosquitoes and blackflies (Land and Miljand
2014). Since 1996, transgenic crop plants expressing entomocidal Cry proteins
from B. thuringiensis have been commercialized, resistant to several insect pests (de
Maagd et al. 1999).
The most common species applied are B. thuringiensis var. kurstaki, var. israe-
liensis, var. tenebrionis, B. popilliae and B. sphaericus (Lysinibacillus sphaericus).
These biological control agents being persistent, cheap, readymade, highly specific
and safe for non-target flora and fauna, certainly contributed to the development of
management practices for agricultural and forest insect pests.
Advancements in the characterization of bacterial pathogens including purifica-
tion and culturing methods, molecular identification of virulence factors and whole
genome characterization and comparisons have prompted the discovery of novel
pest management tools. In this view, insecticidal molecules expressed and secreted
by various EB have been targeted for genetic manipulation to enhance toxicity
(Gatehouse 2008). Recently, other insect pathogenic bacteria with modes of action
similar to Bt have been hailed as agriculturally relevant (Chattopadhyay et al. 2004).
Certainly, EB of diverse taxonomic groups and phylogenetic origins have been
shown to have striking similarities in the virulence factors they produce (Priest et al.
2004). Bacterial virulence factors are often encoded on mobile genetic elements
such as plasmids and can easily be spread through horizontal gene transfer by bac-
teriophages. The discovery that both Gram-negative and Gram-positive bacteria
produce analogous insect-specific toxins infers history of gene transfer between
them (Ochman et al. 2000). For example, Photorhabdus luminescens (Proteobacteria:
Enterobacteriaceae), the bacterial symbiont of the entomopathogenic nematode
Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae), has virulence fac-
tors similar to those of Bt (Nielsen-LeRoux et al. 2012). The first commercial
biopesticide, Sporeine, commercialized in 1938 in France, was based on Bt.
Moreover, the first EB used in a major insect control program was Paenibacillus
popilliae (Dutky, previously Bacillus popilliae; Klein and Jackson 1992), the caus-
ative agent of milky disease in Japanese beetle (Popillia japonica Newman). Despite
successful use, problems related to the mass production of viable P. popilliae spores
(Stahly and Klein 1992) reduced commercial interest in this bacterium. Although
commercial products based on P. popilliae are currently available, their use is lim-
ited to the control of grubs, especially in organic agriculture (Johnson et al. 2001).
Both B. thuringiensis and L. sphaericus are ubiquitous soil microorganisms and
isolates have been obtained from multiple environments worldwide (Guerineau
et al. 1991; Bernhardt et al. 1997). Photorhabdus and Xenorhabdus are the genera
of EB that live in symbiosis with nematodes of the family Heterorhabditis or
Steinernema, respectively, and together they infect insect larvae. As symbionts, the
bacteria are food that supports the bacteriovorous nematode host development, but
also produce toxic natural products (NP) and proteins that kill the insect prey, in
which they actively multiply (Bode 2009).
Bacterial classification began with taxonomic units based solely on the morphologi-
cal characteristics of the organisms, and progressed to the use of a wide variety of
characteristics including physiology, biochemistry and genetic data. Their classifi-
cation was started by Muller (1773) with the discovery of different morphological
forms. Cohn (1872) classified bacteria into 4 major types depending on their shapes
as Cocci (spherical or elliptical), Bacilli (rod or cylindrical), Vibro (curved or
comma) and Spirilla (spiral or spring like). Depending upon the staining reactions
by the Gram stain, bacteria are classified into Gram positive and Gram negative.
Today, the sheer number of species that have been identified and the wide diversity
among them make classification of these organisms into discreet arrangements both
difficult and necessary.
Two main groups of prokaryotic microorganisms are recognized based on the
16S ribosomal RNA sequence: Archaea, containing microorganisms that share
DNA replication, transcription and translation features with eukaryotes; and
Eubacteria or true bacteria. EB are classified within Eubacteria. This group contains
three major divisions, based on the presence or structure of the cell walls: bacteria
with a Gram-negative type cell wall (Gracilicutes), those with a Gram-positive type
cell wall (Firmicutes) and Eubacteria lacking a cell wall (Tenericutes). EB may be
found in all three main Phyla and are mainly classified as spore producers and
non-spore producers (Fig. 3.3). However, when focusing on bacteria with a
demonstrated use in insect control, species of interest occur in the following
families: Bacillaceae, Paenibacillaceae, Enterobacteriaceae, Neisseriaceae,

Pseudomonadaceae, Lactobacillaceae and Micrococcaceae.
Classification of EB into groups on the basis of their pathogenicity presents dif-
ficulties, as most bacteria are facultative pathogens and present virulence variations
depending on the host environment and the specific strain. Bacterial populations,
80 M. Gani et al.
Fig. 3.3 Phenotypic classification of entomopathogenic bacteria
including pathogenic ones, are essentially clonal, implying that exchange of DNA
in nature is infrequent. Many species displaying pathogenicity exist as a number of
distinct lineages, or clones, of which only a few contain the genetic information that
encodes pathogenicity (Selander et al. 1987). For example, genetic diversity among
strains of B. sphaericus explained variation in toxicity against mosquito larvae
(Krych et al. 1980). In some cases, non-pathogenic and disease-causing clonal lin-
eages have been named and classified as diverse species based on their pathogenic
niche (Lan and Reeves 2001). To resolve this issue, a bacterial species concept
based on members sharing a core genome (housekeeping genes) was proposed
(Dykhuizen and Green 1991). In this view, lineages incorrectly described as diverse
are named as clones within a species, based on sequence similarity (Lan and Reeves
2000, 2001).
Advances in DNA sequencing technology with the concomitant increase in
available bacterial genomic data, together with increased characterization of bacte-
rial population genetics, are expected to allow for a more accurate and useful defini-
tion of species that will increase EB understanding. Bergey’s Manual of Systematic
Bacteriology defines the bacterial species as “a collection of strains that share many
features in common and differ considerably from other strains.” It goes on to say
that "a species consists of the type strain and all other strains that are considered to
be sufficiently similar to it as to warrant inclusion with it in the species" .
A more uniform definition of the bacterial species is desirable and can possibly
be obtained through the use of genetic relatedness among bacteria. Established tax-
onomic techniques, including biochemical tests and serological techniques, allow
ready qualitative identification. Bacterial strains are grouped into species based on
sharing of certain distinguishing phenotypes of ecological importance and overall
genomic similarity (Stackebrandt et al. 2002). A bacterial strain is defined as the
descendants of a single isolation in pure culture and is usually derived from succes-
sive cultures, from an initial single colony. Phenotypic screening is usually consid-
ered the first step in the identification of bacterial strains as it allows a rough
comparison and placement into similar or different groups. Most commonly consid-
ered phenotypic traits include: (i) colony morphology (size and shape); (ii) colony
pigmentation; (iii) cell shape and size; (iv) motility and flagellar arrangement; (v)
Gram stain reaction; and (vi) aerobic or/and anaerobic metabolisms. In practice,
more than 70% DNA-DNA hybridization values are considered evidence of a single
species (Wayne et al. 1987). The DNA hybridization method has been then inte-
grated and partially replaced by sequencing methods. Within Eubacteria, current
classification is mostly based on polyphasic (consensus) taxonomy, including anal-
ysis of the nucleotide sequence of the ribosomal small-subunit RNA (16S rRNA),
DNA-DNA hybridization, as well as phenotypic, genotypic and phylogenetic data
(Brenner et al. 2005).
In the past 50 years, numerous bacteria such as B. sphaericus, B. thuringiensis
and Serratia marcescens have been isolated, classified and demonstrated in the lab-
oratory to be pathogenic to various insects (Bahar and Demirbag 2007; Kleespies
et al. 2008). Diverse methods have been used to classify B. thuringiensis and L.
sphaericus isolates, with flagellar H-serotyping being the most widely present in the
literature. For B. thuringiensis, 85 serotypes have been described (Jurat-Fuentes and
Jackson 2012), although most commercial biopesticides are based on serovars (or
subsp.) kurstaki (Btk), aizawai (Bta), israelensis (Bti), and tenebrionis (Btt). Each
of these serovars includes isolates expressing crystal (Cry) parasporal proteins with
entomotoxicity against specific insect taxonomic orders, including Lepidoptera,
Coleoptera and Diptera. Additional noncrystal toxins and other virulence factors
that enhance entomotoxicity may also be produced by diverse isolates. Important
for entomotoxicity of Bti are the cytolytic (Cyt) toxins they produce, which syner-
gize activity of Cry proteins against mosquito larvae (Wu and Chang 1985). On the
other hand, potential secretion during the vegetative stage of some strains of ther-
mostable insecticidal toxins called β-exotoxins is of concern during commercial
production of Bt-based biopesticides. The Bacillus species commonly recognized
as insect pathogens are B. thuringiensis, B. popilliae, B. lentimorbus, B. larvae, and
certain strains of B. sphaericus (Stahly et al. 2006).
Apart from Bacilli, there are further non-Bacillus EB such as Clostridium,
Serratia, Photorhabdus, Xenorahbdus and Pseudomonas (Boemare and Tailliez
2009). The red-pigmented varieties of S. marcescens are most often reported as
pathogens of insects. However, non-chromogenic strains of S. marcescens, for
example S. marcescens Bizio, are also pathogenic. In the last few years, approxi-
mately 60 pathogenic bacterial species and their products have been developed as
biopesticides, worldwide. These various pathogens are being used successfully in
the biological control of insects (Betz et al. 2000).
Bacteria and insects have co-evolved a wide range of complex relationships from
commensalism to parasitism or pathogenesis over more than 250 million years. The
specific host-bacteria relationships that have developed are the outcome of dynamic
co-evolution underpinned by genetic diversity and driven by selection pressure.
While opportunistic bacterial infections occur, often as a result of injury or stress,
most consistent EB have obligate or facultative relationships with their hosts. EB
occupy many niches, functioning as rhizosphere and phyllosphere colonizers, both
82 M. Gani et al.
obligate and facultative pathogens and as endophytes of plants. The main ecological
niche of some species is primarily as pathogen, surviving between hosts as environ-
mentally resistant spores. It has been proposed that Bt evolved in commensal rela-
tionships with plants to provide defence against herbivores’ attacks (Smith and
Couche 1991). This “bodyguard theory” states that microbe–plant symbiosis has
evolved so that microorganisms that are insect antagonistic are recruited and/or
maintained by the plant for protection. It has been supported by recent ecological
and molecular studies on Bt demonstrating rhizosphere competence (Vidal-Quist
et al. 2013), endophytic ability (Tao et al. 2014) and even plant-mediated volatile
attraction.
Genetic variation in bacteria originates from mutation and selection or acquisi-
tion of genetic material from the environment (transformation), bacteriophages
(transduction) or other bacteria (conjugation). These processes, combined with the
rapid generation time of bacteria, result in a high level of variation and wide ranges
of functionality among strains. Horizontal gene transfer of mobile genome frag-
ments known as genomic islands also increases genetic variation among popula-
tions. Genetic information is also sometimes stored in a number of plasmids, small
and usually circular DNA molecules that are self-replicating and that can be trans-
ferred between bacteria through a process known as conjugation. The plasmids usu-
ally contain genes that are crucial for specific functions, including pathogenicity.
For instance, most crystal toxins from Bt are located in plasmids (Held et al. 1982),
which can be exchanged between diverse strains and Bacillus spp. (González et al.
1982). The pathogenicity island of Serratia spp. containing tc genes is also located
on a plasmid (Dodd et al. 2006). Genetic information can also be stored in pro-
phages, DNA from bacteriophages that is inserted into the bacterial chromosome or
plasmid through transduction, which can also confer phenotypes conducive to
pathogenicity. The ecology of B. thuringiensis and L. sphaericus and the evolution-
ary advantage of producing insecticidal crystalline protein inclusions are still mat-
ters of debate. Supporting the identity of these bacteria as bona fide insect pathogens
is their commitment of relevant energy resources to production of high amounts of
insecticidal parasporal proteins. However, although spores of B. thuringiensis are
very persistent in the environment, the bacterium displays low rates of horizontal
transmission between hosts, with rare epizootics. Horizontal transmission of B.
thuringiensis was reported to be tightly related to the production of urease (Martin
et al. 2009), but this is not observed in commercially used strains. Thus, secondary
infections are uncommon after spray with biopesticides based on Btk and subsp.
aizawai (Smith and Barry 1998), which are urease positive (Martin et al. 2010). In
contrast, persistent activity was reported for applications of biopesticides contain-
ing the urease-negative Bti (Tilquin et al. 2008).
Persistence of L. sphaericus biopesticides is related to the low settling of L.
sphaericus spores in the water column (Nicolas et al. 1987) and recycling in the host
(Charles and Nicolas 1986). The biological control paradigm changed when the
potential of EB was discovered, especially associated to species belonging to the
genus Bacillus (Glare and O’Callaghan 2000). Initially, the species Paenibacillus
(former Bacillus) popilliae Dutky was introduced for the management of the
Japanese Beetle Popillia japonica Newman (Steinhaus 1975), but more concrete
results were achieved with the discovery of new Bt strains showing high toxicity
against specific insects at a competitive level, compared to conventional insecti-
cides, in terms of efficacy and costs of production. The strain HD-1, belonging to
subsp. kurstaki (De Barjac and Lemille 1970), soon became the main commercial
focus for the management of lepidopteran pests in agriculture and forestry. Beside
it, other strains are actually commercially available, such as SA-11, SA-12, PB 54,
ABTS-351 and EG2348, all isolated from insects or soil and expressing a range of
different toxins mostly belonging to the Cry1 and Cry2 families. Subsequently, the
discovery of a Bt strain belonging to the subsp. israelensis (Bti) was followed by its
commercialization for the management of mosquitoes and simulids (Goldberg and
Margalit 1977). Then, a particularly active strain of the subsp. tenebrionis was dis-
covered and employed against Coleoptera (Krieg et al. 1983). Homology among
toxins from B. sphaericus and Bt or other EB has been shown, demonstrating their
phylogenetic relationships and revealing a probable common co-evolution (de
Maagd et al. 2003).
Bacteria are characterized by one single chromosome within the nucleoid, whose
size varies among species. For example, the bacterial genomes of B. thuringiensis
(the widely studied insect pathogen), B. cereus and B. anthracis are around 5.4 Mb
(Carlson and Kolstø 1993; Ivanova et al. 2003). Other Bacillus spp. such as B. sub-
tilis and B. licheniformis have a smaller chromosome (4.2 Mb) (Kunst et al. 1997;
Rey et al. 2004). While the phenotypes of these species are different, their intra and
inter phylogenetic relationships are not clear. Several approaches have been used to
classify B. thuringiensis strains, including rRNA gene sequences, amplified frag-
ment length polymorphisms (AFLP), restriction fragment length polymorphisms
(RFLPs) in small subunit (SSU) rRNA sequences, GryB (gyrase subunit B) and
AroE (shikimate-5-dehydrogenase) gene sequences. The results suggest that there is
a high level of sequence homology among strains of B. thuringiensis. Similarly,
overall genetic studies have shown that B. thuringiensis and B. cereus are essentially
identical (Helgason et al. 1998). Bacillus anthracis can only be distinguished from
B. thuringiensis and B. cereus through microbiological and biochemical tests. Since
these genetic methods are not able to easily distinguish different members of B.
thuringiensis, B. anthracis and B. cereus, it becomes necessary to look for some
more easily recognizable markers. With the advent and development of next genera-
tion sequencing technologies, a great deal of sequencing data has been generated in
recent years, as the whole-genome-sequencing gives information on the organiza-
tion of the bacterial genomes. Genome sequencing is informative about the chromo-
some, which can be linear or circular, the presence of plasmids and the genome size
and variation, within a species (Casjens 1998). It also provides information on new
operons, origin of replication and genome polarity. This increases with the growing
84 M. Gani et al.
number of sequenced genomes. Finally, genome sequencing allows the character-

ization of guanine-cytosine content (GC) and its variation, genomic islands and
synteny with others organisms. All these data give information on DNA regions
acquired by horizontal gene transfer and therefore on the evolutionary history of the
bacterium. The rapid accumulation of whole genome data of Bacillus species in
Genbank makes it possible the comparison of genomic differences over the entire
genome that cannot be identified in analyses of specific, single gene sequences.
However, the size of the whole genome data poses great challenges on alignment-
based algorithms, which are effective in dealing with closely related sequences but
are unable to evaluate the recombination, shuffling and rearrangement events of the
whole genomes. Thus, alignment-free sequence analysis approaches, such as
Feature Frequency Profile (FFP), provide attractive alternatives over alignment-
based approaches. FFP is a new method used to study the whole genome phylogeny
based on k–mers (Jun et al. 2010). In this method, the number of features of a par-
ticular length that occur in a particular genome is counted and assembled into a FFP
vector. FFPs from different species are then compared using the Jensen–Shannon
(JS) Divergence (Lin 1991). A neighbor-joining phylogenetic tree can thus be con-
structed based on the resulting distance matrix. Compared to the traditional multiple
sequences alignment (MSA) based method, the alignment free FFP method can
compare both genic and non-genic regions of the whole genome at higher speed. It
can incorporate a wide variety of genomic features into each comparison including
intron deletions, exon sequence indels, transposable element insertions, base trans-
versions in coding sequences and some rare genomic changes such as short inter-
spersed element/long interspersed element (SINE/LINE) insertions (Sims et al.
2009). Wang and Ash (2015) reconstructed the whole-genome phylogeny of Bacillus
species using the FFP approach, with an aim to better understand the phylogenetic
relationships among them. Fifty complete Bacillus genome sequences and associ-
ated plasmids were compared using the FFP method (Fig. 3.4). The resulting whole-
genome phylogeny supports the placement of three Bacillus species (B. thuringiensis,
B. anthracis and B. cereus) as a single clade. These results clearly suggest the close
relationship among B. thuringiensis, B. anthracis and B. cereus species, are in
agreement with earlier results from DNA-DNA hybridization analysis and Multi
Locus Enzyme Electrophoresis (MEE), which showed high identity among them
(Priest et al. 1994). These three species have been grouped under the name of
Bacillus cereus sensu lato (Rasko et al. 2005) despite their obvious difference in
phenotype and pathological effects, which result from the genetic difference in plas-
mid rather than in chromosome. The B. weihenstephanensis strain KBAB4 was
found to be very closely grouped with the major cluster I-d consisting of all B.
thuringiensis isolates and proximal to cluster I-e (B. cereus) and cluster I-c (a clus-
ter containing both B. thuringiensis and B. cereus strains).
FFP proved to be more effective in inferring the phylogeny of Bacillus than
methods based on single gene sequences [16s rRNA gene, GryB (gyrase subunit B)
and AroE (shikimate-5-dehydrogenase)] analyses. Furthermore, the availability and
reduced cost of whole genome sequencing can be used without extensive gene
annotation to provide robust phylogenetic analysis of new isolates as they become
Fig. 3.4 Phylogenetic tree of 50 Bacillus strains, constructed using the NJ algorithm based on the
FFP features of the Whole Genome Data. Escherichia coli Bl21 (DE3) (AM946981.2) was used as
an outgroup in the analysis. The bootstrap confidence values were generated using 1000 permuta-
tions. Different symbols were allocated to represent different species: Blue triangle for B. thuringi-
ensis; Pink diamond for Bacillus cereus; Red circle for Bacillus anthracis; Green Square for
Bacillus subtilis. (Source: Wang and Ash 2015)
86 M. Gani et al.
available. Entomopathogenic nematode symbiotic bacteria Photorhabdus lumines-

cens has a genome size of 5.7 Mb (Duchaud et al. 2003), whereas Xenorhabdus
bovienii and X. nematophila, other nematode symbiotic bacteria, are estimated to
have a genome size of approximately 4.3 Mb (http://www.xenorhabdus.org/). Many
bacteria also contain extrachromosomal elements (plasmids), which are smaller
double-stranded DNA (dsDNA) molecules that replicate independent of the chro-
mosomal DNA. Genes located in bacterial plasmids usually code for proteins that
determine specific phenotypes, but do not code for products needed for bacterial
survival and growth. Bacterial genes are organized in operons or cassettes that con-
sist of a promoter, a series of genes and a transcription terminator. The most impor-
tant genes studied for EB are the Cry genes that code for insecticidal crystal proteins.
They are usually found in large transmissible plasmids or more rarely in the chro-
mosome. These proteins show entomopathogenic properties to insects from orders
Lepidoptera, Diptera and Coleoptera (WHO 1999). Different combinations of Cry
genes are found in various Bt strains including those with one, two or even four dif-
ferent genes (Lereclus et al. 1993).
More than 200 toxin genes with pesticidal activity have been cloned from a wide
range of B. thuringiensis strains (Table 3.5), grouped into 80 classes (Crickmore
et al. 1998). Sequence analysis of these genes is currently considered not only rel-
evant to the classification of bacteria but also for interpreting evolutionary relation-
ships among taxa (Crickmore 2000). Bacterial genome studies have provided
Table 3.5 Some B. thuringiensis toxins active against different insect pest orders
Order and Insects Active Toxin
Lepidoptera
Cydia pomonella Cry1Aa, Cry1Ab, Cry2Aa2, Cry15Aa1
Trichoplusia ni Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca, Cry1Da, Cry1Ea, Cry1Fa,
Cry1Ja, Cry2Aa1, Cry2Ab2, Cry2Ac1
Spodoptera exigua Cry1Ab1, Cry1Ba1, Cry1Bd1, Cry1C, Cry1Ca1, Cry1Fa1, Cry1Gb1,
Ariogeia rapae Cry1Ka1
Bombyx mori Cry1Ba1, Cry1Ka1
Hyphantria cunea Cry1Ba1, Cry1Ia1, Cry1Ka1
Plutella xylostella Cry1Ba1, Cry1Ka1
Manduca sexta Cry1Ba1, Cry1Bd1, Cry1Ea2, Cry1Gb1, Cry1Ia1, Cry1Ka1, Cry2Ab1
Spodoptera Cry1C, Cry1Ea1, Cry1Ea4, Cry2Ac1, Cyt1Aa2, Cyt2Aa1
frugiperda
Spodoptera littoralis Cry1C
Helicoverpa zea Cry1C, Cry1Ca2, Cry1D, Cry1Ea1, Cry1Fa1
Heliothis virescens Cry1Fa1, Cry2Aa1, Cry2Ab2
Ostrinia nubilalis Cry1Fa1, Cry2Aa1, Cry2Ab2, Cry2Ac1
Spodoptera litura Cry1Fa1, Cry2Aa1, Cry2Ab2
Lymantria disper Cry1Ia1
Galleria mellonella Cry2Aa1, Cry2Ab, Cry2Ab2 Cry9Aa1
(continued)

Order and Insects Active Toxin
Coleoptera
Diabrotica Cry3Ba1, Cry3Bb1
undecimpunctata
Leptinotarsa Cry3Ba1, Cry3Bb1, Cry8Aa1
decemlineata
Cotinis spp. Cry8Ba1
Cyclocephala sp. Cry8Ba1
Popillia japonica Cry8Ba1
Chrysomela scripta Cyt1Aa4
Diptera
Musca domestica Cry1Bd1, Cry1Gb1
Aedes aegypti Cry1C, Cry2Aa1, Cry2Ab, Cry2Ab2, Cry2Ac1, Cry4Aa, Cry4Aa1,
Cry4Ba, Cry4Ba1, Cry10Aa1, Cry11Aa, Cry11Aa1, Cry17Aa1, Cry19A,
Cry20Aa1, Cry27Aa1, Cyt1Aa2, Cyt1Aa4, Cyt1Ab1, Cyt2Aa1
Anopheles stephensi Cry4Aa, Cry11Aa, Cry17Aa1, Cry19A, Cry27Aa1, Cyt1Ab1
Culex pipiens Cry4Aa, Cry4Ba, Cry11Aa, Cry17Aa1, Cry19A, Cry27Aa1, Cyt1Aa2,
Cyt1Ab1, Cyt2Aa1
Anopheles gambiae Cry4Aa1, Cry4Ba, Cyt1Aa2, Cyt2Aa1
Culex Cry4Aa1, Cry4Ba, Cry11Aa, Cry11Ba1, Cyt1Aa4
quinquefasciatus
Callíphora stygia Cyt1Aa2, Cyt2Aa
Lucilia cuprina Cyt1Aa2, Cyt2Aa
Lucilia sericata Cyt1Aa2, Cyt2Aa
Hymenoptera
Acromyrmex Cry1, Cry9
Diprion pini Cry5A, Cry5B
Cephacia abietis Cry5A, Cry5B
Nematodes
Caenorhabditis Cry5Aa1, Cry5Ab1
elegans
Pratylenchus spp. Cry5Aa1, Cry5Ab1, Cry6Ba1, Cry12Aa1
Fasciola hepatica Cry5Ab1
Panagrellus Cry6Aa1
redivivus
Pratylenchus Cry6Aa1
scribneri
Source: Hernandez-Fernandez and Lopez-Pazos (2011)
extremely valuable information regarding the genetic diversity of species. Not only
have they assisted in resolving taxonomies at various levels, but also contributed in
assessing evolutionary relationships.
EB are distributed in phylogenetically diverse groups of prokaryotes. Gram-
negative EB include: (i) members of the Enterorbacteriaceae such as Serratia ento-
mophila, S. marcescens, S. proteamaculans and the nematophilic genera
88 M. Gani et al.
Photorhabdus and Xenorhabdus; and (ii) members of the Pseudomonadaceae such

as Pseudomonas entomophila. Gram positive EB comprise the most studied group
of bacteria in insect pathology and include members of Bacillus (Bacillaceae). This
genus has recently been submitted to several taxonomic revisions and a number of
new genera such as Brevibacillus, Lysinibacillus and Paenibacillus have been rec-
ognized from species previously described in Bacillus (Ash et al. 1993; Shida et al.
1996; Ahmed et al. 2007).
Since the 1970s, ribosomal RNA (rRNA) molecules have been considered for
studying bacterial molecular genealogies. These molecules are universally distrib-
uted, easily sequenced and carry generally useful phylogenetic information.
Comparison of rRNA sequences enabled notably the discovery of the domain
Archaea (Balch et al. 1977), a branch of prokaryotes more closely related to Eukarya
than to the other prokaryotes (Eubacteria) (Iwabe et al. 1989).
Sequences of 16S rRNA gene have been considered to assess evolutionary rela-
tionship of Serratia spp. (Dauga et al. 1990; Ashelford et al. 2002) and other lin-
eages. They have also helped to resolve taxonomic conflicts, particularly for
identification of new isolates, previously classified in this genus using basic pheno-
typic characters. Pseudomonas entomophila shares a common ancestor with P.
putida biovar A (bootstrap value of 94% indicating a high robustness of the node).
The genomes of these two species are also very similar (Vodovar et al. 2006), as P.
entomophila may have also acquired insect virulence factors through lateral gene
transfer. Furthermore, analysis of the P. entomophila genome confirmed the pres-
ence of genes that encode insecticidal toxin complexes also found in entomopatho-
genic enterobacteria such as Photorhabdus luminescens, S. entomophila,
Xenorhabdus nematophila and Yersinia spp. (Bowen et al. 1998; Waterfield et al.
2001). Protein-coding genes have been suggested as an alternative option to avoid
some of the problems encountered in phylogenetic studies with prokaryotes
(Hedegaard et al. 1999; Lerat et al. 2003). Recently, two protein-coding gene
sequences, gyrB and recA, have been considered for assessing evolutionary relation-
ships in various groups of bacteria, including the entomopathogenic types (Eisen
1995; Dauga 2002). Akhurst et al. (2004) considered gyrB gene sequences to assess
evolutionary relationships among Photorhabdus spp. Three Photorhabdus groups
can be recognized based on the phylogenetic trees of gyrB gene sequences: the P.
luminescens group, represented by subspecies P. luminescens ssp. laumondii, P.
luminescens ssp. kayaii, P. luminescens ssp. luminescens and P. luminescens ssp.
akhurstii, together with an unidentified strain C8404. The P. asymbiotica group
comprises two subspecies: P. asymbiotica ssp. asymbiotica, P. asymbiotica ssp. aus-
tralis, Photorhabdus strain Q614 isolated from an uncharacterized Heterorhabditis
spp. from Queensland, Australia (Akhurst and Boemare 1986) and strains Cbkj163
and Onlr40 isolated from Heterorhabditis indica isolates from Japan. The P. tempe-
rata group includes two subspecies: P. temperata ssp. temperata and P. luminescens
ssp. thracensis (strain DSM15199T). The phylogenetic position of this latter strain,
whatever the method of reconstruction used, is not congruent with its taxonomic
classification as P. luminescens based on 16S rRNA gene sequences comparison
(Hazir et al. 2004). The distance and maximum likelihood analyses suggest a com-
mon ancestor for P. asymbiotica and the P. luminescens groups; whereas maximum
parsimony analysis suggests a common ancestor for P. asymbiotica and the P. tem-
perata groups.
3.5 Entomopathogenic Fungi (EF)
EF are a group of phylogenetically diverse, heterotrophic, eukaryotic, unicellular or

multicellular (filaments) microorganisms that reproduce via sexual or asexual
spores, or both. They have chitinized cells and are generally non-mobile (Badii and
Abreu 2006). Approximately 80% of the diseases that occur in insects have a fungus
as the causative agent. Practically all insects are susceptible to some diseases caused
by fungi, which can lead to death (Batista 1989).
The kingdom Fungi is one of the major groups of eukaryotic microorganisms in
terrestrial and aquatic ecosystems (Mueller and Schmit 2007), with approximately
100,000 described species (Kirk et al. 2008a), that represent only a fraction of the
estimated diversity, considered to range between 1.5 and 5 million species (Blackwell
2011). The evolution of fungi has been going on since they diverged from other
ancestors around 1.5 billion years ago (Wang et al. 1999). Fungi probably colonized
land during the Cambrian, over 500 million years ago, but terrestrial fossils only
become uncontroversial and common during the Devonian, 400 million years ago
(Lucking et al. 2009). It is considered that animals and fungi evolved separately
from a common ancestor long ago. Although it is believed that fungi first evolved in
aquatic environments, the best fossil evidence suggests that they arose on land.
Analyses with molecular phylogenetics support a monophyletic origin of fungi
(Hibbett et al. 2007).
Insects are known to form intimate relationships with many fungal groups that
include: mutualistic endosymbionts that assist in nutrition (Suh et al. 2005), fungi
as food sources that are farmed as crops by leaf cutter ants (Currie et al. 2003), verti-
cally transmitted parasites (Lucarotti and Klein 1988), commensals (De Kesel 1996)
and pathogens with pronounced effects on host populations (Evans and Samson
1982, 1984). Fungal associations involve members of Coleoptera, Diptera,
Homoptera, Hymenoptera and Isoptera as well as others. The fungi may be clus-
tered taxonomically, as is the case of Ascomycetes in the Hypocreales (e.g.,
Beauveria, Metarhizium, Fusarium), ambrosia fungi in genera Ophiostoma and
Ceratocystis and their asexual relatives, Laboulbeniomycetes, Saccharomycetes
and the basal Microsporidia. Other groups, however, only have occasional members
(e.g., mushrooms cultivated by attine ants and termites) in such associations. These
fungi usually attach to the external body surface of insects in the form of micro-
scopic spores (usually asexual, mitosporic spores also called conidia). Under the
right conditions of temperature and humidity (usually high), these spores germinate,
90 M. Gani et al.
grow as hyphae and colonize the insects' cuticle penetrating through the cuticle or
through body openings, by enzymatic hydrolysis, thus reaching the host body cavity
(hemocoel) ((Tanada and Kaya 1993; Fernandes et al. 2012). Then, the cells prolif-
erate in the host, usually as walled hyphae or in the form of wall-less protoplasts
(depending on the EF species involved). After some time the insect is usually killed
(sometimes by fungal toxins) and new propagules (spores) are formed inside or on
the insect body, if the environmental conditions are again favourable. High humidity
is usually required for sporulation.
EF have evolved specialized mechanisms for the enzymatic degradation of the
integument and for overcoming insect defence compounds. The relationships by
which different fungal species obtain energy from their hosts (i.e., their eco-
nutritional mode) include biotrophy (nutrition derived only from living cells, which
ceases once the cells die), necrotrophy (killing and utilization of dead tissues) and
hemibiotrophy (initially biotrophic and then becoming necrotrophic).
Following entry, some groups (i.e., Metarhizium and Beauveria in the order
Hypocreales, phylum Ascomycota) are known to grow inside the host as yeast-like
hyphal bodies, multiplying by budding (Prasertphon and Tanada 1968). Others, for
example, some species within the Entomophthoromycota, produce protoplasts
(cells without cell walls) instead (Butt et al. 1996). A third group encompassing
some species within Oomycota, Chytridiomycota and the genus Entomophthora,
that infect aphids, are known to grow directly as hyphal filaments inside the host
(Lucarotti and Shoulkamy 2000).
Most EF kill their hosts before the spore production starts (as such they are
termed hemibiotrophic). A few of them, especially some in the phylum
Entomophthoromycota, sporulate from the living body of their hosts (and as such
are termed biotrophic) (Roy et al. 2006). All entomopathogenic oomycetes kill the
host before transmission.
There are about 750 species of fungi that cause infections in insects or mites. As
a group, they attack a wide range of insect and mite species, but individual species
and strains of fungus are very specific. Host death requires between 4 and 10 days,
depending on the type of fungus and the number of infecting spores. After host
death, the fungus produces thousand new spores on the dead body, which disperse
and continue their life cycle on new hosts. Oomycete infections have been recorded
from mosquito larvae in freshwater, primarily in well-aerated streams, rivers, ponds,
lakes (Alexopoulos et al. 1996), and even treeholes (Saunders et al. 1988) or water
collected on leaf axils (Frances et al. 1989). A single example of oomycetes infect-
ing a nondipteran was Crypticola entomophaga, which was described attacking
caddis flies (Trichoptera), which are also aquatic.
The earliest known fossil of an insect pathogenic fungus, an Ophiocordyceps-
like fungus infecting a scale insect (Hemiptera) is from Myanmar amber (100–
110 million years ago) (Sung et al. 2008). Later fossils include an Entomophthora-like
fungus infecting a termite (Isoptera) and a Beauveria infecting an ant (Hymenoptera),
both from Dominican amber (20–30 million years ago) (Poinar and Thomas 1982).
Agostino Bassi described Beauveria bassiana in 1835 as the cause of the devastat-
ing muscardine disease of silkworms and it was instrumental in his development of
the germ theory of disease (Steinhaus 1956b). In 1880, Elie Metchnikoff was among
the first to propose practical methods of microbial control of an insect crop pest,
initiating trials of the fungus Metarhizium anisopliae against grain beetles (Lord
2005).
The approximate number of described insect pathogenic species varies from just
a few in the Chytridiomycota, Blastocladiomycota, Kickxellomycotina and
Basidiomycota to a substantial number in the Ascomycota and almost complete
dominance in Entomophthoromycota. The phylogenetic groups of Ascomycota that
harbor most of the known insect pathogens are the Hypocreales and the Onygenales.
Insect pathogenic life forms evolved repeatedly in the Ascomycota (Spatafora et al.
2007).
Molecular phylogenies have demonstrated that Fungi, which traditionally have
been grouped with plants, are more closely related to the Metazoa or animals
(Baldauf et al. 2000; Berbee and Taylor 2001; Lang et al. 2002). Fungi are thus
likely to have evolved from a flagellated ancestor (Cavalier-Smith 2001), which
bolsters the long-held assumption that Chytrids, which produce flagellated zoo-
spores, represent the earliest surviving lineages of fungi and point to an aquatic
origin for the fungi. Clades of insect pathogens have also originated in both phyla of
the subkingdom Dikarya, the Basidiomycota and Ascomycota. The Basidiomycota
includes only a single group of insect pathogens, the monophyletic
Septobasidiobasidiaceae, which are classified in the subphylum Pucciniomycotina
or rust clade (Aime et al. 2006). Three notable radiations of insect pathogens have
originated within Ascomycota (James et al. 2006), within the Eurotiomycetes and
Sordariomycetes and in the singular class Laboulbeniomycetes. The Eurotiomycete
genus Ascosphaera is an obligate pathogen of bees and A. aphis in the well-known
causal agent of chalkbrood disease of honeybee larvae (Apis mellifera). Interestingly,
honeybee defences towards A. apis appear to have a ‘lock and- key’ dynamic, lead-
ing to suggestions that this pathogen has pushed bees, over evolutionary time
frames, towards lower nest-level relatedness (Tarpy and Seeley 2006; Seeley and
Tarpy 2007). There are 29 described species of Ascosphaera that infect various bee
species, each of which generates pathologies similar to that of A. apis. A ribosomal
phylogeny of selected species of Ascosphaera supports the monophyly of the genus
and provides support for traditional species concepts (Anderson et al. 1998). A
whole genome sequence, the first for any fungal entomopathogen, is available (Qin
et al. 2006) providing an unique opportunity to analyse the genetic architecture of a
specialized entomopathogen.
EF are not a monophyletic group, but rather a heterogeneous assemblage of fungi
independently derived from Ascomycota, Basidiomycota, Entomophthoromycotina,
Blastocladiales, Kickxellomycotina, Neocallimastigomycota and Microsporidia.
There are only a few exceptions, such as the Glomeromycota, which contain only
mycorrhizal clades. Nutritional modes of the earliest fungi remain poorly under-
stood, but the ability to infect insects is clearly represented in several basally diverg-
ing clades, e.g., Blastocladiomycota, Chytridiomycota, and Entomophthoromycota
(Boomsma et al. 2014).
92 M. Gani et al.
EF are particularly well suited for development as biopesticides because, unlike

bacteria and viruses that have to be ingested to elicit disease, fungi are contact
insecticides and typically infect insects by directly penetrating their surface (cuti-
cle) and multiplying in the hemocoel. Approximately 170 pest control products
have been developed based on at least 12 species of fungal entomopathogens (De
Faria and Wraight 2007). Anamorphic EF, such as B. bassiana and M. anisopliae,
are usually developed as inundative control agents which are applied en masse to a
pest population and there is little expectation that they will persist and reproduce
within the biotic environment. As a consequence, research on these fungi has tended
to concentrate on technical aspects of biopesticide development (mass production,
formulation, application, response to abiotic variables etc) with significantly less
work done on understanding their basic ecology. Anamorphic EF are naturally
widespread, particularly in soil and yet little is known about the factors that influ-
ence their distribution, population structure, econutritional behaviour and the evolu-
tion of virulence related characteristics. In contrast, researchers studying
entomophthoralean fungi, which are not easy to mass produce, have focused on the
ecology of these organisms and their role as causative agents of natural epizootics.
Identifying fungal fitness traits and the selective forces that act upon them will
improve our understanding of how and why EF interact with their hosts. Current
theories on the evolution of virulence in micro-parasites, which at present are not
being applied to EF, could provide fresh insights into their ecology and exploitation
for biocontrol. At the same time, the wide arrays of experiments that can be done
with EF are ideal for answering basic questions in parasitology and entomology.
Fungi are more closely related to animals than to plants and are placed with the
animals in the monophyletic group of Opisthokonts (Shalchian-Tabrizi et al. 2008).
Fungal species can have multiple scientific names depending on their life cycle and
mode of reproduction (sexual or asexual). There are fungi that invade dead insects,
called saprotrophs and fungi that infect living insects called entomophagous (Butt
et al. 2006). Of the estimated 1.5 to 5.1 million species of fungi in the world (Hibbett
et al. 2007), approximately 750 to 1,000 are fungal entomopathogens placed in over
100 genera (St. Leger and Wang 2010). Fungal entomopathogens, thus, constitute
the largest number of taxa that are insect pathogens (Ignoffo 1973). De Faria and
Wraight (2007) identified 171 fungal-based products used as biocontrol agents
since the 1960s, most of them based on B. bassiana, B. brongniartii, M. anisopliae
and Isaria fumosorosea. The 2007 classification of Kingdom Fungi is the result of
large-scale collaborative research efforts involving dozens of mycologists and other
scientists working on fungal taxonomy (Hibbett et al. 2007). It recognizes seven
phyla which include Microsporidia, Chytridiomycota, Blastocladiomycota,
Neocallimastigomycota, Glomeromycota, Ascomycota and Basidiomycota (Hibbett
et al. 2007) (Fig. 3.5). Out of these, two phyla - the Ascomycota and the
Fig. 3.5 Phyla of fungi (based on Hibbett et al. 2007) indicate that fungi are more diverse than
previously appreciated. Major changes include separation of groups with flagellated cells
(Chytrids) in three phyla and separation of zygosporic fungi (Zygomycetes) in at least three lin-
eages. Numbers of described fungal phyla are from Kirk et al. (2008b) and for the outgroup from
The IUCN Red List of Threatened Species (http://www.iucnredlist.org/static/stats). (Source: Vega
et al. 2009)
Basidiomycota are contained within a branch representing subkingdom Dikarya,

the most abundant in species and familiar group. Fungal species of different phyla
like Microsporidia, Chytridiomycota, Entomophthoromycota, Basidiomycota and
Ascomycota are known to infect and kill insects (Sung et al. 2007; Shang et al.
2015). The two best-studied groups are the ascomycete entomopathogens and the
Entomophthoromycota. The most well-studied insect ascomycete pathogens fall
into three families within the order Hypocreales: Cordycipitaceae, Clavicipitaceae
and Ophiocordycipitaceae. Many common and important EF is in order Hypocreales
94 M. Gani et al.
of Ascomycota: the asexual (anamorph) phases Beauveria, Isaria (Paecilomyces),

Hirsutella, Metarhizium, Nomuraea and the sexual (teleomorph) Cordyceps; others
such as Entomophthora, Zoophthora, Pandora, Entomophaga belong to the order
Entomophthorales (Zygomycota).
Over the past 400 million years fungi and insects have coevolved a wide array of
intimate interactions (Araujo and Hughes 2016), that include mutualistic endosym-
biosis (Suh et al. 2001), fungi as obligate food sources, such as those found in
fungus-gardening ants (Mueller et al. 2005), sexually and behaviourally transmitted
parasites, such as Laboulbeniales (De Kesel 1996) and the most common disease-
causing agents of insects (Roberts and Leger 2004).
Entomopathogenicity evolved independently and repeatedly in all the major
phyla of the Kingdom Fungi (Araujo and Hughes 2016). The EF have been tradi-
tionally considered as important mortality factors for insects. There are two main
taxonomic orders of entomopathogenic fungi. The Entomophthorales from phylum
Zygomycota include important genera such as Pandora, Entomophthora and
Conidiobolus. Many of their species are co-evolved, obligate pathogens that show
specific eco-morphological adaptations to the life cycles of their hosts, such as the
production of forcibly-ejected infective spores that are produced on insect cadavers
during the night, when environmental conditions are most conducive to infection.
These fungi often cause natural epizootics in insect and mite populations. Recently,
molecular methods have revealed the telemorph connections of these fungi.
Molecular studies are also shedding light on the phylogenetic relationships with
other fungi. Data showed that entomopathogenicity has evolved multiple times in
the ascomycete fungi, characterised by host shifting from an intimate association
with plants to insects and vice versa.
Recently, molecular tools such as DNA sequence analysis have led to a new
phylogenetic classification of fungi that challenged many assumptions about their
relationships with other fungi. This new phylogeny is already leading to significant
new insights that should allow us to better understand the EF ecology. In addition,
it has been discovered recently that many EF play additional roles in nature, as plant
endophytes, antagonists of plant pathogens, beneficial rhizosphere-associates and
possibly even plant growth promoters.
Comparative genomics utilizing the Ophiocordyceps sinensis genome provided
an unparalleled opportunity to develop a deeper understanding of how this unique
pathogen interacts with insects within its ecosystem. It is clear that host-pathogen
interactions are a major driving force for diversification, but the genomic basis for
speciation and host shifting remains still unclear. The genus Metarhizium has been
subdivided into 12 different species according to the sequences of several genes
(Bischoff et al. 2009). Some of these species have a wide host range, whereas others
show specificity for certain insect families and can be used to test hypotheses on
speciation and host specificity. Comparative genomic analyses of seven species
revealed a directional speciation continuum from specialists with narrow host
ranges (i.e. M. album and M. acridum specific to hemipterans and acridids, respec-
tively), to transitional species with intermediate host ranges (Metarhizium majus
and M. guizhouense both have host ranges limited to two insect orders), and then to
generalists (i.e., M. anisopliae, M. robertsii and M. brunneum) (Hu et al. 2014).

Besides host range, generalist and specialist Metarhizium spp. differ in the way they
colonize hosts (Kershaw et al. 1999). Generalists, like M. robertsii, typically kill
hosts quickly via toxins and grow saprophytically in the cadaver. In contrast, the
specialist M. acridum causes a systemic infection of host tissues before the host
dies. This may reflect greater adaptation by the specialists to subverting or evading
the immune systems of their particular hosts so they do not need to kill quickly.
Generalists also have mechanisms for evading host immunity, but appear less able
to subvert immune responses specific to certain hosts. Lack of specific adaptations
could have selected for rapid killing of hosts before the host can mount an enfee-
bling immune response.
Sequencing related entomopathogen species that have evolved specialist or gen-
eralist lifestyles has increased their utility as models and provided insights into the
evolution of pathogenicity. Thus, cross-species comparative analysis identified
novel and specialized virulence mechanisms and, compared to experimental meth-
ods, has allowed for more rapid identification of genes encoding biologically active
molecules and genes responsible for interactions between fungi, plants and insects.
Undoubtedly, the information from comparative genomics will benefit future func-
tional studies of insect-fungus interactions.
Fungi are the most common pathogens of insects and crucially regulate insect popu-
lations. The rapid advance of genome technologies has revolutionized our under-
standing of entomopathogenic fungi with multiple Metarhizium spp. sequenced, as
well as Beauveria bassiana, Cordyceps militaris and Ophiocordyceps sinensis
among others. Comparative genomics offers a way forward by disentangling com-
mon themes of fungal biology, from specific components involved in insect pathol-
ogy and allowing broad host range pathogens to be studied in the context of narrow
host range pathogens (Wang and St. Leger 2014). It is also extremely valuable for
assessing poorly characterized species such as Ophiocordyceps sinensis.
Comparative genomics has facilitated identifying fungal fitness traits and the selec-
tive forces that act upon them to improve our understanding of how and why EF
interact with insects and other components in their environments. Thus, sequence
data provide crucial information on the poorly understood ways that these organ-
isms reproduce and persist in different environments. Alongside the recent avail-
ability of genomic resources, the wide array of experiments that can be performed
with EF make them ideal models for answering basic questions on the genetic and
genomic processes behind adaptive phenotypes. Key challenges for fungi as models
for other eukaryotes include identifying the genes involved in ecologically relevant
traits and understanding the nature, timing and architecture of the genomic changes
governing the origin and processes of local adaptation (Gladieux et al. 2014). As of
February 2016, one published Entomophthoromycota genome (Conidiobolus
96 M. Gani et al.
coronatus (Chang et al. 2015) and nine incomplete Entomophthoromycota genome

sequencing projects were listed in the Genomes OnLine Database (GOLD) (Licht
et al. 2016). To date, there is much more genomic information on ascomycete insect
pathogens, as sequences are available from nine Metarhizium strains (Gao et al.
2011; Hu et al. 2014; Pattemore et al. 2014; Staats et al. 2014), Beauveria bassiana
(Xiao et al. 2012), Cordyceps militaris (Zheng et al. 2011), Ophiocordyceps sinen-
sis (anamorph, Hirsutella sinensis) (Hu et al. 2013), Ophiocordyceps unilateralis
(de Bekker et al. 2015), Tolypocladium inflatum (Bushley et al. 2013) and Hirsutella
thompsonii (Agrawal et al. 2015). Entomophthoromycota boasts one of the largest
fungal genomes ever measured, at 8000 Mb in E. aulicae, which correlates with
microscopic observations of extensive condensed chromatin in the nuclei (Murrin
et al. 1986). Also, Basidiobolus ranarum has a large haploid genome of 350 Mb
(Henk and Fisher 2012), compared to an average genome size of around 40 Mb in
the kingdom Fungi (Gregory et al. 2007; Henk and Fisher 2012). Earlier micro-
scopic analyses have found numerous chromosomes in B. ranarum, ranging from
60 to more than 500 (Sun and Bowen 1972), but it is unclear whether ploidy level
or genome duplication governs genome size in B. ranarum (Henk and Fisher 2012).
In general, entomophthoromycotan genomes are considered to be haploid (Humber
2012; Gryganskyi and Muszewska 2014) and the basal chromosome count in
Entomophthoromycota appears to be 8, whereas 12, 16 and 32 have also been esti-
mated (Riddle 1906; Sawyer 1933; Humber 1982). The large nuclei with numerous
chromosomes and condensed chromatin seen in several species suggest that large
genomes may be an ancestral trait within Entomophthoromycota, but less than 10
out of 280 species have been analyzed for either chromosome count or genome size,
making it unclear whether having a large genome is unusual or the norm within
Entomophthoromycota. However, the first genome assembly (ver 1.0) within the
Entomophthoromycota was the phylogenetically basal Conidiobolus coronatus,
which was sequenced with 454-technology by the US Department of Energy Joint
Genome Initiative (JGI). C. coronatus has 10,635 predicted genes and a genome
size of 39.9 Mb (Chang et al. 2015), which is similar to the average fungal genome
size of 40 Mb. The genus Conidiobolus is paraphyletic, consisting of one clade,
exemplified by C. coronatus, including soil-living saprotrophs that appear to be
facultative insect pathogens and another clade, exemplified by C. thromboides,
which includes primarily insect pathogens (Gryganskyi et al. 2012; Gryganskyi
et al. 2013). This may suggest that the larger genomes reported for E. aulicae
(Murrin et al. 1986) and B. ranarum (Henk and Fisher 2012) could be associated
with specialization to obligate insect pathogenicity. This process has been also
observed in some plant pathogenic fungi, where the expansion of specific gene fam-
ilies and/or whole-genome duplication has been found to be associated with host
specialization (Raffaele and Kamoun 2012). If these inferences are correct it implies
that the evolution of the large haploid genome of 350 Mb in B. ranarum is indepen-
dent from potential genome expansion events within Entomophthorales, and might
be driven by adaptation to amphibian and reptile guts. This is supported by recent
phylogenetic analyses where the genus Basidiobolus is the sister group to all other
Entomophthoromycota and is placed somewhere between Entomophthoromycota
and chytrid fungi (Henk and Fisher 2012; Gryganskyi et al. 2013). From the recon-
structed phylogeny it is evident that entomopathogenicity evolved independently in
these families and that genera of hypocrealean entomopathogens cluster among
closely related phytopathogens, endophytes and mycoparasites. These ancestral
associations are consistent with repeated transitions (host switching) between plant,
fungi and insect hosts, as suggested by Suh et al. (2001) in their study on Cordyceps
spp.
A comparative genome analysis of seven Metarhizium (Clavicipitaceae) genomes
confirmed the genus as a monophyletic lineage that diverged from clavicipetacean
plant pathogens and endophytes about 231 million years ago and placed the
hemipteran-specific M. album as basal to the Metarhizium clade, with an estimated
divergence time about 117 million years ago (Hu et al. 2014). It was suggested that
the close physical proximity of the plant-associated ancestor of M. album to plant-
sap sucking hemipteran bugs may have facilitated this particular host switch to ento-
mopathogenicity (Hu et al. 2014). As part of a wider study on the biocontrol
potential of EF from Central Asia, the phylogenetic characterization of a number of
insect-derived isolates from Uzbekistan was assessed (Ergashev et al. 2009).
Specialization in Metarhizium is associated with retention of sexuality and rapid
evolution of existing protein sequences, whereas generalization is associated with
protein family expansion, loss of genome-defence mechanisms, genome restructur-
ing, horizontal gene transfer and loss of sexuality (Hu et al. 2014). Fungi that are
able to infect insects are not just comprised by a single monophyletic group.
Different groups have arisen independently and repeatedly in many different lin-
eages through fungal evolution (Humber 2008). Recent phylogenetic studies indi-
cate that the ability to utilize insects as a source of nutrition has arisen more than
once among fungi (Spatafora et al. 2007). Scale insects, particularly Coccidae and
Aleyrodidae have the greatest diversity of fungal pathogens documented (Humber
2008). These insects occur in dense and mainly immobile populations feeding on
plants. The sustained proximity between these insects, fungi and other potential
hosts may provide pathogenic fungi with the opportunity to move from plant to
insect and beyond.
3.6 Conclusion
Molecular identification and the phylogenetic analyses of entomopathogens are

basic principles in insect pathology. Molecular phylogenetics applies a combination
of molecular and statistical techniques to recover the order of evolutionary events
and represent them in evolutionary trees that graphically depict relationships among
species or genes over time. Phylogenetics based on sequence data provide us with
more accurate descriptions of patterns of relatedness than was available before the
advent of molecular sequencing. Genome wide studies on gene content, conserved
gene order, gene expression, regulatory networks, metabolic pathways, functional
genome annotation can all be enriched by evolutionary studies based on
98 M. Gani et al.
phylogenetics. Thus, the molecular criteria and bioinformatic tools for pathogen
discrimination and species demarcation is obvious and need of the hour. However,
molecular phylogenies are sensitive to the assumptions and models used for trees
constructing. They face issues such as long-branch attraction, saturation and taxon
sampling problems. This means that strikingly different results can be obtained by
applying different models to the same dataset. Therefore, it is concluded that devel-
oping new experimental approaches to discover the links between these molecular
mechanisms and ecological processes can substantially improve inference in evolu-
tionary biology, in a significant and powerful way.
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Chapter 4
Potential of Entomopathogenic Bacteria
and Fungi
Lav Sharma, Nitin Bohra, Rupesh Kumar Singh, and Guilhermina Marques
Abstract Soil is a reservoir of numerous microorganisms critical for the sustain-

able functioning of natural and managed ecosystems. Entomopathogenic bacteria
and fungi are natural enemies of pest-insects, whose utility in agroecosystems has
been studied since decades. These entomopathogens spend significant time period
in soil, either as saprotrophs, active conidia, resting spores or dormant endospores.
In this chapter, we focus on: (a) the different bacterial and fungal species exhibiting
entomopathogenicity; (b) insect-hosts and pathology; and (c) their survival in soil.
Studying these aspects is of the utmost importance in fully exploiting the potential
of these microorganisms. The bacterium Bacillus thuringiensis and fungi from the
orders Entomophthorales and Hypocreales are discussed in more details, pertaining
to the amount of literature and their dominance in the microbial biopesticide
industry.
Keywords Hypocreales · Entomophthorales · Beauveria · Metarhizium ·

Paenibacillus
L. Sharma (*) · G. Marques

CITAB – Centre for the Research and Technology of Agro-Environmental and Biological
Sciences, University of Trás-os-Montes and Alto Douro, UTAD, Vila Real, Portugal
N. Bohra
School of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, UTAD,
Vila Real, Portugal
Department of Biotechnology, National Institute of Technology Warangal,
Warangal, Telangana, India
R. K. Singh
Centro de Química de Vila Real (CQ-VR), Universidade de Trás-os-Montes e Alto Douro,
Vila Real, Portugal

https://doi.org/10.1007/978-3-030-23045-6_4
116 L. Sharma et al.
4.1 Introduction
Bacteria exhibit different levels of mutualistic relationships with insects, that arose
during the last 250 million years of evolution. Only a few lineages, however, evolved
strategies to invade a host-insects and kill them by overcoming their immune
response, and are known as bacterial entomopathogens or entomopathogenic bacte-
ria (EB). The mechanisms of entomopathogenicity developed through a long co-
evolutionary process (Vilcinskas 2010). Considering the life strategies, EB which
have been commercialised are either obligate (those completing their life cycle
inside the host insect), or facultative (which can grow outside the host). Some bac-
teria, such as Bacillus spp., produce endospores which are resistant/tolerant to
adverse conditions. These endospores prove remarkably beneficial for the bacte-
rium, as they overcome limited temporal availability of the host insect (Jurat-
Fuentes and Jackson 2012). Moreover, bacteria such as Chromobacterium subtsugae
and Bacillus thuringiensis produce toxins which can be exploited even in absence
of the live organisms (Glare et al. 2017). Such organism-free approaches for insect
biological control facilitate the production of stable and non-infectious
biopesticides.
Fungi are commonly found in the terrestrial and aquatic environment, and those
dwelling on land can have various ecological roles ranging from being parasites,
pathogens, endophytes, or symbionts of plants or animals. In a broader sense, those
fungi which cause infections (mycosis) in insects or other arthropods such as mites,
ticks and spiders are known as fungal entomopathogens or entomopathogenic fungi
(EPF). A common difference between EB and EPF is that the former must be
ingested by the insect, whereas the latter generally penetrates their hosts’ cuticle
(Hajek and Meyling 2018). Two fungal orders, Hypocreales and Entomophthorales,
deserve attention due to their applications in various strategies for the biological
control of arthropod pests.
4.2 Bacterial History and Diversity
During the late nineteenth century, the silk industry in Japan observed sudden deaths
of silkworm, Bombyx mori (Beegle and Yamamoto 1992; Milner 1994; Davidson
2012). In 1898, a spore forming bacterium was observed in these caterpillars by the
Japanese scientist Sigetane Ishiwata. This bacterium was lethal enough to kill the
silkworm caterpillars, hours after its ingestion.. The disease was termed as “sotto-
byo-kin” which translates as “collapse-disease-microorganism” (Ishiwata 1901; De
Barjac and Bonnefoi 1968). That led to the discovery of the bacterium Bacillus
sotto. In 1915, Aoki and Chigasaki confirmed the presence of a toxin within the
bacterium (Angus 1954). In another independent study, a similar bacterium was
found in the Mediterranean flour moth larvae by a German scientist Ernst Berliner
in Thuringia, Germany, in 1909, that he named Bacillus thuringiensis (Bt) (Berliner
4 Potential of Entomopathogenic Bacteria and Fungi 117
1915). He also proposed the use of Bt for insect biological control, however, the
strains were lost. Luckily, German scientist Mattes isolated Bt from flour moth and
successfully tested it against European corn borer (Angus 1954). This study later
led to the first commercial product based on B. thuringiensis in 1938 (Beegle and
Yamamoto 1992; Milner 1994).
Bacterial Entomopathogens belong to four phyla: Firmicutes, Actinobacteria,
Proteobacteria and Tenericutes. However, most of the known EB are from families
Neisseriaceae, Enterobacteriaceae, Paenibacillaceae and Bacillaceae. Bacteria
belonging to the genus Bacillus are the most widely studied and used as microbial
insect pathogens. Commonly known EB are considered in this work, and are pre-
sented in Fig. 4.1. Other insect-pathogenic bacteria such as Xenorhabdus and
Photorhabdus from the family Enterobacteriaceae are symbionts of entomopatho-
genic nematodes, and are out of the scope of this chapter.
4.2.1 Gram Positive: Firmicutes and Actinobacteria
Bacteria that exhibit entomopathogenicity in phylum Firmicutes belong to the

orders Bacillales, Lactobacillales, and Clostridiales. Entomopathogenic bacterial
families from these orders are presented in Fig. 4.1. Bacteria from the phylum
Actinobacteria can be pathogenic to plants, animals, and humans. Entomopathogens
from this group belong to the families Streptomycetaceae and Pseudonocardiaceae,
of order Actinomycetales.
4.2.1.1 Bacillaceae
This family consists of some of the bacteria which have been dominating the market
of bacterial entomopathogens for some decades. These bacteria are endospore-
forming, as they undergo sporulation and form one oval-shaped spore per cell dur-
ing adverse conditions.
Bacillus thuringiensis
Bacillus thuringiensis is commonly found in soil, plants, water, dead insects and
stored cereals. It is a gram-positive, facultative anaerobe which produces crystalline
proteinaceous inclusions or “parasporal crystals” during its vegetative and sporulat-
ing growth phases (Argôlo-Filho and Loguercio 2014). Approximately 98% of the
sprayable bacterial biopesticide formulations are from Bt and its subspecies. These
EB collectively cause mortality among six taxonomic orders of insects (van
Frankenhuyzen 2009). Moreover, Bt also infects root-knot nematodes (Jurat-
Fuentes and Jackson 2012). Bacillus thuringiensis generally differs from other bac-
teria as it contains parasporal crystals. The insect specificity of a Bt strain is
determined by the toxins it produces. Crystal (Cry) and cytolytic (Cyt) toxins are the
two proteins present in the parasporal crystals, in general. Besides, Vegetative
118
Fig. 4.1 Classification of entomopathogenic bacteria. (Modified from Jurat-Fuentes and Jackson 2012)
L. Sharma et al.
Insecticidal protein (VIP) toxins are synthesized and secreted by vegetative Bt cells.
At least 732 Cry toxins have been identified from 73 families of crystal toxins.
Moreover, 38 Cyt toxins belonging to three cytolytic protein families, and 125 VIP
toxins belonging to four families, have also been reported (Crickmore et al. 2014).
Toxins from Bt target the receptors of the midgut epithelial cells and disrupt the
natural cell membrane permeability through a pore-forming mechanism. This leads
to the cell lysis, followed by gut paralysis and insect death. The bacteria continue to
grow until nutrition depletion, and ultimately sporulate (Raymond et al. 2010).
Most of the commercially available Bt formulations are spore-crystal mixtures that
are effective against different insects. Bacillus thuringiensis var. kurstaki and B.
thuringiensis var. aizawai are used against lepidopteran larvae. Other strains, such
as B. thuringiensis var. san diego and B. thuringiensis var. tenebrionis are used
against beetle pests, whereas B. thuringiensis var. israelensis is a mosquito patho-
gen. Strategies for integrating cry genes into the crop plants, such as maize and
cotton, to create genetically modified plants, have also been successful (Ruiu 2015).
Lysinibacillus sphaericus
Previously known as Bacillus sphaericus, Lysinibacillus sphaericus is commonly
isolated from soils and aquatic habitats. Based on DNA homology analyses, ento-
mopathogenicity is exhibited only by the IIA sub-group of the species (Krych et al.
1980; Charles et al. 1996). The insecticidal activity varies with the kind of toxin
produced, i.e., Bin (Binary) and/or the Mtx (Mosquitocidal toxin) (Silva Filha et al.
2014). Some bacterial strains also produce other binary toxins which are related to
the crystal toxins of Bt, such as Cry48 and Cry49. These binary toxins are synthe-
sized and stored as parasporal bodies, however, the Mtx toxins are synthesized dur-
ing bacterial vegetative stage (Wirth et al. 2014). Among different mosquito species,
Lysinibacillus infections are more common in Culex, followed by Anopheles and
Mansonia (Berry 2012). L. sphaericus is also toxic to some species of Aedes.
However, several Aedes spp., such as Aedes aegypti, are resistant to L. sphaericus
(Lacey et al. 2015). The bacterium is also applied to control non-biting midges and
black flies (Glare et al. 2017).
After ingestion by the host, Bin and Cry toxins target the midgut cells and induce
hypertrophy. This is followed by an increase in the presences of lysosomes, induc-
tion of apocrine secretion, and damage to the epithelium. Parasitized insects show
the onset of paralysis following damages to neural and skeletal cells. Continual
growth of vegetative cells and sporulation inside the hemocoel sustain the mosqui-
tocidal activity, probably through a synergistic action mediated by the Mtx and Bin
toxins (Berry 2012).
Lysinibacillus sphaericus has some specific advantages over B. thuringiensis var.
israelensis, as the former is quite specific to only some species of mosquitoes, as
mentioned earlier. Furthermore, the bacterium persists longer than Bti in polluted
habitats. However, populations of the mosquito Culex quinquefasciatus from Brazil,
Tunisia, France, Thailand, India, and China exhibit low to very high resistance
against L. sphaericus (Lacey 2007). Approaches such as adding B. thuringiensis
var. israelensis genes to the L. sphaericus genome can overcome insect resistance,
and increase its infectivity spectrum (Federici et al. 2007).
4.2.1.2 Paenibacillaceae
The bacteria from Paenibacillaceae are characterized by a larger spore which can be
oval or ellipsoidal. These bacteria are catalase negative and are difficult to produce
in-vitro, unlike other members of Bacillaceae.
Paenibacillus spp.
Paenibacillus spp. are spore-forming and obligate pathogens attacking larvae of
species within the family Scarabaeidae (order Coleoptera). Paenibacillus spp.
exhibit a limited growth on nutrient media, a characteristic which distinguishes
them from Bacillus spp. These bacteria cause the “milky disease”, named after the
milky hemolymph aspect of infected larvae. Paenibacillus spores can persist in soils
for many years. However, the infection process is induced only when the spores are
ingested (Jackson et al. 2018). Following intake, the spores germinate, and the veg-
etative cells reach the luminal side of the basal membrane, after penetrating the
midgut epithelium, undergoing a primary multiplication cycle (Splittstoesser et al.
1973). The toxins from the parasporal body supposedly disrupt the gut epithelial
barrier and facilitate the invasion of the hemocoel. Vegetative multiplication is fol-
lowed by sporulation, which occur until the refractive spores dominate and confer
the characteristic milky appearance (Zhang et al. 1997). An exception is the infec-
tion by P. lentimorbus in scarab larvae, whichinduces a clotting of the hemolymph,
leading to a brown discoloration of the larvae (Sharpe and Detroy 1979; Glare et al.
2017).
Paenibacillus popilliae was the first microbial insecticide registered in North
America, for the biological control of the Japanese beetle Popillia japonica (Klein
1988). However, in some reports, epizootics caused on P. japonica showed variable
results with limited or no success (Lacey et al. 2015). Its use as a microbial pesticide
was not fruitful as the bacterium required in-vivo culturing, which hampered its
large-scale production. The very narrow host range within Scarabaeidae is also a
limiting factor for commercial exploitation of P. popilliae (Lacey et al. 2015).
Brevibacillus laterosporus
Brevibacillus laterosporus is commonly found in soil and water (Ruiu et al. 2013).
It is aerobic, but can also be a facultative anaerobe. It is reported to infect insects
from at least three orders. Among lepidopterans, B. laterosporus is pathogenic to
the diamondback moth Plutella xylostella and the velvet bean caterpillar Anticarsia
gemmatalis (De Oliveira et al. 2004). Within Coleoptera, B. laterosporus infects the
Mexican cotton boll weevil Anthonomus grandis and the corn rootworm Diabrotica
virgifera virgifera (De Oliveira et al. 2004; Ruiu et al. 2006). Among Diptera, B.
laterosporus has been applied against the larvae of the housefly Musca domestica,
of mosquitoes such as C. quinquefasciatus and A. aegypti, and against the black fly
Simulium vittatum (Favret and Yousten 1985; Ruiu et al. 2007). This bacterium also
infects other invertebrates, such as nematodes and freshwater snails (Ruiu et al.
2013).
Insecticidal toxins of infectious B. laterosporus have been a topic of debate. The
bacterium produces a typical “canoe-shaped” parasporal body (CSPB), and these
crystals were supposed to be required for infections against mosquitoes, e.g. A.
aegypti (Ruiu et al. 2013). However, strains lacking CSPB were also found to be
toxic (Ruiu et al. 2007). Insecticidal secreted proteins (ISPs), with a resemblance to
the Bt VIP proteins, have also been reported from B. laterosporus. Besides, recent
studies have reported the presence of the genes for Cry proteins in B. laterosporus
(Sharma et al. 2012). The progressive symptomatology and the changes that occur
within the midgut are similar to those caused by Bt (Ruiu et al. 2012).
4.2.1.3 Clostridiaceae
These are gram-positive, anaerobic and spore-forming rod-shaped bacteria.

Entomopathogenicity amongst the bacteria from the Clostridiaceae is rarely noticed.
Clostridium bifermentans
Clostridium bifermentans is occasionally isolated as a human pathogen. For insects,
C. bifermentans var. malaysia is a highly toxic entomopathogen of black flies and
mosquitoes. However, C. bifermentans var. malaysia does not exhibit short-term
toxicity against humans, mammals and goldfish (Thiéry et al. 1992; Qureshi et al.
2014). A range of toxins are required for the bacterial infections in mosquitoes. For
example, toxins encoded by Cry operon, Cry16A, Cry17A, Cbm17.1, and Cbm17.2,
are only insecticidal to Aedes spp., however, a different set of proteins are supposed
to cause infection in Anopheles spp. (Qureshi et al. 2014).
4.2.1.4 Streptomycetaceae
Representatives of this family produce extensive mycelium which does not frag-
ment, in general. After maturity, the mycelium forms a typical chain of spores
(Kämpfer et al. 2014).
Streptomyces spp.
Some Streptomyces spp. produce a variety of metabolites that are toxic to insect
pests, such as Aphis gossypii, Chilo partellus, Helicoverpa armigera and S. litura.
These metabolites include antimycin A, avermectin, flavensomycin, macrote-
tralides, piericidins and prasinons (Ruiu et al. 2013). Metabolites like avermectin
inhibit the neurotransmission in insects by binding to their γ-aminobutyric acid
(GABA) receptor, which eventually leads to the paralysis of the insect neuromuscu-
lar systems. Species exhibiting entomopathogenicity include S. albus and S. aver-
mitilis (Glare et al. 2017).
4.2.1.5 Pseudonocardiaceae
Like Streptomycetaceae, the family Pseudonocardiaceae also belongs to the order

Actinomycetales.
Saccharopolyspora spinosa
Saccharopolyspora spinosa produces insecticidal metabolites such as “spinosyns”
during aerobic fermentation. Spinosyns are active towards a wide range of insects.
However, they also exhibit toxicity towards non-target insects, aquatic organisms,
and mammals (Ruiu et al. 2015).
4.2.2 Gram-Negative: Proteobacteria
The Proteobacteria form a phylum which consists of many pathogenic bacteria.

Entomopathogenic species belong to the families Enterobacteriaceae,
Pseudomonadaceae, Coxiellaceae, Neisseriaceae, and Burkholderiaceae.
4.2.2.1 Enterobacteriaceae
Members of the Enterobacteriaceae are rod-shaped facultative anaerobes. Most of

them are pathogenic in nature and do not form spores.
Serratia spp.
Serratia spp. are ubiquitous in the environment, and some are reported from dead or
diseased insects. Serratia marcescens supposedly causes disease in the May beetles
Melolontha melolontha, the tsetse flies Glossina spp. (Poinar et al. 1979), and the
blowfly Lucilia sericata (O’Callaghan et al. 1996; Jurat-Fuentes and Jackson 2012).
Moreover, S. marcescens was reported as toxic against the diamondback moth
(Jeong et al. 2010). Association of Serratia spp. like S. nematodiphila with entomo-
pathogenic nematodes, such as Heterorhabditinoides sp., suggests potential for its
use in the biological control of insects pests (Zhang et al. 2009).
Serratia entomophila, the causal agent of the “Amber disease” in the New
Zealand grass grub Costelytra zealandica, is commonly found in soils (Jackson
et al. 1993). Recently, some S. entomophila strains were found to be pathogenic to
several Phyllophaga and Anomala spp. (Nuñez-Valdez et al. 2008). In general, C.
zealandica larvae stop feeding in a few days after bacterial ingestion. The bacterium
facilitates gut clearance, which is followed by the appearance of characteristic
amber colouration. The diseased larva stays active for 1–3 months in the soil with-
out feeding, and shrinks while consuming its own body fat. This leads to the weak-
ening of the structural tissues, eventually allowing the bacteria to enter the hemocoel
and cause septicaemia leading to the host death (Jackson et al. 1993, 2001).
The entomopathogenicity of S. entomophila and S. proteamaculans is attributed
to two different gene clusters: sep (Serratia entomophila pathogenicity) and afp
(anti-feeding prophage). The sep cluster is composed of three genes, sepA, sepB,
and sepC, and is responsible for the clearance of the gut, and the development of
amber colouration of larvae (Hurst et al. 2007a). The Sep proteins are similar to the
Tc (toxin complex) proteins found in Photorhabdus luminescens. Another gene
cluster, afp, codes for the novel “anti-feeding prophage” which is a toxin delivery
apparatus that makes larvae cease feeding (Hurst et al. 2007b).
Yersinia spp.
The bacterial family Enterobacteriaceae includes some well-known human patho-
gens. Entomopathogenicity among Yersinia spp. have been described only recently.
It was found that the Tc proteins were necessary for the insect pathogenicity of
Yersinia enterocolitica (Bresolin et al. 2006). Later, a few other species, such as
Yersinia mollaretii, Y. pestis, and Y. pseudotuberculosis are presumed entomopatho-
genic due to the presence of Tc genes in their genomes (Fuchs et al. 2008). Recently,
Y. entomophaga was isolated from a diseased C. zealandica larva in New Zealand,
and was found highly pathogenic to the insects from the orders Coleoptera,
Lepidoptera, and Orthoptera (Hurst et al. 2011a). After bacterial ingestion, the larva
stops feeding, this is followed by the regurgitation and clearance of the gut content,
leading to a similar appearance to the amber disease, however for a short duration.
The gut epithelial membrane degrades, allowing the invasion of hemocoel, which
leads to septicemia and death in 2–5 days after bacterial ingestion (Marshall et al.
2012). The pathogenicity of the bacteria is governed by a “Tc protein complex”
which is composed of TcA, TcB and TcC toxins, and two chitinase proteins, col-
lectively constituting the Tc molecule (Hurst et al. 2011b).
4.2.2.2 Pseudomonadaceae
Bacteria belonging to the Pseudomonadaceae are widely distributed in the environ-

ment and include some well-known species, such as Pseudomonas aeruginosa,
which are pathogenic to plants, and animals including humans. Some pseudomo-
nads can also be isolated from diseased and dead insects.
Pseudomonas spp.
A soil isolate of P. entomophila showed high insect pathogenicity against the com-
mon vinegar fly Drosophila melanogaster. With time, additional reports included
the pathogenicity of P. entomophila against the insect orders Diptera, Lepidoptera
and Coleoptera, nematodes, and amoebae (Dieppois et al. 2015). Genome analysis
identified a two-component system, GacS/GacA, that regulates potential virulence
factors, such as toxins, putative hemolysins, hydrogen cyanide, and proteases, and
supposedly assists the entomopathogenicity in P. entomophila. Its genome also con-
sists of genes encoding TcB and TcC toxins, as in the case of Yersinia spp. However,
P. entomophila lacks the genes for TcA component (ffrench-Constant and Waterfield
2005). Another species, P. putida, was reported pathogenic to the Colorado potato
beetle Leptinotarsa decemlineata (Muratoglu et al. 2011).
4.2.2.3 Coxiellaceae
These are gram-negative, obligate intercellular pathogens, and those exhibiting

entomopathogenicity have been reclassified from the order Rickettsiales
(α-proteobacteria) to the Legionellales (γ-proteobacteria) (Leclerque 2008).
Rickettsiella spp.
Rickettsiella popilliae, R. chironomi and R. grylli are the three most widely known
entomopathogens. R. popilliae typically targets the hemolymph cells and the fat
body of the host-insect. The diseased larva is characterised by the development of
white to blue colouration although, in the case of infection in Japanese beetle, the
colonisation causes a greenish-blue color (Jurat-Fuentes and Jackson 2012).
In general, the infected larvae act normal for over a month but with passing time
they loose vigour, become sluggish, and stop feeding before death. In the cases of
infections in crickets by R. grylli, a “behavioural fever” is observed. The diseased
insect tries to reach an environment with a higher temperature, to reduce bacterial
growth. Some concerns, such as the ability of the entomopathogenic Rickettsiella
spp. to cause inflammation and infections in vertebrates, and a very slow insect
death post bacterial ingestion, have raised doubts on their use as microbial agents
for insect pest control (Jurat-Fuentes and Jackson 2012).
4.2.2.4 Neisseriaceae
Most of the bacteria from the Neisseriaceae are animal commensals, although, a few
are human pathogens. Generally, they do not exhibit entomopathogenicity but there
are some exceptions as discussed below.
Chromobacterium subtsugae
A soil isolate of C. subtsugae from Maryland demonstrated high insecticidal activ-
ity against insects from different orders, including Lepidoptera, Hemiptera, and
Coleoptera. The strain produces a chemical “violacein” which confers a violet
colour to its colonies. Pathogenicity of the bacterium is attributed to the various
molecules produced during the infection process, including heat stable insecticidal
toxins (Martin et al. 2007). Recently, another Chromobacterium strain, Csp_P, was
isolated from the midgut of the mosquito A. aegypti, which reduced the larval and
adult survival rates. Furthermore, an anti-pathogen activity of the Csp_P strain
against the malaria-causing Plasmodium falciparum and the dengue virus, was
observed in-vitro. The strain compromised the vector competence of the mosquito
(Ramirez et al. 2014).
4.2.2.5 Burkholderiaceae
Species from the Burkholderiaceae have wider biological roles including human,
animal and plant pathogens.
Burkholderia spp.
Burkholderia spp. have been isolated from insect gut, and can negatively affect the
oviposition time and the number of eggs in the bean bug Riptortus pedestris (Kil
et al. 2014). Recent studies have reported the entomopathogenicity of B. rinojensis
against the two-spotted spider mite Tetranychus urticae and the beet armyworm
Spodoptera exigua (Cordova-Kreylos et al. 2013).
4.3 Soil Habitat and Pathogenesis of Bacteria
Bacillus thuringiensis is ubiquitous, but the numbers of cases of natural insect

infections are rare. Such contradiction has led to an on-going debate: is Bt an oppor-
tunistic pathogen with soil being the primary environment for bacterial reproduc-
tion? It is hypothesized that Bt survives on the decaying organic matter or the root
exudates. The bacterium reaches to the aerial parts of the plants as they grow
(Argôlo-Filho and Loguercio 2014). The ability to grow on a wide range of sub-
strates and a high persistence in soil samples are some of the arguments supporting
this hypothesis (Glare and O’Callaghan 2000, Raymond et al. 2010). However,
other works disagree with Bt growth, and survival of parasporal protein crystals, in
natural soil conditions (West et al. 1985). Some researchers convincingly argue that
Bt is a “bona fide insect pathogen” whose primary reproduction occurs within a
host-insect, and only after multiplication, the bacterium is deposited on plants and
soils, eventually making them its natural reservoirs (Raymond et al. 2010; Argôlo-
Filho and Loguercio 2014). The scarcity of Bt natural insect infections is attributed
to a rare combination of susceptible insects, optimal temperatures, and nutrient
availability. Although, a successful horizontal transfer of Bt spores into the host-
insect after ingesting them from the soil, is quite difficult, as the spores will have to
germinate and undergo at least one cycle of vegetative growth to synthesize Cry
toxins. Therefore, bacterial transmission through plants seems to be more feasible
(Raymond et al. 2010). The strategy of host-seeking by a resistant bacterial spore is
also adopted by the milky disease-causing P. popilliae, as the bacterium produces
ultra-dormant spores which can survive many years. The bacterium gets activated
after the ingestion of the soil organic matter and the plant root by the scarab larvae
(Jackson et al. 2018).
Nonetheless, the soil is an excellent reservoir for Serratia spp., and many meth-
ods have been developed to study the ecology of facultative insect pathogens, S.
entomophila and S. proteamaculans, that cause the amber disease in C. zealandica,
as discussed earlier. Studies within the pasture soils from New Zealand revealed that
both pathogenic as well as non-pathogenic strains of Serratia spp. can co-exist.
Pathogenic strains, however, are generally localised to the areas with grass grub
populations (Jackson et al. 2018). Some bacterial species, such as B. laterosporus,
Bacillus spp. and P. putida may also act as plant endophytes enhancing plant growth,
in the absence of insects.
Different EB undertake distinct mechanisms to infect their host-insects, as
already briefed earlier. A brief pictorial description is presented in Fig. 4.2. There
are two distinct phases in the life cycle of Bt, i.e., vegetative cell development and
spore development. Bacterial vegetative cells measure 2–5 μm long and 1 μm wide,
with short hair-like flagella (Bulla et al. 1980). These cells are divided into two
Fig. 4.2 Mechanism of infection by Bacillus thuringiensis. (Modified from Jean-Michel Vassal,
CIRAD, France)
uniform daughter cells due to the formation of division septa initiated midway
alongside the plasma membrane. Spore development is a typical seven stages pro-
cess that spans from (1) the formation of the axial filament; (2) forespore septum
formation; (3) cell engulfment (4–6) the formation of the exosporium, primordial
cell wall, cortex and spore coats, and subsequent transformation of the spore nucle-
oid; and, lastly, (7) spore maturation and sporangial lysis (Ibrahim et al. 2010).
Bacillus thuringiensis crystals get solubilised in the alkaline environment of the
host gut upon ingestion. These proteins get processed by mid-gut proteases (for e.g.,
tripsin like, or chemotripsin like in case of Lepidoptera) and change from protoxins
to an active toxin core. The processed toxin binds to the cadherin receptors present
on the host epithelial mid-gut cell membrane. Here onwards, two models have been
reported to define the possible mode of action of a Cry toxin. One mode of action is
the “pore formation model” where cadherin initiates the cleavage of helix ɑ1 result-
ing in the oligomerisation of the Cry toxin, which in turn binds to the GPI-anchored
receptors, assisting the toxin to penetrate into the membrane, leading to the forma-
tion of pores and cause colloid osmotic lysis (Bravo et al. 2004). Another mode of
action is the “signal transduction model” where interaction of the Cry toxin with
cadherin activates a guanine nucleotide binding protein (G-protein), which initiates
an adenylyl cyclase activity resulting in the production of the intracellular cyclic
adenosine monophosphate (cAMP). This causes the activation of the protein kinase
A, and the induction of an intracellular pathway triggering cell death (Zhang et al.
2006). Where and when one or the other mechanisms lead to the larval death is still
controversial (Soberón et al. 2009).
Crickmore et al. (1998) defined Cry protein as “A parasporal crystal protein
from B. thuringiensis that shows some experimentally verifiable toxicity against a
target pest”; whereas, Cyt represents “a parasporal crystal protein from B. thuringi-
ensis that exhibits haemolytic activity or any protein which has sequence homology
with a known Cyt protein”. The difference between the Cry and the Cyt proteins
mode of action is that the latter bind to the non-glycosylated lipid portion of the
microvillar bilayer, and therefore do not need binding to a protein receptor (Federici
and Siegel 2007). Since the first cloning of Cry protein was reported by Schnepf and
Whiteley (1981), approximately 200 Bt-proteins have been characterised for their
action against many insects from orders such as Lepidoptera, Coleoptera, Diptera,
Homoptera, Orthoptera, Hymenoptera, and suborder Mallophaga (Schnepf et al.
1998; Baxter et al. 2011). Besides, Bt-proteins have also been reported for their
toxicity against protozoa, mites, and nematodes. In general, the insecticidal prop-
erty is attributed to the crystal proteins. However, recent studies have shown the
utility of the VIP toxins against a wide range of lepidopteran larvae (Estruch et al.
1996). To summarise, some of the criteria which define the insecticidal range and
the efficacy of a particular Cry protein are: (a) a sufficient ingestion by the pest-
insect and an effective solubilisation in the gut media; (b) the efficiency of the
protoxin-toxin conversion, and the specificity of binding to a membrane receptor
(such as Cadherin); and, (c) the membrane pore formation, i.e. membrane insertion
and ion-channel activity (Schnepf et al. 1998).
4.4 Fungal History and Diversity
The earliest evidence of a fungal entomopathogen to date is Paleoophiocordyceps

coccophagus, an Ophiocordyceps-like anamorph, attacking an early Cretaceous
Burmese male scale insect dating 100–110 million years ago (Sung et al. 2008).
Nonetheless, during human civilization, the earlier reports on EPF are also credited,
like for bacteria, to the silk industry. In China, Cordyceps fungi had been observed
on caterpillars and were used in traditional Chinese medicine. However, the first
published report on Cordyceps was provided by a French scientist, René Antoine
Ferchault de Réaumur, as “vegetable growths” (Réaumur 1734–1742, Davidson
2012). In the early nineteenth century, a “white muscardine” disease of silkworms
became a big problem for the silk industry in France. The disease was called “calci-
nacio” because of the white powdery deposit on the body of caterpillars. Agostino
Bassi, who was later renowned as the “Father of Insect Pathology”, investigated the
causal agent, and reported a fungus or a “vegetable parasite” (Bassi 1835–1836).
Giuseppe Gabriel Balsamo-Crivelli then named the fungus Botrytis bassiana in
honour of Bassi. The fungus is now renowned as Beauveria bassiana. These discov-
eries led to the idea of using EPF against other insects, and a few reports emerged
in this direction. In 1878, a similar outbreak of “green muscardine” disease was
analysed near Odessa (Ukraine) by Élie Metchnikoff, on the wheat cockchafers
Anisoplia austriaca. The pathogen was identified as Entomopthora anisopliae
(Metchnikoff 1879). Sorokin (1883) named it as “Metarrhizium” anisopliae, pres-
ently spelt as Metarhizium. Years later, in 1888, the same fungus was used against
the sugar-beet weevil Bothynoderes (Cleonus) punctiventris by Isaak Krassiltstchik

(Krassiltstchik 1888). That was the first large-scale production for a field trial of any
microbial biopesticide.
Whilst the genus Beauveria started gaining importance, Steinhaus (1949) briefed
the process of infection, disease development, and practical usage of B. bassiana
against some pest-insects, such as the chinch bug Blissus leucopterus, the codling
moth Carpocapsa (Cydia) pomonella, and the European corn borer Pyrausta
(Ostrinia) nubilalis (Zimmermann 2007). Similar attention was seen in the usage of
M. anisopliae against numerous pest-insects, such as the sugarcane froghopper
Aeneolamia flavilatera, the wireworms Agriotes obscurus and Agriotes sputator,
the turnip moth Agrotis segetum, the sugarcane white grub Alissonotum impressi-
colle, the sugar-beet weevil, moths from Euxoa spp., the European corn borer, the
coconut rhinoceros beetle Oryctes rhinoceros, P. japonica and the black rice bug
Scotinophara lurida (Müller-Kögler 1965).
However, the continual development of chemical insecticides hampered the stud-
ies on the ecology of the natural biocontrol agents. Besides, the efficacies of micro-
bial insect-pathogens were compared with chemical pesticides, without taking the
ecological requirements of a microbial biopesticide in consideration. This biased
comparison resulted in a disinclination towards the fungal microbial pesticide
agents (Vega et al. 2009). Finally in 1981, after a long delay of a few decades, the
fungus Hirsutella thompsonii was registered as the first fungal insecticide for its use
against the citrus rust mite Phyllocoptruta oleivora, in the USA (Tanada and Kaya
1993). Nonetheless, in terms of biology, entomopathogenicity in fungi is restricted
to a few orders, as showed in the Fig. 4.3. However, occasional insecticidal activities
can be noticed in most fungal phyla.
4.4.1 Oomycota (Kingdom: Chromista)
The oomycetes are eukaryotic microorganisms with filamentous features, and were
thought to be fungi while they are not. They are aquatic microorganisms with a
coenocytic hypha which is mainly aseptate, whose cell walls are mainly composed
of glucan-cellulose. Recent classifications have placed them closer to the diatoms
and the brown algae, in the kingdom Chromista. Asexual reproduction takes place
by “zoospores” exhibiting two flagella of varying lengths. Sexual spores are termed
“oospores” (Wraight et al. 2007). They are able to infect algae, protists, plants,
fungi, vertebrate animals, and arthropods. Although plant pathogens such as
Phytophthora infestans, the potato late blight causal agent, are well known, the
information on entomopathogenic oomycetes is indeed limited. Most of insect
pathogenic oomycetes belong to three different orders: Myzocytiopsidales,
Pythiales, and Saprolegniales. Previous reports showed that a few species from
these groups are facultative pathogens of mosquitoes. In terms of insect pathology,
the species Aphanomyces laevis and Lagenidium giganteum are among the best
studied oomycetes. However, species from genera Leptolegnia, Pythium, Couchia
Fig. 4.3 Classification of entomopathogenic fungi. (Modified from Kirk et al. (2008), Vega et al.
(2009), Gryganskyi et al. (2012) and Boomsma et al. (2014))
and Crypticola have also been spotted occasionally (Scholte et al. 2004). Oomycete
infections on mosquito larvae have been observed on the waters from rivers, ponds,
well-aerated streams, lakes, tree holes, and on the leaf axils (Araújo and Hughes
2016).
4.4.2 Microsporidia
The Microsporidia are among the first diverging lineages of fungi, but in earlier
times they were considered as protozoans (James et al. 2006; Hibbett et al. 2007).
They are ubiquitous, obligate intracellular parasites of animals including inverte-
brates (Wittner and Weiss 1999). They inject spores into the cytoplasm of the host
rapidly, through a thin polar tube (James et al. 2006). Sixty-nine genera have been
reported for entomopathogenicity, and 42 of them infect dipteran insects only.
Amblyospora is the most abundant and the largest genus that alone kills 79 dipteran
species belonging to eight genera (Araújo and Hughes 2016). Amblyospora exhibits
a complex life cycle and requires an intermediate copepod host, and two generations
of insect (mosquito) host for completion of its full lifespan. Nosema is also an
important and widely distributed microsporidian genus. Nosema apis and N. cera-
nae target honey bees and are hence considered quite devastating for apiculture. The
infection occurs as N. apis invades the epithelial layer of the ventriculus and the
midgut of Apis mellifera adult bees. This causes digestive disorders leading to a
shortened bee lifespan, resulting in a decrease in the bee population after winter
conditions (Higes et al. 2006). Studies in the past have also reported microsporidian
pathogenicity against caterpillars, such as the European corn borer, locusts, mosqui-
toes and grasshoppers. However, their utility as biological control agents is marred
by the difficulties in their mass productions (Corradi 2015).
4.4.3 Chytridiomycota
The Chytridiomycota are supposed to be the most primitive lineage of fungi, dating
as back as 400 million years (Taylor et al. 1992; James et al. 2006). They produce
motile zoospores and gametes with smooth whiplash flagellum. The majority of
them are wet soils and freshwater saprotrophs (Wraight et al. 2007). Many chytrid
species are parasitic on protists, tardigrades, fungi, rotifers, plants and animals.
Fewer species, belonging to Myiophagus, have been reported for entomopatho-
genicity, but such cases are rare (Araújo and Hughes 2016).
4.4.4 Blastocladiomycota
Fungi belonging to the phylum Blastocladiomycota can be saprotrophs, plant and

invertebrate pathogens (Longcore and Simmons 2012). They are distinct from the
other fungi as they include an alternation between haploid and diploid generations,
and for the fact that meiosis occurs during the formation of spores within a thick-
walled “meiosporangia”. Entomopathogenic blastocladiomycetous fungi belong to
the genera Catenaria, Coelomycidium and Coelomomyces of the order
Blastocladiales. Catenaria spp. are pathogenic to nematodes, although, some of
them may also infect flies. Coelomycidium spp. are toxic to beetles, scales and some
Diptera (Tanada and Kaya 1993). For example, Coelomycidium simulii is a patho-
gen that occasionally attacks larvae of the black fly Simulium japonicum larvae
(Kim 2011).
The genus Coelomomyces includes over 70 species that are pathogenic to differ-
ent families of Diptera, such as the Chironomidae, Culicidae, and Psychodidae
(Scholte et al. 2004). Coelomomyces are obligate pathogens, and species like
C. psophorae require an intermediate copepod host and a mosquito larva at different
stages of their life cycles (Vega et al. 2012). Some species, such as C. stegomyiae,
do not kill the larva but, on the contrary, reside inside the mosquito, passing through
the larval and pupal stages, and maturing within the ovaries of the adult female.
After the first blood meal, a fungal hypha matures in the zoospore producing “spo-
rangia”. Therefore, as the mosquito reaches any breeding site, instead of eggs, it
lays sporangia full of zoospores, discharging the fungus for a new host (Lucarotti
and Shoulkamy 2000). Despite such infection-causing strategies, problems in the
mass production have rendered these species ineffective for their use on a larger
scale (Chandler 2017).
4.4.5 Zygomycota
The phylum Zygomycota is constituted by two main classes, the Zygomycetes and
the Trichomycetes. Their members are characterised by coenocytic mycelia, asex-
ual reproduction by “sporangiospores”, and absence of flagellate cells and centri-
oles (Alexopoulos et al. 1996; Araújo and Hughes 2016). The Zygomycota differ
from other fungal groups as they produce “zygospores” (thick-walled resting
spores) within the “zygosporangium”, a structure formed when two specialized
hyphae (gametangia) fuse (White et al. 2006). The Zygomycota consist of a wide
group of species which are quite ecologically diverse. These species dwell as sapro-
trophs in soil and dung, and may colonize bread, vegetables, and fruit (Alexopoulos
et al. 1996). Based on their phylogenetic characterisation, the Zygomycota were
classified in five monophyletic taxa, i.e., four subphyla, the Zoopagomycotina, the
Mucoromycotina, the Kickxellomycotina, and the Entomophthoromycotina, and
one phylum, the Glomeromycota, belonging to the arbuscular mycorrhizal fungi
(Hibbett et al. 2007). Later, Humber (2012) described the phylum
Entomophthoromycota, for the Entomophthoromycotina.
Subphyla Zoopagomycotina, Mucoromycotina and Kickxellomycotina are
mainly constituted of saprotrophs. Species belonging to the Zoopagomycotina are
also mycoparasites and nematophagous (Zhang and Hyde 2014). The
Mucoromycotina form the most morphologically diverse and largest subphylum
within zygomycetous fungi. Some orders of Mucoromycotina, such as Mortierellales
and Mucorales, are weak parasites of animals and plants. Just one species i.e.,
Sporodiniella umbellate can be classified as entomopathogenic, as it causes mortali-
ties amongst the lepidopteran genus Acraea in Taiwan, and the hemipteran genus
Umbonia in Ecuador (Araújo and Hughes 2016). The members of the
Kickxellomycotina, from the orders Asellariales and Harpellales, are considered as
gut commensals of arthropods, however, some species, such as Smittium morbosum,
are pathogenic to mosquitoes (Vega et al. 2012).
4.4.6 Entomophthoromycota
The Entomophthoromycota are the most important group of entomopathogens

amongst the basal lineages of fungi. They produce coenocytic hyphae with some
septation, especially in the older mycelial parts. Asexual “azygospores” or sexual
zygospores formed by the Entomophthorales are termed “resting spores”, and are
capable of surviving harsh environmental conditions. The Entomophthoromycota
also produce uninucleate or multinucleate asexual conidiawhich, upon germination
may produce secondary or tertiary conidia (Wraight et al. 2007). This phylum con-
sists of approximately 280 species, and the majority of them are obligate insect or
mite pathogens, with a relatively narrower host range (De Fine Licht et al. 2016).
Other species are pathogens of fern gametophytes, desmid algae, vertebrates, rep-
tiles and macromycetes (Gryganskyi et al. 2012). Some members of families
Basidiobolaceae and Anyclistaceae dwell as saprotrophs (Humber 2008). Most
entomopathogens within the zygosporic fungi belong to the families
Entomophthoraceae and Neozygitaceae. Entomophthora, Entomophaga, Pandora
and Zoophthora are some of the widely known insect pathogenic genera of
Entomophthoraceae. The Neozygitaceae consists of a few genera, such as
Neozygites, which are specialist entomopathogens. Other families, such as
Ancylistaceae and the Basidiobolaceae, also represent fewer insect pathogens, such
as Conidiobolus spp. and Basidiobolus spp., respectively (Vega et al. 2012).
Entomophthoroid fungi generally attack adult insects, although, three species,
Entomophthora conglomerata, Entomophthora aquatica and Erynia aquatica infect
mosquito larvae (Scholte et al. 2004). The Entomophthoromycota target a large
number of insects, however, in small patches within agroecosystems or forests.
Fungal transmission occurs via forcible discharge of spores into the environments,
with an exception of the genus Massospora (Lovett and St. Leger 2016). Moreover,
species from Massospora, Strongwellsea, and a few from the genera Entomophaga,
Entomophthora and Erynia, produce spores while the host is still alive. For exam-
ple, in case of the infections in dipteran insects Hylemya brassicae and Hylemya
platura by Strongwellsea castrans, the flies are seen with a large circular hole on the
lateral side of the abdomen, filled with “conidiophores” (spore-producing cells) and
fungal tissues (Araújo and Hughes 2016). However, the insect can still be seen as
behaving normally. Therefore, these fungi are considered as biotrophs which con-
sume the host while it’s still alive and exhibit no somatic growth after the host’s
death (Roy et al. 2006).
4.4.7 Basidiomycota
The basidiomycetous fungi are quite diverse and exhibit various ecological traits.
They can be saprophytes, and plant pathogens such as the smut and the rust fungi.
Animal pathogens include those attacking nematodes. A few genera are also
recorded as insect parasites. For example, those from order Septobasidiales, i.e.
Auriculoscypha, Coccidiodictyon, Ordonia, Septobasidium and Uredinella, that
infect scale insects. Fibularhizoctonia, a member of order Atheliales, attack termite
eggs (Henk and Vilgalys 2007). The Septobasidiales are the specialist parasites of
scale insects, and use them for their nutrition. However, these fungi seldom kill their
host-insects (Humber 2008).
4.4.8 Ascomycota
Ascomycota is the largest fungal phylum with at least 64,163 species (Kirk et al.
2008). The majority of the ascomycetous fungi are filamentous and produce septate
hyphae. Sexual spores or “ascospores” are packaged in a sac-like structure called
“ascus”. These fungi exhibit very diverse ecology ranging from decomposers to
pathogens of animals, humans, and plants. Lichens are also a part of this phylum
(Money 2016).
The ascomycetous fungi are classified into three subphyla, the Taphrinomycotina,
the Saccharomycotina, and the Pezizomycotina. The Pezizomycotina are the most
numerous and the most complex subphylum in terms of ecology and morphology,
and entomopathogenicity is demonstrated by the members of this group only
(Schoch et al. 2009). These fungi can be “anamorphs” (exhibiting an asexual state);
“teleomorphs” (exhibiting sexual stage); or “synanamorph” (presences of more than
one morphologically distinct asexual stage) (Vega et al. 2012).
Some species belonging to the genera Aspergillus and Penicillium are pathogenic
to insects, besides other members of the order Eurotiales (class Eurotiomycetes)
(Sharma et al. 2018a). Besides, genus Ascosphaera, in the order Ascosphaerales, are
the obligate parasites of the bee larvae, and species like Ascosphaera apis cause the
honeybee “chalkbrood disease” (Maxfield-Taylor et al. 2015). The spores of A. apis
must be ingested for infection.
Amongst the members of the Dothideomycetes incertae sedis, species like
Podonectria (order Pleosporales) are pathogenic to scale insects and cover the body
of the insect with a crust resembling cotton (Roberts and Humber 1981, Kodsueb
et al. 2006). Related anamorphs are from the genera Tetranacrium and Tetracrium.
Around 2000 species of the Laboulbeniomycetes (order Laboulbeniales), are the
obligate haustorial ectoparasites of insects, especially beetles and flies (Weir and
Blackwell 2005). Species from the order Myriangiales, within the class
Dothideomycetes, can also be found on scale insects (Alexopoulos et al. 1996).
Entomopathogenic Myriangiales exhibit perennial growth and the reproduction is
only sexual (Vega et al. 2012).
The hypocrealean fungi from the class Sordariomycetes are probably the most
well-known and the most studied fungal entomopathogens. Most of the entomo-
pathogens were earlier classified within the family Clavicipitaceae, however, some
studies classified the entomopathogens in other families, especially, the
Bionectriaceae, the Hypocreaceae, and the Nectriaceae (Vega et al. 2009; Sosa-
Gómez et al. 2010; Oliveira et al. 2012; Gouli et al. 2013; Sharma et al. 2018a, b, c;
Sharma and Marques 2018). The classical studies on the hypocrealean phylogeny
by Sung et al. (2007a) and Sung et al. (2007b) suggested three families within the
Clavicipitaceae, i.e., the Cordycipitaceae, the Clavicipitaceae sensu stricto (s.s.),
and the Ophiocordycipitaceae. The most specious genus amongst the entomopatho-
genic hypocrealean fungi is Cordyceps sensu lato (s.l.). It comprises at least 400
species (Chandler 2017). Spatafora et al. (2007) provided evidence of host switch-
ing within the hypocrealean families.
The Clavicipitaceae can be characterised by either lilac, pale, or strongly pig-

mented green, yellow or occasionally red stromata (Sung et al. 2007a). Cordyceps
from the family Cordycipitaceae are characterised by brightly coloured and fleshy
stromata, and their anamorphs include species, such as Beauveria, Isaria and
Lecanicillium. Members of the Ophiocordycipitaceae exhibit darker stromata and
mature ascospores. Some of the widely known entomopathogens from this family
include Purpureocillium lilacinum and Ophiocordyceps unilateralis s.l. (Sung et al.
2007a). Recently, Purpureocillium lavendulum was also reported as an EPF from
cultivated soils (Sharma et al. 2018b). Whilst the fungi from the Cordycipitaceae are
mostly entomopathogens, those belonging to the Clavicipitaceae and the
Ophiocordycipitaceae obtain nutrition from animals, plants and fungi (Chandler
2017). Although not specified, it seems that also spider pathogens belong to the
Cordycipitaceae; pathogens of dipteran insects, ants and termites are from
Ophiocordycipitaceae; and scale insect pathogens are from the Clavicipitaceae
(Vega et al. 2012). Ant infections caused by the members of Ophiocordycipitaceae,
such as O. unilateralis, present a spectacular case of a host behaviour manipulation
by the parasite. The ants leave their nests and climb up the plant, and die sticking
themselves onto the leaves. This facilitates fungal dispersal to a longer distance
(Araújo et al. 2018), and can be correlated with “walking zombies”, as the fungus
manipulates the behaviour of the ants to increase its own fitness.
The most important EPF in terms of the usage in agriculture are arguably from
the genera Beauveria and Metarhizium. It is believed that B. bassiana alone can kill
at least 750 different pest-insects species (Ghikas et al. 2010). Besides, there has
been an increase in the literature suggesting the use of EPF, such as B. bassiana, as
endophytes and mycoparasites (Vega et al. 2009, Quesada-Moraga et al. 2014).
Recent multi-gene phylogenies have resolved Beauveria and Metarhizium into
many new species (Rehner and Buckley 2005; Bischoff et al. 2009; Rehner et al.
2011). Moreover, habitat-specific preferences have been noticed for these genera
within different agroecosystems (Vänninen et al. 2000; Meyling and Eilenberg
2006, 2007; Quesada-Moraga et al. 2007; Sun et al. 2008; Meyling et al. 2009;
Goble et al. 2010; Medo and Cagáň 2011; Sánchez-Peña et al. 2011; Schneider et al.
2012; Clifton et al. 2015; Sharma et al. 2018b).
4.5 oil Habitat and Pathogenesis of Entomophthorales

S
and Hypocreales
The soil is a reservoir of many microorganisms, and EPF such as Beauveria,

Metarhizium and Isaria are considered as weak saprophytes in the competitive soil
environment. These fungi spend a considerable part of their life cycle within the
soils, and shown in related literature (Hughes et al. 2004; Sevim et al. 2009; Jaronski
2010; Fisher et al. 2011; Scheepmaker and Butt 2010; Meyling et al. 2011, 2012;
Muñiz-Reyes et al. 2014; Pérez-González et al. 2014; Kepler et al. 2015; Keyser
et al. 2015; Aguilera Sammaritano et al. 2016; Hernández-Domínguez and Guzmán-

Franco 2017; Kirubakaran et al. 2018; Sharma et al. 2018b).
Optimum temperature requirement for Entomophthorales is 15–25 °C, whereas
for Hypocreales it is 20–30 °C. Soil protects EPF from solar radiation and acts as a
buffer against variations in temperatures as well as water availability. It also pro-
vides a habitat to numerous soil-dwelling insects, which serve as potential hosts for
EPF (Quesada-Moraga et al. 2007).
Metabolites secreted by other soil microorganisms may hinder the pathogenicity
of EPF, as they may affect the fungal germination and growth adversely and/or can
be toxic (Sinha et al. 2016). Therefore, it is not surprising that the EPF efficacy and
survival is superior in sterilised soils, when compared with non-sterilised conditions
(Jaronski 2007). However, soil amendments with nutrients help in overcoming this
apparent fungistasis. Nonetheless, EPF continue to germinate and infect the poten-
tial host-insects indicating that, in addition to a reduction in fungistasis, EPF also
rely on host or nutrient derived cues for development (Lacey et al. 2015).
Soil habitat-types can have some significant effects on the occurrences of
EPF. Although there are exceptions, it has been noticed that M. anisopliae is associ-
ated with tilled agricultural soils, and B. bassiana is more frequent in uncultivated
soils, such as hedgerows (Sharma et al. 2018b). This can be explained as B. bassi-
ana is thought to rely more on host-insect, whereas M. anisopliae can also sustain
itself on the plant exudates in the rhizosphere. In return, fungi such as M. anisopliae
translocate nitrogen from the insects directly into the plants (Behie et al. 2012).
Other factors, such as the soil moisture, pH, organic matter, and chemical proper-
ties are also important in determining the abundance and infectivity of EPF (Rath
et al. 1992; Jabbour and Barbercheck 2009; Garrido-Jurado et al. 2011a, b; Sharma
and Marques 2018). Herbicide usage can also have distinct effects of the EPF per-
sistence (Yousef et al. 2015). Nonetheless, numerous abiotic and biotic factors
shape the dynamics of EPF community in the soil, and it is difficult to access the
exact effect of each of these. Main biotic components which influence EPF persis-
tence and efficacy the most are: plants, soil microorganisms, and invertebrates
(Sinha et al. 2016).
Entomopathogenic fungi exhibit diverse lifestyle and mode of action. In pres-
ence of suitable conditions, EPF invade percutaneously into the host-insect body
and cause mycosis. A brief pictorial description of the mechanism of infection of
EPF is provided in Fig. 4.4. In the majority of cases, the entire infection cycle
requires these following steps: (1) spore attachment to the cuticle; (2) germination
of the spore, which initiates cascades of many reactions related to recognition and
enzyme activation, both by the EPF and by the host-insect; (3) the penetration of
host’s integument through hyphal tubes; (4) the suppression of host immune defence
mechanisms by the fungus; (5) the alteration of the fungal morphology into a yeast-
like phase, leading to the formation of hyphal bodies or “blastospores”, which cir-
culate within the insect’s hemolymph leading to the insect death; and, (6) the
attainment of the previous saprophytic phase, the typical hyphal form, which comes
out of the dead body to produce new conidia (Vega et al. 2009; Vega et al. 2012;
Chandler 2017).
Fig. 4.4 Life cycle and mode of action of entomopathogenic fungi (saprotrophic and parasitic
cycle). (Modified from Ortiz-Urquiza et al. (2015) and Lu and St. Leger (2016))
As the majority of the research has focussed on the entomophthoralean and the
hypocrealean fungi, we will only discuss these two fungal groups in more detail.
Entomophthoralean fungi are obligate insect pathogens and do not grow outside
their hosts. Therefore, they are quite host-specific and restricted to only one family
or genus. For the same reason, they are difficult to mass produce in an artificial
culture medium. These fungi eject spores actively and rapidly upon the realisation
of favourable conditions. This property makes them different from their hypocrea-
lean counterparts (Steinkraus 2007). The life cycle of the entomophthoralean fun-
gus is indeed quite complex and often involves at least two different types of
spore-forms: resting and conidia.
Condia spore-forms are responsible for infecting insect-hosts, especially when
the hosts are active. Conidia are actively discharged from the dead host, although
there are few exceptions, for e.g., Entomophthora thripidum is discharged by living
thrips (Pell et al. 2001). Conidia are usually coated with preformed mucous, and
they adhere to the integument upon landing on the insect cuticle. After conidial
attachment, the formation of an “appressorium” (a specialised attachment cells
below which the EPF penetrates the cuticle) facilitates fungal penetration. However,
some entomophthoralean fungi such as Conidiobolus obscurus, Entomophthora

planchoniana, and Pandora neoaphidis do not form appressoria, and penetrate the
insect cuticle directly through the germ-tube (Vega et al. 2012). The conidia are
fragile and short-lived but germinate quite rapidly. In the cases when the primary
conidia do not land on the host-insect surface, they can give rise to the secondary
conidia, which can further produce the tertiary conidia. These supernumerary
conidia increase the chances of fungal infection, although, after every passing gen-
eration, they become smaller in size. Interestingly, only secondary conidia are infec-
tive in Neozygites spp. (Pell et al. 2001).
Mechanical pressure and cuticle-degrading enzymes are indispensable for cuti-
cle penetration. Thereafter, some Entomophthorales initially grow as wall-less pro-
toplasts, to avoid the insect immune response, and later turn into hyphal bodies.
Others grow either as hyphal bodies or protoplasts within the insect host. Host
ceases eating as the infection progresses, and several behavioural changes are
noticed prior to death. These behavioural changes can be noticed in Entomophaga
infecting acridid hosts, Pandora infecting the ant genus Formica, and those infect-
ing flies such as M. domestica and Scatophaga stercoraria. The fungus multiplies
within the host, and manipulates its behaviour by making the host climb to an ele-
vated position (referred as “summit disease”) and kills it. In other cases, for e.g.,
infections by Erynia spp., some fungal structures (rhizoids) are formed to fasten the
hosts more securely to the substrate (Małagocka et al. 2015). Some physiological
changes, such as behavioural fever (an increase in the body temperature of infected
hosts) generally originated when reaching warmer locations, is noticed in the house
flies M. domestica infected by Entomophthora schizophorae or E. muscae.
Furthermore, changes in the mate-seeking behaviour for healthy males is also
noticed, which is characterised by an increased attraction towards the infected
females. The fungi attack the tissues once the hemolymph has significant numbers
of fungal cells, in order to fill the host body cavity with cells by the time the insect
dies (Hajek and Meyling 2018). The Entomophthorales kill the host-insect by
depleting most of the resources inside the hemolymph, probably trehalose sugar,
and sporulate. This leaves host with no time for toxin production (De Fine Licht
et al. 2016). As the host stays alive for some time before sporulation, it may even
succeed to complete a reproductive cycle before dying (Gryganskyi et al. 2017).
Most entomophthoralean species also produce resting spores for survival, when
hosts are either not active or present. For example, during lower temperature peri-
ods, or at a late instar larval stage, or during a decreased photoperiod. The resting
spores are produced inside the cadaver after host death and remain dormant. Resting
spores can either be azygospores (formed when one hyphal cell rounds up at one
end) or zygospore (when two hyphae unite) (Pell et al. 2001). These spores possess
a thick wall, and are deposited in the soil when cadavers disintegrate. Resting spores
may germinate if they find the hosts, however, many of them do not, at least in the
first year of their genesis (Butt et al. 2001). Moreover, in some cases, for e.g.
Entomophaga maimaiga, only a part of the total resting spores germinate in a year.
This is done to ensure fungal infectivity in the cases of prolonged seasons of host
unavailability or inactivity. Resting spores may retain the capacity of germination

for many years (Hajek and Meyling 2018).
The hypocrealean fungi represent some of the well-known, easy to culture, ana-
morphic EPF, such as Metarhizium and Beauveria (Meyling and Eilenberg 2006,
2007; Quesada-Moraga et al. 2007; Garrido-Jurado et al. 2011a; Carlos et al. 2013;
Rudeen et al. 2013; Garrido-Jurado et al. 2015; Steinwender et al. 2015; Castro
et al. 2016; Fernández-Salas et al. 2017; Gan and Wickings 2017; Sharma et al.
2018b). The host range varies from being specialist pathogens, such as Hemiptera-
specific M. album and white grubs specialist B. brongniartii, to generalists, such as
M. robertsii and B. bassiana (Enkerli et al. 2004; Hu et al. 2014). Hypocrealean
teleomorphs show a narrower host range (Boomsma et al. 2014).
Infection occurs either through: (a) an actively discharged ascospore or (b) a pas-
sively transmitted conidia. Passive transmission is facilitated by rain splash or wind
currents, contact with an infected individual, or dispersal by the bodies of arthro-
pods (Boomsma et al. 2014). Conidia of some species are hydrophobic and have a
proteinaceous rodlet layer, whereas, some species have hydrophilic conidia with a
smoother surface (Hajek and Meyling 2018). Conidial adhesion of M. anisopliae is
mediated by “Metarhizium adhesion-like proteins”, for e.g., MAD1 and MAD2,
which are also responsible for conidial germination and blastospore formation.
Carbon and nitrogen sources are required for spore germination and germ-tube for-
mation in case of B. bassiana. Other EPF, such as Isaria farinosa and M. anisopliae,
produce mucilage for fungal adhesion during the germ-tube formation. Cuticle pen-
etration requires a set of enzymes, such as proteases, chitinases, and lipases (Vega
et al. 2012).
Hypocrealean anamorphs often grow inside the insect hemocoel as blastospores
or hyphal bodies, by utilising available nutrients for growth and reproduction. Host
death by EPF, such as M. anisopliae, is supposed to be facilitated by the fungi
undergoing either a “toxin strategy” or a “growth strategy”. The toxin strategy sug-
gests that the fungus employs the production of the secondary metabolites, such as
destruxins, to kill the host, while maintaining very little vegetative growth. On the
contrary, the growth strategy suggests that the fungus relies on a profuse growth
inside the hemolymph, leading to the host starvation and death (Kershaw et al.
1999). Nonetheless, a fungal hypha emerges from within the insect and sporulates
to produce aerial conidia.
4.6 Conclusion
With the growing concerns about the use of chemical pesticides, the use of micro-
bial biopesticides does exhibit a great potential. The majority of the microbial pes-
ticides either spend some part of their life cycle in soils, or somehow survive the
harsher conditions in the soil environment as spores. Hence, understanding the
diversity and ecology of the EB and EPF is of utmost importance. More studies on
the ecology of entomopathogens will provide a better understanding of insect
population cycles and pest outbreak. Strategies considering the bacterial and fungal
ecologies within the agroecosystems may provide a better application design for the
use of microbial entomopathogens in controlling pest-insects.
Attributes such as higher efficacy, low production cost, desired specificity and
enhanced persistence are always crucial in commercialising microbial agents for
biological control. Moreover, the evolution of resistance in the target insect pest and
the deaths of the non-target arthropods are some of the challenges needed to be
addressed. Constant screening of toxins and virulence factors is always underway,
and the discoveries of newer species, approaches for directed evolution, and genetic
engineering provide enormous opportunities to be looked upon in the development
of a biopesticide.
Acknowledgement This work is a part of L. Sharma’s PhD dissertation at the ‘University of Trás-
os-Montes and Alto Douro’, Vila Real, Portugal. The funding was provided by the ‘Centre for the
Research and Technology of Agro-Environmental and Biological Sciences’ (CITAB) through the
fellowship: BIM/UTAD/16/2018; and by the EcoVitis project. National Funds by FCT – Portuguese
Foundation for Science and Technology, UID/AGR/04033/2013; and the European Investment
Funds by FEDER/COMPETE/POCI Operacional Competitiveness and Internationalization
Programme, under the Project POCI-01-0145-FEDER-006958, are also acknowledged. Due to the
limited space, we could not cite many relevant articles. However, we deeply acknowledge those
works.
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Field manual of techniques in invertebrate pathology: Application and evaluation of pathogens
for control of insects and other invertebrate pests (pp. 223–248). Dordrecht: Springer.
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analysis of the first cry gene from Bacillus popilliae. Journal of Bacteriology, 179, 4336–4341.
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Chapter 5
Ascomycota and Integrated Pest
Management
Tariq Ahmad, Ajaz Rasool, Shaziya Gull, Dietrich Stephan,

and Shabnum Nabi
Abstract Employing fungal pathogens to combat insect pests of agricultural

importance has gained momentum due to its ecofriendly approach, availability and
host specificity. Successful future prospects include efficiency and improvements in
the research methods, proper selection of strains, mass production, genetic manipu-
lations and other innovative techniques. Further aspects are the preparation of for-
mulations that will increase persistence, longer shelf life and ease of application,
pathogen virulence and spectrum of action. A description of Ascomycetes biology,
taxonomic characters and mode of action is presented here with a focus on its role
in Intergrated Pest Management strategies. Recent studies on genetic modifications
for improving their virulence are also discussed.
Keywords Ascomycota · Fungi · Entomopathogens · Pest · IPM · Biological

control
T. Ahmad (*)
Federal Research Centre for Cultivated Plants, Institute for Biological Control, Julius Kühn
Institute, Darmstadt, Germany
Entomology Research Laboratory, Postgraduate Department of Zoology, University of
Kashmir, Srinagar, Jammu and Kashmir, India
A. Rasool · S. Gull
Entomology Research Laboratory, Postgraduate Department of Zoology, University of
Kashmir, Srinagar, Jammu and Kashmir, India
D. Stephan
Federal Research Centre for Cultivated Plants, Institute for Biological Control, Julius Kühn
Institute, Darmstadt, Germany
S. Nabi
Interdisciplinary Brain Research Centre, J. N. Medical College, Aligarh Muslim University,

https://doi.org/10.1007/978-3-030-23045-6_5
152 T. Ahmad et al.
5.1 Introduction
With the world population expected to reach 10–12 billion by 2100 (UN Report
2011) the agricultural sector is facing the biggest challenge of providing enough
food to meet the pressing demands of nations. To meet this ever increasing demand,
efforts have been done towards improving crop varieties for higher quality and
yields, shorter durations of life cycle and resistance to insect pests and diseases,
which are responsible for 20–40% crop losses, annually (FAO 2012). Integrated
pest management (IPM) is a broad-based approach that integrates practices for eco-
nomic control of pests and aims at suppressing pest populations below economic
injury level (EIL). It emphasizes the growth of a healthy crop with least possible
disturbance to agro-ecosystems, and encourages natural pest control mechanisms.
IPM makes use of a range of actions including cultural controls, physical barriers,
chemical control or pesticides, and finally biological control, including, adding and
conserving natural pest predators and enemies. For conventional farms, IPM reduces
human and environmental exposure to noxious chemicals, and potentially lowers
overall costs. It extends the concept of integrated control to all classes of pests and
includes all tactics. Other tactics, such as host-plant resistance and cultural manipu-
lations also became part of the IPM framework while uniting Entomologists, Plant
Pathologists, Nematologists and Weed Scientists at one platform to define organized
and integrated strategies against a particular pest species.
Entomopathogenic fungi (EF) trials were started by Russian Microbiologist, Elie
Metchnikoff way back in 1888 (Samson et al. 1988; Glare and Milner 1991), by
mass producing fungal conidia on sterilized brewer’s mash and combined cultures
with sand granules for spreading on field crops. Although his results were inconsis-
tent, the work of Metchnikoff (1888) ignited inquisitiveness around the world and
led to programs in Europe and United States for experimentation with “friendly
fungi” against insect pests (Lord 2005). Boverin, a Beauveria bassiana-based
mycoinsecticide was developed in 1965 for the control of Colorado potato beetle
and codling moth in the former USSR (Kendrick 2000). The first formal and pub-
lished proposal of microsporidia to control grape phylloxera, Daktulosphaira vitifo-
liae (Fitch) was suggested by LeConte (1874). The study offered imaginative ideas
for an effective and economic alternative method of pest control and paved the way
for future scientists to study EF. Owing to greater environmental awareness, food
safety concerns and insecticide resistant species, the application of EF is growing
by leaps and bounds (Shahid et al. 2012).
Ascomycota, the largest and main phylum of the Kingdom Fungi with over
64,000 species (Kirk et al. 2008), is one of the most diverse eukaryotic phyla,
encompassing some important species in biological control programmes throughout
the world. Ascomycetes are very varied and can be identified from the fruiting bod-
ies and the way in which the asci develop. They either act in nature per se or have
been manipulated and used worldwide to control various pest species, with some
level of success in Asia and South America (Fuxa 1987). The distribution of some
species is variable and limited while some are cosmopolitan such as the white
5 Ascomycota and Integrated Pest Management 153
truffle, Tuber magnatum found in isolated locations in Italy and Eastern Europe
(Mello et al. 2006).
Around 90% of microbial pesticides are used as insecticides, accounting for the
1.3% of the world’s total pesticide market (Hajek and Leger 1994), with few com-
mercial formulations of ascomycetes developed for crop pest management. Among
171 products of EF developed, products based on B. bassiana and M. anisopliae
represent 33.9%, while Isaria fumosorosea and B. brongniartii represent 5.8 and
4.1% respectively (Moorhouse et al. 1992; De Faria and Wraight 2007). Although
mycoinsecticides have been found effective in controlling various insect pests of
economic importance, successful marketing and utilization of these products have
been somewhat slow, largely due to low production efficiency, high cost, low per-
formance under testing environmental conditions and lack of awareness programs.
Nonetheless, these mycoinsecticides have potential to play a key and defining role
in IPM programs while focusing on formulations with increased persistence, longer
shelf life and pathogen virulence, ease of application and spectrum of action.
5.2 Biology and Taxonomy
Ascomycetes comprise 75% of all the described fungi speciesThey are heterotrophs
and obtain nutrients from dead or living organisms (Carroll and Wicklow 1992)
while as biotrophs they form symbioses with algae (lichens), the leaves and stems
of plants (endophytes), and roots (mycorrhizae). Ascomycota are either single-
celled (yeasts), filamentous (hyphal) or both (dimorphic). While most yeasts and
filamentous Ascomycota are haploid, some species such as Saccharomyces cerevi-
siae can also be diploid. Ascomycetes are mostly terrestrial or parasitic, however,
few have adapted to marine or freshwater environments whilst in most land-based
ecosystems, and act as decomposers facilitating detrivores to obtain nutrients.
The key character that defines Ascomycota is the ascus, a special, elongated cell
or sac within which nuclear fusion and meiosis take place, and that give the group
its name. Ascomycetes are ‘spore shooters’ and produce microscopic spores
insidethe asci. Asexual reproduction is the dominant form of propagation, a part of
reproducing by means of haploid conidiospores and through budding. The ascus,
which is the result of the sexual reproduction, is a tube-shaped vessel, a meiospo-
rangium, containing the sexual spores called ascospores. It starts with the develop-
ment of special hyphae from either one of two types of mating strains. The “male”
strain produces an antheridium while the “female” strain develops an ascogonium
which combine in plasmogamy at fertilization. Later on, special ascogenous hyphae
arise, in which pairs of nuclei migrate: one from the “male” strain and one from the
“female” strain. In each ascus, two or more haploid ascospores fuse their nuclei in
karyogamy, giving rise to diploid nuclei which, by the process of meiosis, give rise
to haploid nuclei. The ascospores are then released, germinate forming hyphae and
are disseminated in the environment to start new mycelia.
154 T. Ahmad et al.
Fungi and fungus-like organisms are usually defined by their heterotrophic,

absorptive mode of nutrition and their apical hyphal growth (Deacon 1997). The
classification of fungi has been traditionally defined upon morphology (e.g.
Conidiogenesis) and ultrastructure (e.g. cell wall and septum structure). The Phylum
Ascomycota, together with the Basidomycocetes, forms Sub-Kingdom Dikarya
which includes members that form septate haploid hyphae and yeasts. More precise
placement of EF into defined taxonomic groups has been achieved by approaches
such as metabolisms comparison (e.g. nutrient utilization, enzyme and toxin pro-
duction) and/or using genetic analyses (e.g. rRNA sequences, karyotyping, mtDNA
restriction length polymorphism (Khachatourians 1992).
Actually, true fungi are placed in four Phyla (Ascomycota, Basidiomycota,
Chytridiomycota, and Zygomycota) plus one artificial form, the Phylum
Deuteromycota, that contains filamentous fungi. The Deuteromycetes, also known
as Fungi imperfecti, are actually thought as ascomycetes or basidiomycetes which
have either lost their potential for sexual reproduction or comprise undescribed
sexual forms (Barr 1992).
There are only three subphyla in the phylum Ascomycota that have been
described (Berbee and Taylor 1992):
• The Pezizomycotina is the largest subphylum of Ascomycetes producing asco-
carps (fruiting bodies), except for the genus Neolecta which includes most mac-
roscopic “ascos” such as truffles, ascolichens, ergot, cup-fungi (discomycetes),
pyrenomycetes, lorchels, powdery mildews, dermatophytic fungi, and
Laboulbeniales.
• The Saccharomycotina comprising “true” yeasts such as baker’s yeast and
Candida, that reproduce vegetatively by budding.
• The Taphrinomycotina originally named Archiascomycetes (or
Archaeascomycetes), including a disparate and basal group within the
Ascomycota, and recognized by molecular analyses. It includes hyphal fungi
(Neolecta, Taphrina, and Archaeorhizomyces), mammalian lung parasite
Pneumocystis and fission yeasts (Schizosaccharomyces).
Supraordinal fungal classification in Fungal Tree of Life (AFTOL) listed 3 Subphyla,
14 Classes, and 60 Orders of Ascomycota (Hibbett et al. 2007). Of the five classes
that contain EF, most species are found in Laboulbeniomycetes and Pyrenomycetes
(Tanada and Kaya 1993). The class Hemiascomycetes, consisting of yeast like ento-
mopathogens, includes species typically slow acting and cause chronic infections,
such as infection of the biting midge (Dasyhelea obscura) by Monosporella unicus-
pidata. Members of the class Plectomycetes are responsible for chalkbrood disease
in bee colonies. The class Loculoascomycetes is characterized by bitunicate asci,
which are released in specialized stomatic compartments called locules. Two ento-
mopathogenic orders of this class, Myriangiales and Pleosporales are considered
important pathogens of scale insects.
The Pyrenomycetes, a major class in Ascomycota, possess distinguishing unitu-
nicate cylindroid asci and consist of all EF species in order Sphaeriales, most of
which are described within the genus Cordyceps including over 250 species. Both
Exopteygota and Endopterygota insect orders are infected by members of this

genus, and some are even pathogens of spiders. Cordyceps spp., abundant in tropi-
cal forest ecosystems, is an important insect pathogen (Samson et al. 1988) infect-
ing numerous soil dwelling pests.
Laboulbeniomycetes is the other major class of Ascomycota composed of min-
ute, ostensibly inconspicuous fungi that were originally thought to be external com-
mensals of insects (Tanada and Kaya 1993). An estimated 115 genera of this class
are known and only few entomopathogenic species have been found in a wide vari-
ety of habitats (Carruthers and Hural 1990). Although Coleoptera come into view to
be the most common targets, these are known to be pathogenic to members of at
least 11 insect orders with most species reported from Aspergillus, Lecanicillium
(formerly Verticillium), Metarhizium, Beauveria, Nomuraea, Aschersonia,
Hirsutella, Culicinomyces, Paecilomyces and Sorosporella. Most of these genera
have been linked to one or several ascomycete genera. Such linkages can be demon-
strated either biologically (an insect infected with an anamorph dies and a teleo-
morph is produced) or by using molecular tools showing the genetic relationship
between anamorphs and teleomorphs (Huang and Erickson 2002; Liu et al. 2002).
5.3 Mode of Action
Entomopathogens typically have unique mode of action and infection while coming
in contact with their arthropod host. With ideal conditions of moderate temperature
and relative humidity, their spores germinate and breach the host cuticle with the
combined aid of enzymes and mechanical pressure. Later on the germ peg invades
the tissues, multiply yielding a mycelium that produces more spores, killing the host
by a variety of means such as starvation through multiplication or production of
toxins. The mode of action of various ascomycete species is discussed as follows.
5.3.1 Beauveria spp.
Beauveria is an EF that dwells in soil all over the world. Some strains are exceed-
ingly adapted to specific host insects with high potential to be used as biological
insecticides against pests. The conidiospores germinate on the superficial area of the
cuticle, resulting in the formation of long hyphal tubes directly piercing the insect
host integument. After crossing this layer, the fungus shows morphological changes,
becomes yeast like and produces hyphal bodies which move in the haemolymph and
start fast budding. This yeast-like phase is essential for the pathogenicity and subse-
quent propagation by conidia by emerging mycelia. Attachment of fungal spores to
the host is mostly passive and represents the first event for mycosis development.
Spores are randomly transferred by various agents like air and water.
156 T. Ahmad et al.
Dry spores of B. bassiana have an outer layer made of interwoven fascicles of

hydrophobic rodlets. Each conidial stage has a unique rodlet layer which is never
found on the vegetative cells. Rodlets exert non-specific hydrophobic forces which
result in hold of dry spores to the cuticle (Boucias and Pendland 1988). Nevertheless,
the mechanism, that is yet to be known in detail, results in yielding adhesive forces
between conidia and the insect cuticle (Latge and Monsigny 1988). When the fungal
spores reach the haemocoel, it shows rapid germination and growth which is
enhanced by nutrient availability, water, oxygen and temperature.
Beauveria enters through the cuticle into the insect body to acquire nutrients for
reproduction and growth. It results in both an enzymatic activity as well as a
mechanical pressure allowing entering the host, owing to physical separation by
inserted hyphae. Different pathogenic enzymes such as chitinases, lipases, esterases
and at least four different classes of proteases, are responsible for breaking of host
cuticle, degrading its major components. Enzymes such as endoproteases (PR1 and
PR2) and aminopeptidases are produced on the cuticle, coincident with formation
of appressoria. Proteolytic enzymes are formed at faster pace in comparison to
N-acetylglucosaminidase (St Leger and Butt 1989). The most important factor for
of cuticle breaking is the activity of endoproteases, although other enzymes syner-
gistically work together for penetration. Beauveria spp. also release lethal fungal
toxins leading to cellular disturbances in the host. Many insects show changes in
behaviour such as overall paralysis, sloth appearance and decreased petulance, due
to neuromuscular toxins. Toxins extracted from the infected host body include beau-
vericin, beauverolides, bassianolide and isarolides (Hamill and Sullivan 1969;
Elsworth and Grove 1977), lethal to tissues leading to cellular disturbances and
death.
5.3.2 Isaria spp.
Isaria (I. farinosa and I. fumosorosea), includes filamentous fungi attacking various
insect pests, worldwide. Isaria spp. can be used as biocontrol agents for controlling
predominantly white flies. They produce cuticle destroying enzymes viz. proteases
(Pr1 and Pr2), chitinases, chitosanases and lipases. They act as a strong antifeedant
decreasing the host feeding potential and body weight (Shaukat et al. 2010). They
target the host cuticle through the enzymatic action of chitinases. The chitinase gene
isolated from the genome of I. fumosorosea (Ifchit1) encodes a polypeptide of 423
amino acids. Ifchit1 chitinase has proved to be a virulent pathogenic factor towards
the host (Zhen et al. 2016). When in contact with a specific host, Isaria becomes
rapidly pathogenic when the conidia are inoculated on the hind wings. The fungus
shows a higher response in comparison to the conidia attachment to other part of the
body, due to a germination rate which is higher on wings.
5.3.3 Metarhizium spp.
Metarhizium, formerly known as Entomophthora anisopliae, is an EF having world-

wide distribution, acting as an insect parasite. Metarhizium spp., like other entomo-
pathogens, penetrate the cuticle by adhesion of asexual, dry spores and get attached
to the upper waxy insect coating by hydrophobic, electrostatic forces and protein
interactions between conidia and host integument. Different species have specific
genes for pathogenicity, such as Mad 1 kinase gene of M. anisopliae. Asexual spores
of Metarhizium spp. contain hydrophobins, that increase adhesion properties rein-
forcing attachment (Ment et al. 2010a). The adhesion is largely dependent on envi-
ronmental factors and on the chemical composition of hosts (Santi et al. 2010). At
the onset of germination, concentration of trehalase, that uses the sugar trehalose
present in the haemolymph, increases. The process is initiated by the presence of
exogenous carbon and nitrogen (Ment et al. 2010b). Trehalase gives energy for
spores development and swell up, germ tubes formation and subsequent develop-
ment of the appressorium (Lovett and Leger 2015). Mad 1 and Mad 2 are formed by
replacing hydrophobins so that conidia attach to the integument in a firm way.
Besides, some fatty acid esters, amino acids, glucose and peptides are also required
during germination. After germination, appressorium formation takes place with the
help of two genes, ODC1 and Mpl1. Through the conidial germination, ODC1
translates for ornithine decarboxylase and appressorium is formed from the germ
tube (Wang and St Leger 2007a, b; Pulido et al. 2011). Mechanical and turgor pres-
sure is generated by conidia penetrating inside the insect cuticle, a process enhanced
by a mucilage layer (Greenfield et al. 2014; Staats et al. 2014). In M. anisopliae,
another enzyme MAPK (Mitogen-Activated Protein Kinase) is present that helps in
regulation of the expression of Mad1 gene for adhesion, while appressorium devel-
opment is highly enhanced by the gene MaMK1 which is an extracellular signal-
regulated kinase. Tetraspanins determined by the MaPls1 gene also helps in turgor
pressure and in the development of the appressorium in a specific pathway (Luo
et al. 2013). Penetration of conidia is the next stage in M. anisopliae that results in
the secretion of proteins such as chymotrypsins, trypsins, carboxypeptidases and
subtilisins by which the protein rich procuticle of arthropods is digested (Wang
et al. 2008). M. anisopliae produces many types of proteins which affect different
hosts and are very specific. Enzymes such as subtilisin proteases (Prl) are found to
be degrading cuticle by hydrolysing the integument, while other enzymes such as
chitinases work with other proteases to pierce the cuticle (Butt et al. 2013). On the
surface of conidia, lipases are also present that enhance adhesion of conidia by lipo-
lytic activity and results in the release of free fatty acids (Beys da Silva et al. 2011).
All these enzymes work synergistically to enhance penetration and are mostly
dependent on the amount of nutrients present in the host haemocoel . The chi2 gene,
which encrypts for an endochitinase, is also decisive for the infiltration of the host
cuticle by plummeting the lethal time required to kill a specific insect host (Boldo
et al. 2009). Certain substances, such as tetraspanins, help in regulation of apsA and
kinesin, responsible for the structure and migration of the cytoskeleton. It uses
158 T. Ahmad et al.
nutrients proceeding from the host cuticle to start germination. All the cellular activ-
ities are under the control of GTPases and tetraspanins, that code MaPlsl while
helping the fungus to thrive in the host environment. Colonization is the most
important stage in the pathogenicity caused by M. ansiopliae in the haemolymph of
host insect. Dextruxin A and dextruxin E, natural insecticidal molecules, act on the
immune system of the host suppressing all the cellular activities and encapsulated
spores are released. The gene dtxS1 is responsible for the synthesis of dextruxin and
is mostly found in Metarhizium (Wang et al. 2012). However, some other fungal
proteins elude the host immune system, such as the evasion proteins Mcl1 The glu-
cose required for their activity is provided by trehalose from the trehalases released
in the haemolymph (Ment et al. 2010b). When germinated conidia enter the host
body, there is a fast multiplication and hyphal differentiation step, controlled by the
Mad1 protein and in turn, these proteins regulate every activity in the cell cycle
(Wang and St Leger 2007a).
Cytochrome P504s genes are also found in M. acridium, which reduces the anti-
microbial host activity. Finally during sporulation, hyphal growth is enhanced due
to the action of a Hog 1 kinase, resulting in a thick layer of green spores formed on
the dead body of infected insects (Ocampo and Caoili 2013).
5.3.4 Lecanicillium spp.
Lecanicillium, earlier identified as Verticillium (i.e. V. lecanii), is one of the most

important EF with a high potential to be used as commercial biopesticide. It relies
on hydrolytic enzymes and mechanical forces to pierce into the integument of the
host. They act as mycoparasites to plant pests (Goettel et al. 2008) being highly
virulent against aphids and white flies. Lecanicillium spp. have a wide host range
and are known as white halo fungi as their mycelial growth is white in colour. Their
potential and virulence have been reported on pests such as thrips, mealy bugs, scale
insects, white flies (Horn 1915; Ekbom 1979a, b; Kanagaratnam 1982). These fungi
can be grown on potato dextrose medium and are initially white in colour and later
develop light yellow nuances in the medium (Naejrech 1973). Lecanicillium spp.
act on the host while contacting its integument and get attached to the outer, waxy,
epicuticle coating. Afterwards, they start germinating with hyphae formation that
penetrate the cuticle directly (Hughes and Gillesipie 1985). However, at times they
may grow on the external surface of the cuticle as well. Various extracellular
enzymes are responsible for piercing the integument such as chitinase, protease,
esterase, n-acetyl glucosamine, endoprotease, carboxypeptidase A, PR 1- chymo-
elastase serine protease and endoprotease (St Leger and Cooper 1986, 1987). All
these enzymes work synergistically for breaking the cuticle wall, while PR-1 acts as
the major cuticle breaking enzyme, by increasing its concentration at the point of
penetration. Later on they start forming blastospores in the form of hyphal growth.
Infested insects die due to combined effect of mycotoxins and outer mechanical
pressure (Ferron 1981). Beauvericin, dipicolinic acid (Claydon and Grove 1982),
bassianolide (Kanaoka 1978), vertilecanin-A1, decenedioic acid and 10-hydroxy

8-decenoic acid (Soman 2001) are some of the chemicals present in the process.
When the nutrient concentration is reduced inside the host body, hyphae are pro-
truded outside to form a mycelial mat on the external body surface, which rapidly
leads to the host insect death (Yeo 2000).
5.3.5 Nomuraea rileyi
Nomuraea, a potential dimorphic hyphomycete EF, is effectual on various pests,

mostly Lepidoptera and Coleoptera (Ignoffo 1981). It is a versatile fungus, with a
broad host range, widespread occurrence and infecting any developmental stage. Its
mode of action starts with the attachment of conidia to the insect body (Srisukchayakul
et al. 2005). On the host epicuticle, tube-like structures are formed, puncturing the
endocuticle and producing hyphal bodies inside the haemocoel, leading to the insect
death. Conidiophores are formed at the end of the infection cycle, when mycelia
develop from the insect cuticle surface (Srisukchayakul et al. 2005). The fungus
penetrates the body wall by both mechanical pressure and enzymatic activity, as
well as by secreting on the epicuticle various proteinaceous substances such as pro-
teinases, lipases and chitinases. It starts budding in the insect haemocoel, when
hyphal bodies are formed. It has been reported that N. rileyi produces one specific
insecticidal toxin (Ye et al. 1993) which causes infection to sap sucking insects and
several noctuid pests, with a potential to infect any developmental stage including
pupae (Ferron 1978; Anand et al. 2009).
5.3.6 Hirsutella thompsonii
Hirsutella thompsonii is cosmopolitan in distribution and considered a very impor-

tant entomopathogen. It is highly effective on mites, penetrating through the legs
and forms hyphal bodies inside the haemolymph. Spores which are formed inside
the body show their emergence through openings such as mouth and genitalia, and
then from all over the body. The insects which are infected by this fungus in in-vivo
conditions show the same general mechanism, i.e. conidia gets adhered to the cuti-
cle, followed by germination and body penetration (Liu et al. 1996). Hirsutella
thomopsonii is highly effective towards various arthropods such as mosquito larvae,
aphids and mites (Krasnoff and Gupta 1994). It secretes an insecticidal polypeptide
chain named Hirsutelin A (Ht A), showing ribosomal inhibiting activity on the pests
when infected. Ht A has insecticidal properties (Liu et al. 1995; Mazet et al. 1995).
Its toxicity was observed in vivo against adult citrus rust mite, Phyllocoptruta oleiv-
ora, which is its natural host (Omoto and McCoy 1998). Ht A has an ability to form
a ribonucleolytic enzyme and other ribotoxins (Liu et al. 1995) and acts on insect
cells at increasing concentration of phosphatidyl ethanolamine and
160 T. Ahmad et al.
phosphatidylinositol (Marheineke et al. 1998), increasing permeability due to the

presence of thin membranes and ribotoxin targets. Ht A causes death of larvae upon
infection, albeit infection rates depend on the ribotoxin concentration (Lacadena
et al. 1995). In addition, H. thompsoni causes cellular damage by generating oxida-
tive stress in the insects and by the action of enzymes such as CAT, GPx and SOD
(Fornazier et al. 2002).
5.3.7 Aschersonia spp.
Species of Aschersonia are predominantly effective biocontrol agents of whiteflies.

Immature stages, especially the first, second and third nymphal instars of whiteflies
are susceptible to infection by Aschersonia while all other substages, from fourth
nymphal instar to adults, are usually less susceptible (Fransen 1987). Its action
starts with the attachement of conidia to the host insect cuticle. Changing the attach-
ment from passive to active induces an enzymatic process of secretion of various
mucilaginous substances, by germinating conidia (Fargues 1984). There is a visible
colour change in the host at early infection stages, as germ tubes are produced on
the cuticle which start penetration or form an appressorium. All these events involve
enzymatic and physical activities whilst hyphal bodies are produced inside the host.
They circulate in the haemolymph before germinating to yield a mycelium on which
masses of spores are produced. The fungus pervades all body organs while protrud-
ing outside, resulting in the host death (Roberts and Humber 1981). Under optimal
conditions, the first symptoms occur within 24–48 h. A vigorous hyphal growth
takes place in 4–6 days and production of conidia occurs usually from 7–9 days
after initiation of infection (Fransen 1987). Relative humidity plays an important
role in the infection cycle. Conidial germination is retarded below 98% RH and is
impaired below 90% (Fransen 1987).
5.4 Ascomycetes in IPM Programmes
EF are known to infect insects of almost all orders, and especially Hemiptera,
Lepidoptera, Diptera, Coleoptera, Orthoptera and Hymenoptera (Ramanujam et al.
2014; Khan and Ahmad 2015). Nymphal and larval stages are more susceptible to
EF attacks than adults, while in others the reverse may be true. Some fungi such as
Aschersonia aleyrodis have restricted host ranges and infect only whiteflies, whereas
N.rileyi infects only lepidopteran larvae. Others like B. bassiana and M. anisopliae
infect more than 700 species in several insect orders and have been tested against
aphids, whiteflies, thrips, and a few against coleopteran and lepidopteran pests.
Ascomycota harbour most number of species, some of which play a key role in
IPM and natural regulation. Various species in Ascomycota may provide effective
long term and short term control including members of Beauveria, Verticillium,
Isaria, Aschersonia, Hirsutella, Metarhizium and Nomuraea, that are integral com-
ponents of IPM used in myco-biocontrol of insect pests.
5.4.1 Beauveria spp.
Beauveria bassiana (family Cordycipitaceae), is one of the widely and popularly

used entomopathogens. It is the anamorph (asexual form) of Cordyceps bassiana,
the sexually reproducing form (teleomorph) collected only in eastern Asia (Li et al.
2001). In cultures, this fungus grows as a white mould producing dry, powdery dis-
tinctive conidia in white spore balls. Rehner and Buckley (2005) argued that B.
bassiana consists of many diverse lineages that should be recognized as distinct
phylogenetic species. For a long time B. bassiana has been known as the most com-
mon causal agent of the disease related to dead and moribund insects in nature
(Mcleod 1954). It has also been scrutinized worldwide as a microbial control agent
of hypogeous insect species (Ferron 1981). Many curculionid weevils with subter-
ranean larval stages are highly susceptible to it (Beavers et al. 1983). Growing natu-
rally in soils throughout the world, B. bassiana acts as a pathogen on various insect
species, inflicting diseases such as the white muscardine (Sandhu and Vikrant 2004;
Thakur et al. 2005; Jain et al. 2008).
Some of the hosts of agricultural and forest importance of Beauveria include
Codling moth, the Colorado potato beetle and several genera of termites, the
American bollworm, Helicoverpa armigera, the teak defoliator, Hyblaeapara and
teak skeletonizer, Eutectona machaeralis (Thakur and Sandhu 2010). With world-
wide distribution, B. bassiana is also used as a biological insecticide to control a
number of pests such as termites, whiteflies, and malaria-transmitting mosquitoes
(Hamlen 1979). When used as an insecticide, spores are sprayed on affected crops
as an emulsified suspension or wettable powder. While acting as a non-selective
biological insecticide, it parasitizes a wide range of arthropod hosts. It has been
effective against pine caterpillars, Dendrolimus spp., the European corn borer,
Ostrinia nubilalis and green leafhoppers, Nephotettix spp. Pathogenity studies of
two isolates of B. bassiana, M. anisopliae and Paecilomyces farinosus against sol-
dier ants under laboratory conditions revealed high mortality rates around 80%,
killing the ants in the first four days after inoculation (Loureiro and Monteiro 2005).
In China, one of the major forest pests, the pine caterpillars Dendrolimus spp., have
been successfully controlled through aerial applications of B. bassiana in oil, spray-
ing up to 300,000 ha of forest cover over a 5-year period, resulting in mortality
ranging from 43% and 93% (Pan and Zheng 1988). Our recent studies on walnut
pests and their management by entomopathogens showed efficacy of Beauveria
bassiana on Myllocerous fotedari. (Fig. 5.1a).
162 T. Ahmad et al.
Fig. 5.1 (a) Myllocerous fotedari infested with Beauveria bassiana (b) Verticillium lacanii infect-
ing whitefly (c) Bagrada bug killed by the green fungus Metarhizium anisopliae (d) Spodoptera
larvae infected with Nomuraea sp. (Elangbam et al. 2016)
5.4.2 Lecanicillium lecanii
Lecanicillum lecanii (previously known as Verticillium lecanii) is another widely

distributed EF with broad host ranges, affecting insects of the orders Homoptera
(Milner and Lutton 1986; Etzell and petitt 1992), Coleoptera (Barson 1976)
Lepidoptera (Gopalakrishnan 1989) and Orthoptera (Khachatourians 1992). Some
plant disease pathogens such as cucumber powdery mildew (Verhaar et al. 1996)
and Chrysanthemum rust fungi (Whipps 1993) may be also affected by this fungus.
Kim et al. (2002) reported that L. lecanii was an effective biological control agent
against the greenhouse whitefly, Trialeurodes vaporariorum in South Korean green-
houses (Fig. 5.1b). While attacking nymphs and adults, it sticks to the leaf underside
by means of a filamentous mycelium (Nunez et al. 2008). In the ‘70s, L. lecanii was
used to control whiteflies and several aphid species, including the green peach
aphids (Myzus persicae) in greenhouse cultivated Chrysanthemum (Hamlen 1979).
Aspergillus flavus and L. lecanii were applied to control Helopeltis spp.
(Hemiptera: Miridae) under laboratory conditions (De Faria and Wraight 2007).
Pathogenicity test revealed that the average mortality of Helopeltis spp. occurred
from the second until the seventh day after inoculation, and that mortalities were
90% with Aspergillus sp., 80% with A. flavus and 77% with L. lecanii. The latter has
been also reported to inflict natural epizootics in aphid and scale populations in
tropical and sub-tropical regions. It was also the first fungus to be studied and
developed for use as an inundative mycoinsecticide in glasshouses and available in
the form of two products manufactured by Koppert Biological Systems in the
Netherlands (De Faria and Wraight 2007). The product contains different isolates as
active ingredients such as “Vertalec” against aphids and “Mycotal” against white-
flies and thrips and is registered in Denmark, Finland, Netherlands, Norway and UK.
5.4.3 Metarhizium spp.
Metarhizium anisopliae is explored for IPM of various notorious insect pests

(Sandhu and Mishra 1994). Several strains of M. anisopliae have been developed as
biological control agents for grasshoppers, locusts, cockchafers, spittlebugs, grubs,
bagrada bugs and borers (Fig. 5.1c). A complete bioactivity of M. anisopliae tested
on teak skeletonizer, Eutectona machaeralis has confirmed its potential as a myco-
biocontrol agent also for this pest (Sandhu et al. 2000).
Between 1985 and 1989, due to outbreaks of the Desert locust, Schistocerca
gregaria, a devastating and destructive pest of crops and pasture grasses in many
parts of Africa, extensive chemical insecticides were applied for control. Later on,
looking into the possibility of using EF as biological control agents, a collaborative
research programme, prompted by international concern on adverse environmental
impacts of insecticide applications, was initiated between research institutes in the
UK, the Netherlands, and the Republics of Benin and Niger (Prior and Greathead
1989). During the course of the study, M. anisopliae var. acridum was found to be
an important natural pathogen of locusts and grasshoppers (Shah et al. 1997; Driver
et al. 2000). Currently, a Metarhizium-based mycoinsecticide is supplied in sachets
containing dried conidia which can be mixed with diesel or kerosene oil before
spraying for pest control (Bateman et al. 1998). Spraying this mycoinsecticide led
to infection and death rates around 70–90% of treated locusts or grasshoppers
within 14–20 days after application, without negative effects on non-target organ-
isms (Lomer et al. 2001). “Green Muscle”, a patented product of M. anisopliae, is
recommended for locust and grasshopper control by the Food and Agriculture
Organisation of the United Nations (Lomer et al. 2001).
A number of entomopathogenic Hyphomycetes, including M. anisopliae and
B. bassiana, commonly isolated from termite colonies demonstrated considerable
potential for controlling termites (Milner et al. 1998a). Application of M. anisopliae
conidia to mound- and tree-nesting termites in Australia showed substantial mortal-
ity (Milner and Staples 1996; Milner et al. 1998b). In Brazil, a high prevalence of
termite mortality (100%) was observed in 19 of 20 nests treated with M. anisopliae.
Brazil is the single largest country where commercial biopesticides based on
M. anisopliae are used against spittlebugs on sugarcane and grassland, annually (Li
et al. 2010). Rangaswami et al. (1968) reported 100% mortality by Metarhizium sp.
in Pyrilla purpusilla in field conditions, besides controlling spittle bugs (Mahanarva
posticata) in sugarcane (Ferron 1978). Mweke et al. (2018) evaluated the
164 T. Ahmad et al.
p athogenicity of 23 fungal isolates including M. anisopliae, B. bassiana and Isaria

sp. against adults of Aphis craccivora in the laboratory. All the fungal isolates were
pathogenic to A. craccivora with mortality rate between 34.5 and 90%. Adults of the
coconut-palm beetle, Oryctes rhinoceros, one of the major pests of Asian- and
Pacific-grown coconut and oil-palms, feed on palm fronds, boring into the axils and
destroying plant tissues (Bedford 1980). Its larvae are naturally infected by M.
anisopliae which considered as an indispensable natural mortality factor (Carruthers
and Soper 1987).
5.4.4 Nomuraea sp.
Nomuraea rileyi is responsible for epizootics in various insects populations. Many

insect species belonging to Lepidoptera, including Spodoptera litura and some
Coleoptera, are susceptible to N. rileyi (Ignoffo 1981) (Fig. 5.1d). The host specific-
ity of N. rileyi and its ecofriendly behaviour encourages its use in insect pest man-
agement, as shown by epizootics in populations of several noctuid pests (Tang and
Hou 1998). Its infection and development have been reported for several insect
hosts such as Trichoplusia ni, Plathypena scabra, Heliothis zea, Bombyx mori,
Pseudoplusia, Helicoverpa armigera and Anticarsia gemmatalis. Spilosoma moths
are severely attacked by N. rileyi and various species were studied in detail for
mycobiocontrol properties (Mathew et al. 1998). In addition, an epizootic of N.
rileyi observed on Hedge plant eater, Junonia orithya proved to be the best alterna-
tive for management (Rajak et al. 1991).
5.4.5 Isaria spp.
Formerly known as Paecilomyces, Isaria spp. have the ability to grow extensively
over leaf surface under humid conditions thus enhancing their spread through white-
fly populations (Wraight et al. 2000). Natural epizootics of these fungi suppressed
Bemisia tabaci populations (Seryczynska and Bajan 1975) while its closed taxon,
Paecilomyces fumosoroseus (Wize), one of the key natural enemies of whiteflies
worldwide, causes the disease known as “Yellow muscardine” (Nunez et al. 2008).
It has strong epizootic potential against Bemisia and Trialeurodes spp. in green-
house and open field environments. It has been reported to cause considerable reduc-
tions in B. tabaci populations during prolonged periods of cool and humid conditions
in the field or greenhouse or immediately following rainy seasons (De Faria and
Wraight 2007). Kim et al. (2002) reported that P. fumosoroseus is paramount for
controlling the nymphs of whitefly, while it covers their body with mycelial threads
and sticks them to the leaves underside (Nunez et al. 2008). Paecilomyces furiosus
is also used to control Culex pipiens (Sandhu and Mishra 1994). Furthermore, iso-
lates of Paecilomyces induce highest mortality rates in European pepper moth,
Duponchelia fovealis (Lepidoptera: Crambidae), a greenhouse pest of cut flowers,

vegetables, and aquatic plants in northern Europe and Canada (Matuzzia et al. 2016).
5.4.6 Aschersonia sp.
Aschersonia is characterised by bright coloured stromata, filiform ascospores and

pycnidial to acervular anamorphs. It was recognised as an effective biocontrol
agents in Florida for control of whitefly, Aleyrodes citri (Fawcett 1908) and of citrus
white fly, Dialeurodis citri in USSR. Aschersonia spp. were introduced from India,
China, Japan, Vietnam, USA and Cuba. Protsenko (1967) reported 80% larval mor-
tality of white flies with foliar spray of conidia. Experiments carried out by Uchida
(1970) reported fall in D. citri effectiveness. Similar experiments carried out by
Solovey and Koltsov (1976) found 83% mortality of orange white fly, Aleurocanthus
spiniferus. The pathogenicity and effectiveness of Aschersonia spp. was carried out
on a wide range of insects, indicating their potential role as biocontrol agents
(Spassova et al. 1980; Ramakers 1983 & Ellis et al. 2002).
5.4.7 Hirsutella spp.
The entomopathogenic Hirsutella species have a worldwide distribution and are

known to infect larval, pupal, and adult stages of insects living in diverse habitats.
The genus Hirsutella include members which represent mesothermic mycopathoge-
nens of pest and non pest mites, insects and nematodes. These are known to be host
specific and regulate host populations in a density dependent relationship. Many
members within the genus Hirsutella are anamorphs (asexual state) of telomorphs
(sexual state) within genera Cordyceps and Torrubiella (Ascomycota: Hypocreales)
(Hywell-Jones 1995, 1997), or synanamorphs (second anamorph state) of genus
Harposporium (Hodge et al. 1997; Evans and Whitehead 2005; Li et al. 2005).
Presently, the genus Hirsutella includes more than ninety species attacking a wide
range of mites, insects, and nematode hosts.
Hemipteran vectors of plant diseases are the major group of economically-
important insects that harbors Hirsutella infections. The highly polyphagous,
xylem-feeding glassy winged sharpshooter, Homalodisca coagulata (Hemiptera:
Cicadellidae) are one of the regulated pests. This insect harbours the phytopatho-
genic bacterium Xylella fastidiosa, the causal agent of Pierce’s disease of grape,
almond leaf scorch, plum leafscald, oleander leafscorch, citrus variegated chlorosis
and many other plant diseases (Purcell 1989; Hopkins and Purcell 2002). Hirsutella
spp. have been also reported to be specific towards mites inhabiting foliar substrates.
Species such as H. nodulosa, H. kirchneri, H. necatrix, H. gregis, and H. thompsonii
are known to attack eriophyoid mites (McCoy et al. 1988; Maimala 2004). Likewise,
Rossi-Zalaf and Alves (2006) reported that H. thompsonii isolated from the rubber
166 T. Ahmad et al.
tree mite Calacarus heveae was highly lethal (>90%) to the false spider mite,
Brevipalpus phoenicis. Recent laboratory bioassays revealed that H. kirchneri, iso-
lated from the cereal rust mite, Abacarus hystrix was infectious to prostigmatids,
including various spider mites and an astigmatid parasitic mite (Sztejnberg et al.
1997). Moreover, well characterized H. thompsonii strains isolated from eriophyoid
mites have been reported to infect and kill the mesostigmatid Varroa destructor, the
devastating ectoparasitic mite of the honeybee, Apis mellifera (Muma 1958; Gerson
et al. 1979; Shaw et al. 2002).
5.5 Ascomycetes as Mycoinsectides
Thanks to the more than 750 species of fungi pathogenic to insects, offering great
potential for IPM, about 171 products—based on at least 12 Ascomycete species—
have been developed worldwide (De Faria and Wraight 2007; Ramanujam et al.
2014; Hu et al. 2016). Some species. Pathogenic to insect pests of agricultural crops.
Already used in formulated mycoinsecticides include: Beauveria bassiana, B.
brogniartii, Lecanicillium spp., M. anisopliae, H. thompsonii and I. fumosorosea
(Maina et al. 2018) (Table 5.1). These Entomopathogenic fungi infect insects of
almost all orders with most common targets being Hemiptera, Diptera, Lepidoptera,
Coleoptera, Orthoptera and Hymenoptera (Ramanujam et al. 2014). Some fungi
have restricted host ranges viz., Aschersonia aleyrodis infects only whiteflies while
others such as B. bassiana and M. anisopliae infect more than 700 species in several
Table 5.1 Fungi pathogenic to insect pests of agricultural crops (Maina et al. 2018)
Fungus Target pest Crop
Beauveria bassiana BB-01 Schizaphis graminum, Rhopalosiphum Laboratory
padi, Brevicoryne brassicae and Lipaphis
erysimi
Beauveria bassiana Mustard Ahpid, Lipaphis erysimi, Aphis Canola (Brassica
PDRL1187 craccivora Koch napus L.)
Beauvaria bassiana Whiteflies Melon
Beauvaria bassiana Myzus percsicae Cabbage
Verticillium lecanii V17, Cabbage aphid, mustard Ahpid (Myzus Cabbage, canola
PDRL922 persicae, Lipaphis erysimi) (Brassica napus L.)
Metarhizium anisopliae L6, Cabbage aphid, mustard Ahpids, Lipaphis Cabbage, canola
M440, PDRL711, PDRL526 erysimi, Aphis gossypii, Aphis craccivora (Brassica napus L
Koch
Paecilomyces fumosoroseus Mustard aphids, diamondback moth Cabbage, canola
n32, Lipaphis erysimi, Plutella xylostella (Brassica napus L.)
Verticillium lecanii Myzus persicae, Aphis craccivora Koch Chili
Peacilomyces lilcinus Mustard Ahpids, Lipaphis erysimi Canola (Brassica
PDRL812 napus L.)
Hirsutella thompsonii Aphis craccivora Koch Cowpea
insect orders. Due to their insecticidal properties, many EF have been formulated
into commercial products for controlling insects of economic importance even
though temperature and humidity limit their efficacy in field applications. Some of
the well known products include, Metarhizium 50, Biogreen and Green Guard,
Cryptogram and Bb plus, BIO 1020 and Green Muscle (Bidochka and Small 2005).
These mycoinsecticides have been tested and used to control a number of insect
pests in glasshouse and field crops worldwide. A list of some commercially avail-
able mycoinsecticides, with brand names, target pests and country of production are
presented in Table 5.2.
Mycoinsecticides, being part of biopesticides, are produced and marketed for
pest management strategies while representing only 3% of the global crop protec-
tion business. Nonetheless, owing to successes and ecofriendly approach, its growth
rate is high, reaching 10% per year (Patrick and Kaskey 2012). Of the total global
biopesticide market, mycoinsecticides are second (27%) to Bacillus thuringiensis
products, mostly produced in America, Europe and Asia (Kabaluk et al. 2010). In
China, at least 30 mycoinsecticides have been registered with B. bassiana being the
most popular. Among them up to 14 products have been used for the control of
locusts, pine moth and diamond back moth (Hu et al. 2016). Furthurmore, M. aniso-
pliae and Paecilomyces lilacinus with eight seven products, respectively, are regis-
tered for application on grubs, corn borer, aphids and whiteflies.
Commercially available B. bassiana is the most widely used species. Its products
are available for a very wide range of insect pests, including pine caterpillars
(Dendrolimus spp.) in China (Feng et al. 1994), banana weevils (Cosmopolites sor-
didus) in Brazil (Alves et al. 2003) and the European corn borer and greenhouse
aphids in the Western world (Shah and Goettel 1999; Copping 2001). Formulations
based on Beauveria consist of aerially produced conidia as wettable powders or in
emulsifiable oil. Products based on B. brongniartii are available against a wide vari-
ety of Coleopteran, Lepidopteran, Homopteran and Dipteran pests in flowers, oil
palms, vegetables, and other crops in Colombia (Alves et al. 2003), against the
European cockchafer (Melolontha melolontha L.) and other white grubs in Europe
(Copping 2001) and against cerambicid beetles in Japan (Wraight et al. 2001).
Clay granules or barley kernels of L. lecanii have been used to produce conidia
in solid-state and marketed for control of greenhouse aphids, thrips and whiteflies
(Copping 2001; Wraight et al. 2001) as well as for the control of lepidopteran,
homopteran, and dipteran pests of flowers, vegetables, and other crops (Shah and
Goettel 1999; Alves et al. 2003). Products with blastospores produced in submerged
fermentation or conidia in solid-state fermentation are commercially available.
Products based on M. anisopliae are available for a wide range of pests, including
red-headed cockchafer in Australia (Shah and Goettel 1999), sugarcane spittlebugs
(Mahanarva spp.) in Brazil (Alves et al. 2003), termites in USA (Copping 2001),
grasshoppers and locusts in Africa (Lomer et al. 2001) and Australia (Copping 2001).
Formulations of P. fumosoroseus are primarily used in greenhouse applications
and marketed against whiteflies, thrips, aphids and spider mites in Latin America,
Europe, and North America (Copping 2001; Alves et al. 2003). In addition, numer-
ous other products are currently available in many countries such as Entomophthora
168 T. Ahmad et al.
Table 5.2 Some familiar commercial mycoinsecticides with their brand names, target pests and
country of production (De Faria and Wraight (2007), Kachhawa (2017), Mishra et al. (2015))
Fungus Brand name Target pest Country
Beauveria bassiana Mycotrol WP Whiteflies/aphids/Thrips USA
Myco-Jaal Diamondback moth India
Conidia Coffee berry borer Germany
Naturalis L Whiteflies/thrips/aphids/ USA
white grub
Boverol Leaf beetles Czech
Republic
Ostrinil Moths France
BioGuard rich Moths, Thrips, weeils, India
aphids, scarab beetles
Boverin Whiteflies, Thrips,Mites Russia
Metarhizium flavoviride Biogreen Scarab larvae Australia
Metarhizium anisopliae Bioblast Termites USA
Metaquino Spittle bugs Brazil
Metabiol Froghoppers Venezuela
Bio-magic Weeils, scarab beetles, India
plant hoppers
BIO 1020 Weevils Germany
Metarhizium Scarab beetles Switzerland
Andermatt
Fitosan-M Scrab beetles, locusts, Mexico
grasshoppers
DeepGreen Scrab beetles, grubs bugs Colombia
Metarhizium anisopliae (var. Green muscle Locust, grasshoppers South Africa
acridum) Green muscle Locusts China
PFR-97 Whitefly USA
Isaria fumosoroseus Pae-sin Whitely Mexico
Fumosil Aphids, Thrips, Colombia
mealybugs whiteflies
Baeuvaria brongniartii Betel Scarab beetle larvae France
Nomuraea rileyi Numoraea 50 Lepidoptera Colombia
Hirsutella thompsonii Mycohit Acari India
Conidiobolus thromboides Vektor 25SL Aphids/Thrips/whiteflies South Africa
Lecanicillium longisporum Vertalec Aphids Netherlands
L. Muscarium Mycotal Whiteflies/Thrips Netherlands
Verticillin Whiteflies, Thrips, mites Russia
B. bassiana; M. anisopliae; I. Tri-sin Psyllid Mexico
fumosorosea
Aschersonia aleyrodis Aseronia Whiteflies Former
USSR
B. bassiana; M. Anisopliae; N. Micobiol Mites, beetles, bugs, Colombia
rileyi I. fumosorosea; Bacillus Completo aphids, flies, locusts
thuringiensis
virulenta for control of whiteflies and N. rileyi for management of Lepidoptera in

Colombia (Shah and Goettel 1999; Alves et al. 2003), P. lilacinus against plant para-
sitic nematodes in Australia (Copping 2001) and Hirsutella thompsonii for control
of mite species (Kumar and Singh 2001; Copping 2004).
5.6 Genetic Modifications to Enhance Virulence
5.6.1 Augmenting Virulence
Owing to their eco-friendly approach and efficiency against agricultural pests, EF

have been developed as an alternative to chemical insecticides and a vital compo-
nent in biocontrol programmes around the globe. However, use has been marred by
low virulence and inconsistencies in performance against various pests, which in
turn has led to small market shares (Fang et al. 2012) being the prime reason the
environmental stresses (Lovett and St. Leger 2015). Traditional approaches to
improve pathogen efficacy are based on physiological manipulation or development
of better formulation and application strategies (Butt et al. 2016). On the other hand,
genetic engineering has given a new lease of life to these entomopathogens (Zhao
et al. 2016a), chiefly Ascomycetes, to significantly improve their virulence and
adaptations in adverse conditions prevailing in various habitats. Genetic engineer-
ing, in concert with a better understanding of fungal pathogenesis and ecology, has
provided a myriad of opportunities to improve the efficacy and thus cost-effectiveness
of mycoinsecticides by recuperating their tolerance to environmental stresses and
enhancing their virulence. These methods have resulted in the development of
strains displaying: (1) increased cuticular degradation and faster ingress into the
host; (2) engineered delimited insect host range; (3) rapid cessation of feeding and
paralysis via expression of insecticidal toxins; (4) increased resistance to abiotic
stress; and (5) ability to block transmission of human disease-causing agents.
Various fungi have been engineered to express insecticidal proteins or peptides
or by over expressing the pathogen’s own genes for virulence enhancement.
Functional domains from diverse genes of pathogenic fungi and other organisms
have also been matched and engineered for producing insecticidal proteins with
novel characteristics. Furthermore, fungal tolerance to abiotic stresses, especially
UV radiation, has been improved by introducing a photoreactivation system from an
Archaean and pigment synthesis pathways from non-entomopathogenic fungi
(Zhao et al. 2016b).
Genetic engineering to increase virulence has focused on reducing both lethal
conidial dosage and time to kill, while improving infection rates. Studying molecu-
lar mechanisms of fungal pathogenesis (primarily Metarhizium spp. and B. bassi-
ana), has opened new avenues while allowing many pathogenicity-related genes to
be characterized, and used as a resource for enhancing EF performance. For exam-
ple, notwithstanding insect cuticle that acts as a barrier, entomopathogens produce
170 T. Ahmad et al.
proteases and chitinases to overcome this obstacle and grow on insect pests. Under
normal regulation, expression of most of these genes is under tight control (Fang
et al. 2009a, b). Constitutively overexpressing the gene encoding the subtilisin-like
protease Pr1A increased the virulence of Metarhizium anisopliae towards Manduca
sexta the recombinant strain showed a higher performance in survival time towards
the insect as compared to the parent wild-type (WT) strain (St. Leger et al. 1996).
Similarly, constitutive overproduction of B. bassiana chitinase (CHIT1) also
resulted in improvement of virulence by 23% (Fang et al. 2005). Expression of M.
anisopliae Pr1A into B. bassiana also increased its killing efficiency (Gongora
2004). These examples illustrate that pathogenicity-related genes from one fungus
can be used to improve the virulence of other fungi. Likewise, these fungi need
carbohydrate sources for growth and proliferation in the haemolymph. As trehalose
is the main sugar in the insect hemolymph, EF must utilize it by secreting the
enzyme trehalase (Thompson and Borchardt 2003; Jin et al. 2015). Overexpression
of the acid trehalase gene ATM1 accelerated the growth of M. acridum in the hemo-
coel of locusts, reducing the number of conidia and causing 50% mortality (Peng
and Xia 2015). Therefore, modifying the way entomopathogens exploit hosts for
nutrition is another reasonable way to improve virulence with many possible
mechanisms.
The range of endogenous genes suitable for genetic engineering is likely to be in
high numbers, including species-specific toxin encoding genes, and gene required
to evade the host immune response (Fang and St. Leger 2010; Gao et al. 2011; Lin
et al. 2011; Wang and St. Leger 2006, 2007a, b). Prospectively, combining the avail-
able genomes from B. bassiana and the several Metarhizium species with robust
genetic manipulation technologies, might open doors for characterization of the full
range of pathogenicity and host-specificity-related genes (Fang et al. 2004; Fang
et al. 2006; Gao et al. 2011; Hu et al. 2014; Xiao et al. 2012; Xu et al. 2014). Wang
et al. (2011) demonstrated that transfer of an esterase gene (Mest1) from the gener-
alist Metarhizium robertsii to the locust specialist M. acridum enabled it to expand
its range ad potential to infect caterpillars.
Genome-wide analyses of horizontal gene transfer events in Fungi have revealed
that Metarhizium species acquired diverse genes from bacteria, archaea or even
arthropods, plants and vertebrates (Hu et al. 2014). For instance, sterol transporter
(Mr-NPC2a) which allows fungus to compete with host for the growth-limiting ste-
rols present in the haemolymph hace been acquired by horizontal gene transfer
(Zhao et al. 2014). This evolutionary event has been reproduced in B. bassiana
which lacks an endogenous (Mr-NPC2a) homolog, thus improving its pathogenicity
(Zhao et al. 2014). Inspired by this, virulence of B. bassiana was improved by trans-
genic expression of several insect molecules (Ortiz-Urquiza et al. 2015). Similarly,
expression of the M. sexta diuretic hormone (MSDH) considerably increased the
virulence of B. bassiana against various lepidopteran targets (e.g., M. sexta and
Galleria mellonella) as well as mosquitoes (Anopheles aegypti) (Fan et al. 2012a).
Expression of an inhibitory regulator of toll signalling pathway, a key immune-
related signal pathway, also increased B. bassiana virulence against two taxonomi-
cally different insect species, G. mellonella (wax moth) and adult Myzus persicae
(green peach aphid) (Yang et al. 2014). Hence, a conserved molecule among taxo-
nomically distant insect species can add to the EF virulence against various target
species. In addition, expression of the species-specific pyrokinin b-neuropeptide
from fire ants (Solenopsis invicta) increased the virulence of B. bassiana against fire
ants. However, it was ineffective against lepidopteran hosts such as G. mellonella
and M. sexta (Fan et al. 2012b).
To date, insect molecules that have been reported to increase EF virulence are
involved in five types of biological processes: sterol hemostasis, food digestion,
osmotic balance, immunity, and neural system. However, in theory, any biological
process in insects could be a potential target for disruption by mixing and matching
different insect molecules, thus making it possible to create more virulent fungal
strains with higher specificity against target pests.
Bacterial and viral pathogens of insects could benefit by additional toxins by
means of novel modes of action. Besides crystal proteins, Bt vegetative insecticidal
proteins having insecticidal activities have proven to kill a broad spectrum of lepi-
dopteran insects by lysing the midgut epithelium. Agrobacterium-mediated trans-
formation has progressed to a high state of efficiency in Metarhizium and Beauveria
spp. (Xu et al. 2014) while genome-wide functional screening using Agrobacterium
insertional mutagenesis in Metarhizium robertsii identified genes involved in sporu-
lation (Fang et al. 2010) and enhancement of virulence (Zhao et al. 2014).
5.6.2 Tolerance to Abiotic Stresses – UV Radiation and Heat
The efficacy of mycoinsecticides is often limited by their susceptibility to abiotic

stresses viz., UV radiation and temperature (Lovett and St. Leger 2015; Ortiz-
Urquiza and Keyhani 2015). Screening for increased growth has been effectively
used to identify stress tolerance and hypervirulence in UV-induced mutants of M.
anisopliae (Zhao et al. 2016b). However, genetic engineering has been used to
increase tolerance to environmental stresses such as UV radiations, producing lines
that reliably perform for longer durations in variable conditions and diverse
ecosystems.
UV radiation is the most challenging environmental factor for solar-exposed
mycopesticides that primarily damages DNA through the generation of chemical
modifications, most of which are cyclobutane pyrimidine dimers (CPDs) (Sinha and
Häder 2002). Expression of photolyases from highly UV-tolerant Halobacterium
improved the survivability of M. robertsii and B. bassiana by more than 30-fold,
maintaining virulence against Anopheles gambiae even after exposure to several
hours of sunlight (McCready and Marcello 2003; Fang and Leger 2012).
Besides directly damaging DNA, UV radiation elevates oxidative stress through
the production of reactive oxidative species (ROS) (Lesser 1996). Superoxide
dismutase (SOD) overexpression improves the ability of B. bassiana to detoxify
ROS, enhancing UV tolerance (Ying and Feng 2011). Expression of a tyrosinase
from Aspergilllus fumigatus activated the production of pigments in B. bassiana,
172 T. Ahmad et al.
thus increasing their conidial tolerance to UV radiation (Shang et al. 2012). The
dihydroxynaphthalene-melanin (DHN-melanin) synthesis pathway of Alternaria
alternata, that increases UV tolerance in many fungi, has been transferred to M.
anisopliae, resulting in doubling its tolerance to UV radiation, with increased toler-
ance to thermal stress (35 °C) and low water activity (Tseng et al. 2011).
Temperature extremes can also limit the effectiveness of pest control agents. As
with UV radiation, heat stress produces ROS and small heat-shock proteins (HSPs)
bestowing thermotolerance in many organisms. Overexpressing HSP25 in M. rob-
ertsii also increased thermotolerance and survival under heat stress (Liao et al.
2014). Exposing M. robertsii to continuous culture with increasing heat stress, pro-
duced thermotolerant variants (De Crecy et al. 2009). Consequently, tools of genetic
engineering and synthetic biology could offer ample scope to overcome the low
tolerance to abiotic stresses and low virulence that constrained the development of
biocontrol agents. This approach could generate an unlimited arsenal of anti-insect
proteins at pace with the accelerating discoveries of virulence genes and insect
vulnerabilities.
5.7 Conclusion and Future Prospects
Agricultural pests including arthropods, plant pathogens, weeds and other inverte-
brates together with ever changing environmental conditions pose serious threat to
crop production and dwindling crop yields, worldwide. Entomopathogenic fungi,
being naturally available biological control agents, play a key role in population
dynamics of pest populations while limiting their economic injury level. Various
species of EF have been applied to control pests in different countries and in diverse
environmental settings, while some commercial products acting as mycoinsecti-
cides have also been developed and registered. Nonetheless, compared to other
technologies and insect management techniques, use of entomopathogens and
mycoinsecticides for regulating pest populations is still in its infancy. Need of the
hour is to draw attention of Agricultural Scientists, Entomologists, Researchers,
Farmers, NGO’s, Goverment agencies and other stakeholders, who are directly or
indirectly involved, with a focus on extensive research, innovation, awareness, com-
patibility, availability and ecofriendly approaches.
A range of entomopathogens particularly in the Phylum Ascomycota such as
Metarhizium, Lecanicillium, Beauveria and Isaria are grown at large scale while
playing a role in IPM strategies. Mycoinsecticides involving M. anisopliae and B.
bassiana have been used to control a wide range of pests like mealy bugs, aphids
and thrips. Nonetheless, their utilization has not yet reached its zenith in view of the
fact that only 3% crop protection globally relies on entomopathogens, with an
annual increase around 10% (Patrick and Kaskey 2012). Therefore, paying attention
to ecofriendly entomopathogens principally in terms of formulations, strain selec-
tivity, stability in long term use and efficacy under field conditions, is of paramount
importance.
Future prospects of entomopathogenic fungi depend on ecological niches where

they dwell for better production and utility. Focus should be given to finding alterna-
tive methods for easy use, longer shelf-life and, most importantly, exploitation
towards broad spectrum pests. On the contrary, inadequate production of mycotox-
ins in some species, carcinogenic mycotoxicosis in non-target organisms and slow
effectiveness of conidia are some of the hindrances to EF application in IPM.
Nonetheless, to overcome this issue, strategies based on synergistic approaches and
genetic manipulation have been developed by researchers. Lethal chemical pesti-
cides in low doses have been combined with fungi to kill the pests, synergy applied
for example using imidacloprid and B. bassiana against the Colorado beetle
(Furlong and Groden 2001) and Spilarctia obliqua caterpillars (Purwar and Sachan
2006).
Likewise in genetic manipulation, improvements of pathogens have been
attempted through parasexual crossing and protoplast fusion as well as by conven-
tional mutagenesis. Even though great strides have been made in improving meth-
ods of production, formulation, and application, many promising innovations and
improvements are still possible. Production of “cocktails” especially combining
microbials from different classes of entomopathogens or even with chemical or
botanical antagonists, is only just beginning. Whatever strategies are contemplated,
they should be considered with an intention of not just replacing chemical pesticides
by EF, but contributing to the overall development of sustainable agriculture, horti-
culture, and forestry as well as the preservation of biodiversity in the long run.
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Chapter 6
Thermotolerance of Fungal Conidia
Flávia R. S. Paixão, Éverton K. K. Fernandes, and Nicolás Pedrini
Abstract Conidia of entomopathogenic fungi (EF) are the propagules most fre-
quently used in arthropod biocontrol programs. This anamorphic form is essential
for the infection process, including spore germination, penetration, vegetative
growth, conidiogenesis and dissemination. Most EF are mesophilic and can develop
between 10 and 40 °C, but optimal growth is between 25 and 35 °C. Abiotic factors,
especially temperature (high or low) can determine their viability, virulence and
success or failure of infection process. Temperature has the highest impact on
conidial stress inhibiting metabolic processes, such as decreased morphogenesis
during germination, protein denaturation and membrane disorganization. Several
studies show that some strains of Beauveria spp., Metarhizium spp., and Isaria spp.
exhibit conidial survival even when grown at high temperatures, indicating a rela-
tionship between conidial thermotolerance and their geographical isolation origin.
Moreover, the high variability in fungal thermotolerance is also dependent of the
culture media composition and growth condition. EF that grow at high temperatures
do not grow at low temperatures and vice versa. Moreover, when growth conditions
are not set at optimal temperatures, EF development is affected and their effective-
ness in biological control programs of arthropods is reduced. Thermal stress directly
impacts on fungal strains ability to target arthropods and their environmental activ-
ity performance. The screening for fungal strains with a higher thermotolerance and
the improvement on conidial formulations may aid in optimizing the conditions for
biocontrol agent application.
Keywords Temperature · Conidia · Germination · Biological control
F. R. S. Paixão · N. Pedrini (*)

Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), Consejo Nacional de
Investigaciones Científicas y Técnicas (CONICET)-UNLP, Universidad Nacional de La Plata
(UNLP), La Plata, Argentina
É. K. K. Fernandes
Instituto de Patologia Tropical e Saúde Pública (IPTSP), Universidade Federal de Goiás
(UFG), Goiânia, GO, Brazil

https://doi.org/10.1007/978-3-030-23045-6_6
186 F. R. S. Paixão et al.
6.1 Introduction
Entomopathogenic fungi (EF) are responsible for epizootics that often regulate
insect pest populations. The genera Metarhizium (Hypocreales: Clavicipitaceae),
Beauveria (Hypocreales: Cordycipitaceae), and Isaria (Hypocreales:
Cordycipitaceae) are the fungi most frequently used in biological control programs.
The asexual spore, named conidium, is an anamorphic and primary form essential
in the life cycle of many filamentous fungi (Osherov and May 2001). Mostly, ento-
mopathogenic fungal infection starts with the attachment of conidia to the insect
cuticle, and then progresses to conidial germination, penetration, vegetative growth
(as hyphal bodies), conidiogenesis and, finally, dissemination (Lacey et al. 2001;
Pedrini 2018).
Temperature is an abiotic factor that influences all fungal development stages,
from primary processes such as biochemical and cellular reactions in conidia to
overall infection and fungus-host interaction (Zimmermann 1982; Cabanillas and
Jones 2009) (Fig. 6.1). Generally, three thermal conditions are considered for fungal
development, i.e., low (below 11 °C), intermediate (11–28 °C) and high tempera-
tures (above 28 °C) (Vidal et al. 1997). Under this classification, EF are considered
mesophilic because they develop well between 10 and 40 °C, with optimal growth
between 25 and 35 °C (Crisan 1973; Fargues et al. 1997; Vidal et al. 1997; Dimbi
et al. 2004). Metarhizium rileyi (= Nomurea rileyi) presented optimum growth at
25 °C (Ignoffo et al. 1976). Similar studies with Metarhizium spp. strains other than
M. rileyi showed optimum growth between 11 and 35 °C (Ouedraogo et al. 1997),
whereas B. bassiana strains grew over a wider temperature range, from 8 to 35 °C,
exhibiting optimal growth at temperatures as low as 20 °C and as high as 30 °C
(Fargues et al. 1997).
Inhibiting
metabolic processes
Decreasing:
Inducing protein
Viability
denaturation
Morphogenesis
Stressful Germination
conidia
Temperature Host interaction
Inducing membrane
disorganization Virulence
Sporulation
Increasing
oxidative stress
Fig. 6.1 Effect of non-optimal temperatures for fungal growth in various processes of conidia life
cycle
6 Thermotolerance of Fungal Conidia 187
Thermotolerance is defined as the ability to withstand relatively hot (or cold)

conditions. Viability in thermotolerance above 40 °C was observed in several strains
of Metarhizium spp. (Hedgecock et al. 1995; Li and Feng 2009). However, the
growth of some strains of Beauveria spp. was inhibited after 2 h at 45 °C. On the
other hand, cold activity was reported for both Beauveria spp. and Metarhizium
spp., B. bassiana being able to grow at temperatures as low as 5 °C and some M.
anisopliae strains at 8 °C, due to their cold adaptation (McCammon and Rath 1994;
Croos and Bidochka 2001; Fernandes et al. 2008; Santos et al. 2011).
This physical factor is very important to regulate all development processes,
since the beginning of germination up to conidial sporulation (Edelstein et al. 2005;
Keyser et al. 2014) (Fig. 6.1). Thus, the screening of fungal strains thermotolerant
to high and low temperatures, together with the molecular and genetic characteriza-
tion, and the investigation on formulations to increase fungal performance are key
factors to distinguish strains with high potential to be used in biological control
programs of arthropods (Dillon and Charnley 1990; Fernandes et al. 2010; Oliveira
et al. 2018).
6.2 Thermal Characteristics and Geographical Origin
Interactions between different environmental abiotic factors, spore germination and

other physiological traits in fungi were early reported by Gottlieb (1950). EF are
ubiquitous in soils worldwide, from the Arctic to the tropics (Zimmermann 2007),
thus different thermal behavior in fungi isolated from different geoclimatic origins
and/or from diverse hosts might be expected. In fact, Isaria fumosorosea
(= Paecilomyces fumosoroseus) strains from tropical or subtropical origin (Cuba,
USA, India, Nepal, and Pakistan) demonstrated high tolerance to upper limits (opti-
mal development at 32–35 °C) than European strains (optimal growth at 25–28 °C).
Conversely, European strains isolated from temperate areas are able to grow at 8 °C
and show to be more tolerant to low temperature than those fungal strains originated
from tropical or subtropical regions (Vidal et al. 1997; Fargues and Bon 2004).
Some studies reported no thermal variability between B. bassiana strains iso-
lated from temperate or tropical areas from Europe, Africa, Asia and America
(Fargues et al. 1997, Devi et al. 2005, Rangel et al. 2005, Borisade and Magan
2014). Although M. anisopliae strains from the tropical region (Africa) were sus-
ceptible to germinate at 15 °C (Dimbi et al. 2004), M. anisopliae isolates from
Ontario (Canada) showed large variation in both growth rate and conidial produc-
tion at temperatures between 8 and 22 °C. This latter study shows that M. anisopliae
isolated from forested areas were cold-active, while the isolates from agricultural
areas showed an ability for growth at high temperatures and resilience to UV expo-
sure (Bidochka et al. 2001).
Regarding cold activity, fungal strains isolated far from the equator presented
higher relative germination under cold conditions than strains originated from near
the equator. Beauveria bassiana isolated from higher latitudes were cold-active,
however, there was not a similar correlation for heat (Fernandes et al. 2008).
Thermal characteristics and geographical origins coincide with conditions during
natural epizootics between EF and hosts. Persistence in the environment indicates
certain adaptation as it was reported for Isaria spp., which showed to be well
adapted to semiarid region (Cabanillas and Jones 2009). Finally, the specific patho-
gen M. acridum, virulent against desert locusts, is able to tolerate temperatures up
to 42 °C, and thus to avoid the behavioral fever developed by host in an attempt to
stop the development of the fungus, i.e., a successful strategy to avoid infection by
thermosensitive EF such as B. bassiana (Elliot et al. 2002).
6.3 Culture Conditions and Conidial Thermotolerance
The production of conidia by EF is influenced mostly by culture conditions (viz.,

temperature, pH, water activity, aeration) and media composition (viz., carbon and
nitrogen sources, metal ions) (Hallsworth and Magan 1996; Ibrahim and Jenkinson
2002; Ying and Feng 2006; Rangel et al. 2008). Elevated temperatures reduce spore
viability, growth, germination and virulence (Anderson and Smith 1972; McCammon
and Rath 1994; Inglis et al. 1996; Rangel et al. 2010; Tumuhaise et al. 2018). It is
possible, however, to optimize both conidial production and thermotolerance by
efficient culture conditions and/or supply of culture media (Ouedraogo et al. 1997;
Cabanillas and Jones 2009; Kim et al. 2010c). Accordingly, Isaria spp. showed
greater tolerance (from 20 to 30 °C) after growing on Sabouraud Dextrose Agar
Yeast extract (SDAY) than fungi grown on Sabouraud Maltose Agar (SMA)
(Cabanillas and Jones 2009). Submerged cultures of I. fumosorosea grown in
Sabouraud Dextrose Broth (SDB) were able to develop well from 20 to 34 °C,
showing optimal growth at 28 °C. In solid fermentations, however, these strains
grew optimally at 25 °C (Esther et al. 2013). Although the excess of heat on conidia
causes unviability in the derived fungal propagules, it is possible to improve ther-
motolerance from 26 to 30 °C by increasing the sucrose content in the culture media
(McClatchie et al. 1994). Conidia of M. robertsii produced on potato, dextrose, agar
and yeast extract (PDAY) medium, containing low concentrations of salicylic acid,
demonstrated increased tolerance to heat (Rangel et al. 2012). Millet grain was used
as a substrate to produce conidia by B. bassiana and M. anisopliae, potentially
enhancing conidial thermotolerance of fungi grown on a massive production system
(Kim et al. 2011).
Metarhizium acridum grows on agar medium and produces both aerial and
microcycle conidia. Tolerance of both propagules was compared at 40–45 °C, show-
ing that microcycle conidia were more heat resistant than normal aerial conidia
(Zhang et al. 2010). Microcycle conidiation is defined as a process in which the
germination of spores directly produces the formation of conidia, without the inter-
vention of an intermediate mycelial growth. This microcycle conidiation can be
induced by manipulation of environmental conditions, especially culture conditions
that are stressful to fungi (Hanlin, 1994; Bosch and Yantorno 1999; Zhang et al.
2010; Jung et al. 2014).
A relationship between thermotolerance and hydrophobicity can be traced.
Employing a siloxane-mediated conidial collection method based on hydrophobic-
ity, it is possible to classify conidia from B. bassiana and M. anisopliae into two
groups with different thermotolerance (Kim et al. 2010b). Similar results were
observed for species with hydrophobic conidia such as B. bassiana, M. brunneum,
M. robertsii, M. anisopliae and I. fumosorosea, which were more thermotolerant
than species with hydrophilic conidia such as Tolypocladium cylindrosporum, T.
inflatum, Simplicillium lanosoniveum, Lecanicillium aphanocladii, Aschersonia
placenta and A. aleyrodis (Souza et al. 2014). The sugar content (types and concen-
trations) used as carbon source in culture media for conidial production may affect
both conidial thermotolerance and hydrophobin-like or formic-acid-extractable
(FAE) protein content (Ying and Feng 2004), (see Sect. 6.5).
6.4 Effect of Abiotic Factors on Conidial Germination
The interaction of abiotic factors (temperature, humidity, light, pH) are important to
germination, dispersion, and development of fungal conidia (Glare et al. 1986;
Jaronski 2010; Osherov and May 2001; Oliveira et al. 2018). Water is fundamental
to start conidial germination. However, the interaction between water and tempera-
ture can reduce the viability and/or limit conidial viability of some EF, e.g., B.
bassiana, M. anisopliae, I. farinosa and I. fumosorosea require humidity and opti-
mum temperature conditions to their development (Hallsworth and Magan 1999;
Devi et al. 2005; Borisade and Magan 2014).
Humidity and temperature are key factors in activation of metabolic pathways
allowing the nutrients mobilization required for conidial germination, but are also
important during storage periods, longevity and persistence of quiescent conidia
(Daoust and Roberts 1983; Dillon and Charnley 1990, Bouamama et al. 2010).
These factors also affect physiological interactions between the host and pathogen
(Walstad et al. 1970; Luz and Fargues 1997; James et al. 1998). The ultraviolet
(UV) radiation (UV-A and UV-B) also affects conidial germination of M. acridum
and M. robertsii, limiting entomopathogenic fungal development, however increas-
ing thermotolerance by accumulating trehalose and mannitol (Braga et al. 2001;
Pereira-Junior et al. 2018; Rangel and Roberts 2018).
6.5 Thermal Effects and Metabolic Processes
EF have mechanisms to overcome and circumvent thermal effects (Rangel et al.

2010; Tseng et al. 2011; Keyser et al. 2014), triggering signal transduction and
metabolic pathways that synthesize the molecules that will ultimately protect the
fungal cells from damage caused by heat exposure (Farrell and Rose 1967; Ying and
Feng 2004; Zhang et al. 2011; Liao et al. 2014; Wang et al. 2017, 2018). Among
them, fungal proteins of hydrophobic nature associated to cell walls are often linked
with thermal protecting functions. Hydrophobins are small proteins important for
fungal growth and development (Wösten and Vocht 2000). Hydrophobin-like or
formic-acid-extractable (FAE) proteins were studied in aerial conidia of B. bassiana
and I. fumosorosea based on thermotolerance. FAE proteins provide hydrophobicity
to conidia, exhibiting different composition between B. bassiana and I. fumosoro-
sea. For both fungi, conidial viabilities decreased after exposure to heat stress
(48 °C for up to 150 min), perhaps as a result of different conidial structure related
to FAE proteins (Ying and Feng 2004). Zhang et al. (2011) characterized structur-
ally the cell wall carbohydrates in B. bassiana, and demonstrated that targeted gene
knockouts lacking β-1,3-glucanosyltransferase destabilize the cell wall and
decreased germination after 1 to 4 h of heat shock at temperatures >40 °C.
Trehalose is a disaccharide that accumulates in fungi during stress situations,
such as adverse growth conditions, heat, and hyperoxidative shock. Thus, along
with other polyols these molecules are known as stress metabolites (Van Laere
1989; Fillinger et al. 2001; Liu et al. 2009). Polyols accumulation is associated with
thermotolerance by helping in the stabilization of structure (and function) of pro-
teins and enzymes at high temperatures (Kim and Lee 1993). Accumulation of glyc-
erol, erythritol, arabitol, mannitol, and trehalose in conidia of M. anisopliae, B.
bassiana, and I. farinosa under different culture age (up to 120 days), temperature
(5–35 °C) and pH (2.9–11.1) were reported by Hallsworth and Magan (1996). Also,
high accumulation of both trehalose and mannitol were observed in abiotic stressed
conidia of M. acridum, suggesting they are part of a mechanism that the fungus uses
to attain its high tolerance to UV-B radiation and heat (Rangel and Roberts 2018).
The heat stress also triggers the production of toxic reactive oxygen species
(ROS), favoring oxidative stress in fungal propagules (Zhang and Feng 2018).
Catalase is an antioxidant enzyme characterized in B. bassiana (Pedrini et al. 2006)
that showed to be an important regulator of conidial thermotolerance (Wang et al.
2013). The relationship between oxidative stress and elevated culture temperature
also was reported for Aspergillus niger (Bai et al. 2003). The heat shock proteins
(HSPs) are also associated with tolerance to heat: overexpressing the gene encoding
for HSP25 in M. robertsii increased fungal growth under heat stress either in
nutrient-rich medium or on insect wings, and also enhanced the tolerance of heat
shock-treated conidia to osmotic stress (Liao et al. 2014).
6.6 Conidial Formulation and Thermotolerance
Formulations preserve the viability of conidia exposed to environmental stresses,

improving the efficiency of fungal propagules in microbial control (Faria and
Wraight 2007). Conidial formulations based on oil or oil-in-water emulsions are
investigated because the combination of conidia with oils improved their
performance against heat stress (Malsam et al. 2002; Mendonça et al. 2007; Barreto
et al. 2016; Paixao et al. 2017; Oliveira et al. 2018).
Oil-based formulations of M. anisopliae s.s. were used to improve both germina-
tion and appressorium production in conidia used for tick control (Barreto et al.
2016; Alves et al. 2017), and also to protect conidia against the effect of solar radia-
tion (Alves et al. 1998) and high temperatures (McClatchie et al. 1994). Conidia of
M. anisopliae s.l. and M. robertsii formulated on either vegetable or mineral oils
were more tolerant to heat stress than those either unformulated or formulated on
carboxymethylcellulose gel (Paixao et al. 2017). M. anisopliae and B. bassiana
viability also increases when fungi are formulated in emulsifiable oil (Oliveira et al.
2018), and vegetable oil improved both performance and thermotolerance of
B. bassiana (Kim et al. 2010a). Thus, formulation is considering a very important
tool to manage heat stress on conidia.
6.7 Conclusion
Temperature is a key factor that limits survival of entomopathogenic fungal conidia

used in biological control programs. As detailed in this chapter, most of the investi-
gations in this area have concentrated in: (i) fungal screening for thermotolerance,
based on geographical origin of the strains (McCammon and Rath 1994; Morley-
Davies et al. 1996; Fargues et al. 1997; De Croos and Bidochka 1999; Devi et al.
2005), (ii) test of tolerance to low or high temperature (Fernandes et al. 2008; Paixão
et al. 2017), (iii) appropriate culture medium for conidial production (Hallsworth
and Magan 1999; Cabanillas and Jones 2009; Esther et al. 2013), (iv) formulations
to increase conidia thermotolerance and protection (Hedgecock et al. 1995; Barreto
et al. 2016; Paixão et at. 2017), and (v) biological/molecular characteristics and
mechanisms that mediate stress tolerance (Liu et al. 2009; Fernandes et al. 2010;
Rangel et al. 2018). On the basis of the literature available, we can conclude that EF
are promising tools against many arthropods (Zimmermann 2007; Faria and Wraight
2007). However, additional research is still needed mostly in both screening of ther-
motolerant strains and formulation types of fungal propagules, to circumvent the
negative effects of abiotic factors that potentially limits their efficacy, thus improv-
ing the use of EF in biological control programs.
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Chapter 7
Oxidative Stress in Entomopathogenic
Fungi and Its Potential Role
on Mycoinsecticide Enhancement
Carla Huarte-Bonnet, M. Constanza Mannino, and Nicolás Pedrini
Abstract Entomopathogenic fungi (EF) are used worldwide as environmentally

friendly mycoinsecticides. A successful invasion process depends on the fungal
ability to cope with several stress factors, such as osmotic stress, temperature, UV
radiation, and oxidative stress. Reactive oxygen species (ROS) can appear due to
either previous environmental stresses or endogenous metabolic changes. Moreover,
ROS may be either part of the host defense against fungi or the fungus itself can
release ROS in the hemolymph to overcome insect defenses. Regardless of its
source, fungi must mitigate ROS damage in their cells. Antioxidant response in
fungi involves the action of enzymes as well as non-enzymatic compounds.
Oxidative stress and antioxidant responses are known to have several direct and/or
indirect consequences in fungal adaptation. Nutritive stress produced by non-
preferred carbohydrate sources in conidia production can increase ROS scavengers
consequently enhancing UV tolerance. Additionally, growth in long chain cuticular
hydrocarbons triggers ROS production and antioxidant gene induction, leading to
more virulent conidia. Also, ROS can act as signaling molecules for cell differentia-
tion into new propagules such as microsclerotia and mycelial pellets that tolerate
desiccation and produce new infective conidia in the field. In this chapter we will
summarize ROS sources and antioxidant scavengers during conidial production and
fungal invasion into their hosts, and the beneficial consequences for stress tolerance,
virulence and cell differentiation that can arise from these initial drawbacks.
Keywords Beauveria bassiana · Metarhizium anisopliae · ROS · Stress tolerance

· Cell differentiation
C. Huarte-Bonnet · M. C. Mannino · N. Pedrini (*)

Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), Consejo Nacional de
Investigaciones Científicas y Técnicas (CCT La Plata CONICET-UNLP), Universidad
Nacional de La Plata (UNLP), La Plata, Argentina

https://doi.org/10.1007/978-3-030-23045-6_7
198 C. Huarte-Bonnet et al.
7.1 Introduction
Entomopathogenic fungi (EF) are currently used as biocontrol agents of a large

number of pests in agricultural systems, including aphids, beetles, grasshoppers,
moths, butterflies, termites, weevils, whiteflies (Lacey et al. 2015; Deshayes et al.
2017), as well as for arthropods of medical and veterinary importance, such as mos-
quitoes, kissing bugs, bed bugs, ticks and tsetse flies (Blanford 2005; Scholte et al.
2006; Pedrini et al. 2009; Fang and St. Leger 2012; Maniania and Ekesi 2013;
Barbarin et al. 2017). Unlike any other insect pathogen, EF do not need to be
ingested to invade their hosts since they can initiate the infection cycle by penetrat-
ing the insect cuticle. Infective fungal cells meet the host surface and subsequently
start a series of physical, molecular and physiological changes allowing adhesion to
the epicuticle, penetration through the cuticle into the insect haemocoel, coloniza-
tion and sporulation throughout the cadaver (Pedrini 2018).
In all those stages, fungi are exposed to both external and internal sources of
stress that can increase reactive oxygen species (ROS), e.g. singlet oxygen, perox-
ide ion, hydrogen peroxide, and superoxide anion, hydroxyl and peroxide radicals.
In these scenario, a hyperoxidant state in which ROS exceed the antioxidant capa-
bility of the cell can transiently appear, endangering the integrity of cellular compo-
nents such as DNA, proteins and lipids (Georgiou et al. 2006; Gessler et al. 2007;
Hernandez et al. 2010; Zhang and Feng 2018). Fungal antioxidant responses include
antioxidant enzymes and other antioxidant compounds, which have been reviewed
by Gessler et al. (2007). In this chapter we will summarize some ROS sources dur-
ing fungal infection and in vitro fungal production and its potential role in further
stress resistance, cell differentiation and virulence enhancement.
7.2 ROS During Fungal Infection
During host colonization, fungi must mitigate the effect of several stresses to rap-
idly and efficiently kill the target insect. Before penetration into the host, fungal
conidia are exposed to solar radiation and temperature fluctuations. Ultraviolet radi-
ation causes several changes in the cell. UV-B radiation mainly provokes DNA
modifications such as pyrimidin dimers and 6–4 photoproducts, whereas UV-A
induces the formation of ROS in the cell (Griffiths et al. 1998). Temperature fluctua-
tions can result in osmotic imbalance as well as free radicals generation (Ortiz-
Urquiza and Keyhani 2015). Penetrating inside the host is also challenging. Fungi
need to trespass the hydrophobic epicuticle and the protein-chitin procuticle to
reach the hemolymph. The epicuticle is a thin layer mainly composed of long chain
hydrocarbons (Pedrini et al. 2007). Alkane degradation by fungi involves ROS for-
mation in peroxisomes through β-oxidation, as well as antioxidant stress response
induction in Beauveria bassiana and other EF (Huarte-Bonnet et al. 2015). Once
inside the hemocoel, fungi must struggle with the host immune response as well as
7 Oxidative Stress in Entomopathogenic Fungi and Its Potential Role… 199
osmotic changes due to hemolymph solutes. Cellular and humoral insect immune
responses includes phagocytosis, pathogen encapsulation, induction of a variety of
antimicrobial peptides, lectins, and the prophenoloxidase cascade. The latter induces
the release of insect ROS into the cavity and melanin production, toxic to both
insect and fungi. Insect can also subject themselves to a behavioral fever by select-
ing environments warmer than their normal preference, to delay or inhibit fungal
pathogenicity (Lovett and St. Leger 2014; Ortiz-Urquiza and Keyhani 2015; Pedrini
2018). Subsequently, invading fungi use hemolymph substrates to produce hyphal
bodies until the nutrient availability is exhausted. At this stage, they return to starva-
tion, under osmotic and oxidative stress. Finally, the pathogenic fungi transit through
the host in the opposite direction and proliferate on the cadaver surface, being once
again vulnerable to environmental stress (Lovett and St. Leger 2014; Rangel et al.
2015).
Several studies indicate that antioxidant stress enzymes, as well as the protectant
compounds produced are crucial to the fungal fitness, virulence and tolerance to
stress. The catalase (cat) gene family of B. bassiana consists of catA (spore-
specific), catB (secreted), catP (peroxisomal), catC (cytoplasmic) and catD
(secreted peroxidase/catalase). Single deletion knockouts of these genes shed light
into their contribution to stress tolerance and virulence: BbcatP, BbcatA and BbcatD
are influential virulence factors against insect pest whereas BbcatA and BbcatD are
equally crucial in regulating conidial UV-B resistance, followed by BbcatB.
Moreover, BbcatA also acts as the most important regulator of conidial thermotoler-
ance and BbcatP is crucial to oxidative stress management (Wang et al. 2013a).
Superoxide dismutase gene (sod) single knockouts and knockdowns were also
constructed for five sod in B. bassiana (Xie et al. 2012; Li et al. 2015). Whereas all
transgenic strains were more sensitive to UV stress and less virulent, only Bbsod1,
Bbsod2, Bbsod3 and Bbsod5 knockout strains delayed germination, but none of
them showed differences in thermotolerance. Interestingly, Bbsod5 is involved in
oxidative stress response, more specifically in the fungal response to the host
immune defense after penetrating the cuticle. Additionally, six thioredoxins (trx),
four glutaredoxins (grx) and one glutathione reductase (glr) were disrupted in B.
bassiana. The six ∆Bbtrx mutants, ∆BbGrx3 and ∆BbGlr displayed one or more
phenotypic changes associated with the fungal biocontrol potential, i.e., conidiation
and germination, thermotolerance, UV-B resistance and virulence of their conidia
(Zhang et al. 2015, 2016).
Melanins present in Metarhizium spp. strains are involved in heat stress and UV
tolerance. In M. anisopliae, the laccase MLAC1 is involved in melanin biosynthesis
and contributes to conidial pigmentation, tolerance to abiotic stresses and pathoge-
nicity (Fang et al. 2010). Moreover, overexpression of antioxidant stress enzymes
improved EF biocontrol potential: Bbsod2 overexpression led to a significant
increase in superoxide dismutase activity, accompanied by increased oxidative and
UV stress tolerance, as well as an increased virulence against larvae of Spodoptera
litura (Xie et al. 2010). A transgenic strain of M. anisopliae overexpressing Macat1
and a B. bassiana transgenic strain overexpressing BbcatE7 showed increased resis-
tance to hydrogen peroxide, decreased germination times and improved virulence
against Plutella xylostella and S. exigua larvaes, respectively (Hernandez et al.

2010; Chantasingh et al. 2013).
7.3 ROS During Conidial Production
EF are produced and commercialized worldwide as mycoinsecticides. Production

and formulation processes are crucial to obtain fungal propagules that are more
virulent and more tolerant to stress (Faria and Wraight 2007; Jackson et al. 2010).
In this regard, several studies have changed nutritional conditions to enhance germi-
nation rates, tolerance to abiotic and oxidative stress, and virulence.
Conidia of M. robertsii produced in media without a carbon source, known as
minimal media (MM), were found to be more tolerant to UV-B radiation.
Additionally, fungi grown in MM supplemented with non-preferred carbon sources
such as arabinose, fructose, galactose, inositol, and lactose, also increased UV-B
tolerance, and germinated faster with superior cuticle adherence (Rangel et al. 2006,
2008). Fungal growth in absence of glucose is a nutritional stressor that triggers dif-
ferent cell responses, including apoptosis, macroautophagy, production of second-
ary metabolites and antioxidant stress responses alteration (Rúa et al. 2014). During
nutritional stress (and other stresses) trehalose accumulation in the cell was shown
to be increased (Rangel et al. 2015). Trehalose functions as a free radical scavenger
and protects cellular constituents from oxidative stress. Therefore, trehalose can
protect fungal cells from further oxidative stress, and its levels can be considered as
stress indicators. Thus, trehalose and other stress responses can provoke “cross-
protection” in the cell: some stress responses can be beneficial to overcome further
stress, since response pathways are common to different stress sources (Hallsworth
2018).
Since EF penetrate the wax-rich epicuticle, supplementation with insect-like
hydrocarbons has also been tested. Napolitano and Juárez (1997) first described the
ability of B. bassiana and M. anisopliae to degrade hydrocarbons. Isolates of B.
bassiana grown on MM supplemented with hydrocarbons were more virulent than
glucose-grown fungi (Crespo et al. 2002; Pedrini et al. 2009). Moreover, alkane-
grown cells showed peroxisome proliferation (Crespo et al. 2000), and an oxidative
stress scenario was triggered (Huarte-Bonnet et al. 2015). In Isaria fumosorosea,
conidia produced with hydrocarbons showed higher antioxidazing enzymes activ-
ity, lower germination times, increased resistance to exogenous hydrogen peroxide
and higher virulence against S. exigua (Ali et al. 2013).
The effect of different oxygen concentrations during production of I. fumorosea
conidia was described by Miranda-Hernández et al. (2014). Oxygen pulses during
fungal growth provoked greater germination rates and resistance to thermal and
osmotic stresses, as well as higher virulence against Galleria mellonera larvae. This
was presumably due to the induction of an antioxidative stress response, as catalase
activity in these conidia was increased. Furthermore, oxidative stress can also be
generated by exposing conidia to UV-A radiation, hydrogen peroxide
s upplementation in the culture media, as well as other oxidants such as menadione.

Rangel et al. (2011) induced cross-protection in M. robertsii conidia to other stress
conditions after exposure to visible light, including UV-A wavelengths. However,
not all oxidative stress generators are capable to induce tolerance against other types
of stress (Rangel et al. 2015).
7.4 ROS and Cell Differentiation
A hyperoxidant state in the cell is a transient condition that needs to be battled to

prevent harmful damage that could lead to the cell death. Cell differentiation in
eukaryotic microorganisms has been proposed as a process triggered by hyperoxi-
dant states, allowing cell isolation from molecular oxygen (Hansberg and Aguirre
1990). However, isolation from O2 also isolates the cell from water and other nutri-
ents, forcing the exploitation of endogenous sources to survive and maintain such a
condition (Georgiou et al. 2006).
In some filamentous fungi, cell differentiation can induce sclerotial biogenesis.
Sclerotia are macroscopic, pigmented aggregates that can survive long periods in
adverse environmental conditions. They have ben previously reposted in several
plant pathogenic fungi. In particular, oxidative stress has shown to be involved in
cell differentiation into sclerotia in Sclerotium rolfsii, Sclerotinia minor, Sclerotinia
sclerotiorum and Rhizoctonia solani (Sideri and Georgiou 2000; Georgiou et al.
2006; Papapostolou and Georgiou 2010a,b). EF, however, produce smaller sclerotia
known as microsclerotia (MS). Up to date MS formation was described in
Metarhizium anisopliae, M. acridum, M. robertsii, M. brunneum, M. rileyi,
Lecanicillium lecanii, Beauveria bassiana, B. brogniartii and B. pseudobassiana
(Jaronski and Jackson 2008; Jackson and Jaronski 2012; Wang et al. 2013b;
Mascarin et al. 2014; Song et al. 2014; Villamizar et al. 2018). These propagules
have been explored as potential propagules for biocontrol strategies since they can
tolerate desiccation and germinate without any exogenous carbon source, producing
new conidia in the field. These characteristics make MS promising propagules since
they can be formulated as solid granules, stored for long periods of time for later use
and produce great quantities of conidia once they are applied and spread (Jaronski
and Jackson 2008). To date, oxidative stress involvement in MS differentiation was
only studied in M. rileyi. First, upregulation of antioxidant enzymes during MS
formation was found by transcriptome analysis (Song et al. 2013). During culture
optimization, Fe2+ was found to promote MS formation (Song et al. 2014). Iron ions
are known to increase ROS concentration via Fenton reaction (Georgiou et al. 2006;
Song et al. 2013). Additionally, whereas exogenous menadione and peroxide hydro-
gen in the culture media induced MS biogenesis, ascorbic acid, a ROS scavenger,
inhibited MS formation (Liu et al. 2014).
It was recently described that alkane-cultured B. bassiana differentiated into
mycelial pellets, compact spherical aggregates previously described only in non-
entomopathogenic filamentous fungi (Huarte-Bonnet et al. 2018). These hyphal
aggregates tolerated desiccation, sporulated and produced viable conidia after rehy-
dration. They were found to be pathogenic against larvae of Tenebrio molitor and
Tribolium castaneum, making them also a promising propagule for mycoinsecticide
production. Electron microscopy imaging showed that cells forming the pellets had
high peroxidase activity, with peroxisomal proliferation. Several antioxidant
response genes encoding for catalases and superoxide dismutases were induced in
these pellets, compared to mycelia from glucose-grown fungi. These results sug-
gested that oxidative stress previously found in alkane-grown conidia (Huarte-
Bonnet et al. 2015) could have triggered B. bassiana differentiation into mycelial
pellets. Although not yet tested, as conidia produced by hydrocarbon-grown fungi
were more virulent than those produced with glucose, it is possible that conidia
produced after pellet rehydration would result more virulent as well.
7.5 Conclusion
Microbes are constantly under stress. Stress biology involves all microbial organi-
zational levels. Therefore, the study of stress is crucial to better understand and
successfully implement microorganism-based technologies (Hallsworth 2018). At
this regard, EF do not represent an exception. In particular, oxidative stress can
appear either as a direct or indirect consequence of ROS formation inside the cell,
due to other stresses, or ROS can be produced as metabolic subproducts. If ROS
accumulation is higher than the amount a fungal cell can handle, induction of anti-
oxidant enzymes and production of antioxidant compounds are activated to defend
the cell integrity. In some other cases, supposedly when antioxidant stress responses
are not sufficient, cell differentiation can be triggered to isolate fungal cells from
subsequent stress. If fungi can finally overcome stress, molecular and phenotypic
changes transformed formerly stressed cells into better pathogens, improving their
germination rates, tolerance to more harmful stress levels or to other stresses
sources, enhancing virulence against insects, or even forming new propagules more
tolerant to environmental stress and more suitable to be formulated and spread in
the field. In conclusion, if sublethal, stress apears as an useful improvement
stimulant.
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Chapter 8
Effects of Cytotoxic Factors Produced
by Entomopathogenic Bacteria on Insect
Haemocytes
Carlos Ribeiro and Amélia Vaz
Abstract Members of genera Xenorhabdus and Photorhabdus are entomopatho-

genic bacteria (EB) that associate with nematode hosts from the genera Steinernema
spp. and Heterorhabditis spp., respectively, providing them with entomopathogenic
services through exceptional physiological and metabolic mechanisms. Both sym-
biotic pairs infect and kill insects, with the bacteria contributing to host pathogen-
esis and death, supplying nutrition for the nematode from available insect-derived
nutrients. In turn, the nematode provides the bacteria with protection from soil
predators, access to nutrients, and a dispersal mechanism. These EB are able to
produce and secrete a broad range of cytotoxic metabolites, aimed not only at cir-
cumventing and disabling the insect’s complex web of humoral and cellular
defences, but also at killing it quickly, eliminating all potential competitors for the
newly acquired food source.
An overview of most studied compounds and of their impact on the immune
response effectors is provided, with emphasis on the cellular components of the
insect immune system. The different cell types found in the haemolymph of lepi-
dopterans are described and characterised, in comparison to the cell types recog-
nized in Drosophila.
Keywords Xenorhabdus · Photorhabdus · Immunity · Haemocytes · Cytotoxins ·

Cytolysins · Haemolysins
C. Ribeiro (*) · A. Vaz

Departamento de Biologia, Faculdade de Ciências e Tecnologia, Universidade dos Açores,
Ponta Delgada, Portugal

https://doi.org/10.1007/978-3-030-23045-6_8
208 C. Ribeiro and A. Vaz
8.1 Introduction
Insects are ubiquitous organisms, and a small but significant part of them are con-
sidered pests, despite the subjectivity of that classification, because what qualifies
as “pests” varies greatly from case to case. In general, this designation is reserved
for organisms that cause potential damage in several domains, namely to animal
and/or human health, to natural habitats and ecosystems, and agricultural crops,
wherein they cause a series of well-documented socio-economic losses.
The current review deals, primarily, with the effects of entomopathogenic bacte-
ria (EB) on the cellular immune responses of insect pests, when challenged with
two entomopathogenic nematodes (EPNs) living in soil, Steinernema carpocapsae
(Nematoda: Steinernematidae) and Heterorhabditis bacteriophora (Nematoda:
Heterorhabditidae), strictly associated with a monospecific natural symbiont from
the bacterial genera Xenorhabdus (Akhurst and Boemare 1988; Akhurst 1993;
Nishimura et al. 1994) and Photorhabdus (Boemare et al. 1992; Akhurst et al. 1996;
Szállás et al. 1997; Fisher-Le-Saux et al. 1999), respectively. Both genera comprise
Gram-negative motile rods from the Enterobacteriaceae family of γ-Proteobacteria.
These bacteria are carried in the nematode gut, where they find shelter from soil
stressors and protection from antagonists, namely telluric bacterial consortia and
bacteria present in the insect gut. In fact, neither has ever been isolated from soil
samples in the absence of their specific nematode host. These symbiotic pairs are
pathogenic, and capable of parasitizing and killing the larval stages of their hosts,
namely Lepidoptera, Diptera, Coleoptera, Orthoptera, Hymenoptera and Isoptera
(Laumond et al. 1979; Poinar 1979; Akhurst and Boemare 1990; Boemare 2002;
Khan et al. 2016).
EPNs from genera Steinernema and Heterorhabditis are among the most widely
known biological control agents, being often used in classical, conservative and
augmentative biological control approaches (Bedding 2006). In this symbiosis, the
eukaryote partner provides not only safe shelter, but also a mean of entering the
insect body cavities where the bacteria are released, first in the insect gut and then
in the haemocoel. Upon release, the bacteria start to produce and secrete (1) a large
spectrum of antibiotics and/or antimicrobial metabolites, in order to begin dealing
with the bacterial consortia in the insect gut, annihilating all the competitors, (2)
several toxins, secondary metabolites and enzymes, that help in the destabilization
of the intestinal epithelium and, once in the haemocoel, circumvent and inhibit the
humoral and cellular defence reactions of the insect, ultimately killing it, and (3)
bacterial enzymes, initiating the biotransformation of the macromolecular contents
of the insect cadaver, preventing its putrefaction and making available the nutrients
necessary for both nematodes and bacteria to develop and grow, completing their
life cycle (Forst and Nealson 1996; Forst and Clarke 2002). This interaction between
the nematobacterial complex and the insect is somewhere between host-parasite and
predator-prey. The penetration of the insect, coupled with the rapid multiplication of
the bacteria and nematodes once inside the larvae, are close to a parasitic behaviour,
while the speed with which the complex kills the victim evokes the actions of a
8 Effects of Cytotoxic Factors Produced by Entomopathogenic Bacteria on Insect… 209
predator (Ribeiro 2002). In the end, it must be pointed out that the bacterial symbi-
ont inhabits and influences the life cycles of two host animals, helping one to feed
and reproduce optimally in a controlled environment (the nematode), while killing
the other (the insect) (Herbert and Goodrich-Blair 2007).
As far as the host insects are concerned, they defend themselves against the nem-
atodes and their symbiotic bacteria with cellular and humoral innate immune
responses. The cellular responses against bacteria include phagocytosis and/or nod-
ule formation, whereas against EPNs these reactions are melanisation and encapsu-
lation. The major humoral response factors in lepidopteran larvae include the
synthesis of antimicrobial peptides (AMPs, e.g. cecropins, defensins and attacins),
lysozyme and phospholipase A2, and the prophenoloxidase/melanisation and coagu-
lation cascades.
8.2 Life Cycles of the Nematode-Bacteria Symbionts
The life cycles of the two nematobacterial mutualistic symbionts, Steinernema:

Xenorhabdus and Heterorhabditis: Photorhabdus are generally described as one,
despite the fact that there are four living entities involved, that the nematodes have
different life cycles and that the molecular components of the regulatory networks
controlling pathogenicity and mutualism in Photorhabdus and Xenorhabdus are
very different (Goodrich-Blair and Clarke 2007). Indeed, the life cycles of these
entities appear to be quite similar at the macroscopic level, with only slight differ-
ences, namely for the foraging strategies and reproductive cycles (Poinar 1975;
Johnigk and Ehlers 1999a, 1999b). However, at the cellular and molecular levels,
there are significant differences that are important to consider and study, both related
to the pecularities of the specific interactions of the symbiotic pairs, during mutual-
ism (Herbert and Goodrich-Blair 2007). They include symbionts evolution, leading
to two convergent life styles originated from two divergent genomes (Chaston et al.
2011), and the interactions with the insect preys during parasitism (Chapuis et al.
2009; Lee and Stock 2010; Stilwell et al. 2018). Furthermore, coevolution with the
symbiotic partners has led to a high level of mutualism specialization, which effec-
tively prevents horizontal transmission of bacterial symbionts between different
nematode species (Sicard et al. 2004a).
The free-living infective juvenile (IJ) nematode stages are soil-dwelling and not-
feeding, and protected by a double cuticle. They rely upon their energy reserves for
survival up to several months, while seeking their insect preys, using different for-
aging strategies that vary from ambush to cruise (Lortkipanidze et al. 2016). At this
point, the IJs harbour their bacterial symbiont either disseminated in their intestinal
lumen, in the case of Heterorhabditis: Photorhabdus (Boemare 2002) or, in the case
of Steinernema: Xenorhabdus, specifically in a vesicle (Bird and Akhurst 1983).
Once a suitable host is encountered, the nematode enters the insect via its natural
openings, namely the mouth and/or anus and, during this process, sheds its outer
cuticle (Poinar and Himsworth 1967; Sicard et al. 2004b). Nematodes may also
enter the body cavity via the tracheal system, provided that the aeropyles of the
spiracles are wider than their body diameter. Direct penetration of the insect cuticle
by Steinernema has never been described. However, Heterorhabditis is capable of
puncturing the larval cuticle, entering the host’s body this way (Bedding and
Molyneux 1982).
Once in the insect gut, and as they begin to pass through the ectoperitrophic
matrix and the intestinal epithelium on their way to the haemocoel, the nematodes
begin releasing the bacteria. During infestation of the cotton leafworm Spodoptera
littoralis larvae with EPNs harbouring GFP-labelled Xenorhabdus (Sicard et al.
2004b), the authors described in detail all the sequential steps of the parasitic path.
In particular, the visualization of Xenorhabdus in IJ nematodes provided new
insights on the stimuli triggering the bacterial release in the insect midgut. Moreover,
in the early stages of colonization, as the bacteria reach the haemocoel, living bacte-
rial cells were found in two places: in the connective tissues of the hematopoietic
organ, and in the haemolymph plasma. These results gave new insight into the
behaviour of Xenorhabdus during the interaction with host tissues. It seems that
these bacteria need to find a connective tissue to ensure stability for growing suc-
cessfully in the early steps of haemocoel colonization. It would be interesting to
investigate which putative molecular components present in the cellular matrix of
the basal membranes (e. g. laminin, collagen, fibronectin or other proteinaceous
and/or glycoproteinaceous materials) are the most suitable for bacteria to interact
with.
After the pair reaches the insect haemolymph and the nematodes release their
symbionts, the bacteria begin the task of overcoming the insect immune response
and killing it, by toxaemia and/or septicaemia, through the production and secretion
of an array of toxic molecules (Akhurst and Boemare 1990; Forst et al. 1997). After
the onset of generalized toxaemia and/or septicaemia, and the concomitant monoax-
enic colonization by the nematobacterial complex, the insect cadaver undergoes
substantial changes, starting with the digestion and biotransformation of the entire
molecular and tissue content, providing a rich nutritional environment for nema-
todes and bacteria to feed and reproduce. Ultimately, the massive amount of bacte-
rial biomass produced serves also as a source of nutrition for both organisms. In
fact, the bacteria grow exponentially in a way that makes it impossible to fit not only
in the cadaver, but also in the intestine of the next generation of nematodes and so,
it is assumed that a significant part of their biomass will also serve as nutrient con-
tent, after the stationary and declining growth stages are reached. In fact, Ribeiro
(1994, 2002) reported the presence of live Xenorhabdus rods alongside bacterial
spheroplasts and debris from dead bacteria in microscopic observations of greater
wax moth, Galleria mellonella, cadaver contents. Spheroplasts appear in the last
third of exponential growth in artificial medium (Akhurst and Boemare 2015), as
they do inside the insect cadaver.
In this nutrient-rich environment, the nematodes begin several rounds of repro-
duction cycles, sexually in Steinernema and hermaphroditically in Heterorhabditis
(Johnigk and Ehlers 1999a, 1999b). Concomitantly, with their growing numbers
being limited by nutrient scarceness, they successively stop their reproductive cycle
in the L3 instar larvae. At this stage, the nematode progeny receives uncharacterized
environmental cues in response to a signal, possibly nutrient deprivation or space
limitation (Popiel et al. 1989; Herbert and Goodrich-Blair 2007) which stimulate
the development of a new generation of IJs, that are then colonized by their symbi-
ont partner, before gaining the protection of a new double cuticle and emerging by
the thousands from the insect cadaver.
The success of the symbiotic pair entails a high degree of specialization, namely
in the specificity of the re-association and the high efficiency in vertical transmis-
sion, that are potentially under a strong selective pressure. In fact, in Steinernema:
Xenorhabdus, it has been found that an association with bacteria other than their
natural symbiont is clearly detrimental for the nematode’s fitness and only symbi-
onts that were cognate to the nematode were associated and transmitted (Sicard
et al. 2004a). Therefore, an effectively axenic environment in the insect cadaver is
paramount in order to exclude any bacteria other than the natural symbiont, since
this is the only suitable biological reservoir for the entomopathogenic symbionts to
complete their life cycles.
Looking at the bacterial symbiont life cycle, it can be divided into three separate
stages: infection, after the nematode enters the insect and the bacteria are expelled
from its gut into the insect gut and/or haemocoel; reproduction, after the insect is
killed and its biomass is converted by the bacteria into a nutrient-rich soup enabling
the growth and development of both organisms; and re-association, as the nema-
todes stop reproducing, and the bacteria regain shelter de novo in the nematode gut
before the pair emerges. To successfully complete these stages, the bacteria must
accomplish five distinct tasks: (i) within the nematode gut, they must keep a stabi-
lized sessile condition, in order to maintain a successful symbiosis, (ii) once in the
haemocoel, they must overcome the insect’s immune response and kill it, (iii) estab-
lish an axenic environment, (iv) produce nutrients from the insect cadaver to facili-
tate the development of the nematode and their own growth, and (v) colonize the
next generation of nematode IJs.
In the specific case of X. nematophila, three regulons (Lrp, LrhA and CpxR) are
thought to be responsible for the modulation of activities necessary for the fitness of
both symbionts in response to the shifting environment throughout the bacteria’s
life cycle. Their activation possibly responds to nutritional cues, expressing viru-
lence factors, degradative enzymes or nematode colonization factors accordingly
(Richards and Goodrich-Blair 2009).
LrhA is a LysR-type regulator heavily involved in the modulation of virulence,
flagellar motility and lipase activity through positive regulation of FlhDC. This is a
transcription factor controlling flagellar synthesis (Givaudan and Lanois 2000),
transcription and secretion through the flagellar apparatus of the XlpA lipase, sup-
porting Steinernema reproduction (Park and Forst 2006; Richards and Goodrich-
Blair 2010), and the expression of several virulence effectors such as the
cell-associated haemolysin, encoded by xhlA, and the secreted haemolysin, encoded
by xaxAB (Cowles and Goodrich-Blair 2005; Vigneux et al. 2007).
Lrp, the leucine-responsive regulatory protein encoded by lpr, is involved in all
stages of the X. nematophila life cycle. It regulates both pathogenic and mutualistic
host interactions, and is thought to be responsible for coordinating the so called

“feast or famine” adaptation to fluctuations in nutrient availability, found ubiqui-
tously in both bacteria and archaea (Yokoyama et al. 2006; Cowles et al. 2007). Lrp
positively regulates motility, antibiotic production, protease activity and the haemo-
lysins encoded by xhlA and xaxAB, as well as the production of LrhA and, by exten-
sion, of XlpA (Richards and Goodrich-Blair 2009). It negatively regulates the
expression of the nematode colonization genes nil, working synergistically with
NilR in their repression (Cowles and Goodrich-Blair 2006).
CpxR, the response regulator of the two-component system CpxRA, positively
regulates motility and production of LrhA and XlpA, as well as the expression of
the nil colonization genes, and is thought to have an opposite, negative effect on
antibiotic, protease and haemolysin activities (Herbert et al. 2007). Like Lrp, it is
implicated in mutualistic and pathogenic host interactions, with both of them
appearing to regulate the transition between hosts, via their opposed influence on
the nil genes associated with nematode colonization (Richards and Goodrich-Blair
2009). Lrp may aid in the repression of nil gene expression both in the early stages
of infection and during the reproduction stage, when they are not needed, as the
mutualistic relationship with the nematode host is interrupted and the bacteria are
engaged in a pathogenic relationship with the insect host. Once the insect is depleted
of nutrients, and transmission to a new host becomes beneficial, CpxR may then
activate nil gene expression, triggering the re-association process and therefore
inducing the shift from a pathogenic relationship to a mutualistic one.
8.3 Insect Immunity
The insects defence mechanisms can be classified into two broad strategies based on
their specificity. The first one relies on non-specific structural and passive barriers
such as the cuticle, designed to either keep the pathogens from entering the insect
body altogether or, if the pathogens are ingested and therefore already inside, to
prevent them from accessing the haemocoel through the gut wall and ease their
destruction. This action is carried out by means of the peritropic membrane that
lines the gut, and its physicochemical properties, respectively.
The second strategy, much more complex, is specific and comprises the immune
system as a whole, with its concomitant cellular and humoral immune responses,
that interact in a coordinated way to respond to the pathogen challenges. Insects lack
the adaptive immune defence mechanisms that vertebrates have. They instead rely
entirely on the so-called innate immune system. In fact, this is the most ancient and
common system of defence against microbes and parasites, and its elements have
been conserved throughout evolution in the animal kingdom. The innate immune
system in vertebrates and invertebrates is mediated by genes that are homologous or
very closely related (Hoffmann 2003; Lemaitre and Hoffmann 2007). Both utilize a
similar set of receptors, signalling pathways, transcription factors, humoral factors
and cell-mediated mechanisms, in the course of the immune response.
Although the cellular and humoral defence reactions are usually studied and con-
sidered separately in immunology, this division is somewhat artificial. The detection
of a pathogen elicits in vivo a large array of interconnected and synergistic defence
reactions in the immune-responsive connective tissue (i.e. haemolymph). The two
systems clearly overlap, as the immune response involves a combination of soluble
and cell-derived factors that are activated in conjunction (Theopold et al. 2002;
Jiravanichpaisal et al. 2006). In the case of Drosophila, these switches between
humoral and cellular responses in the course of the immune reaction are mediated
by cytokine growth-blocking peptides (Tsuzuki et al. 2014).
Depending on the final effector that attacks the foreign body, the innate immune
reactions are characterized as either humoral or cellular. The humoral factors include
activation of i) the prophenoloxidase/melanisation cascade, leading to wound heal-
ing and opsonisation of invading pathogens, ii) the coagulation cascade that immo-
bilize foreign bodies, and iii) the induction of immune proteins such as lysozymes,
lectins and AMPs (anti-bacterial and anti-fungal proteins). The cellular mechanisms
are the haemocyte-mediated immune responses, interconnected with and stimulated
by the previously mentioned humoral factors, and include coagulation, melanisa-
tion, phagocytosis of microbes and apoptotic tissues, nodulation and encapsulation
of larger particles, such as parasitoid eggs, protozoa and nematodes, and production
of reactive intermediates of oxygen and nitrogen.
8.3.1 Insect Haemocytes
Like Arabidopsis thaliana, Caenorhabditis elegans or Danio rerio, Drosophila

melanogaster is a model system, one that is extensively used for studies on the
development of metazoans. Most studies in immunology have been devoted to the
production of AMPs, and the reactions of Drosophila humoral immunity are now
well understood. Cellular immune reactions have not been explored so extensively,
although there is a growing tendency in studies investigating them, mostly being per-
formed on lepidopterans (Liu et al. 2013), making comparisons between Drosophila
and other insects increasingly necessary.
Unfortunately, while the bulk of haemocyte types (plasmatocytes, granulocytes,
oenocytoids and spherulocytes) are recognized and accepted in most insect species
(Lavine and Strand 2002; Ribeiro and Brehélin 2006), Drosophila emerges as a
peculiar exception in the class Insecta, as far as the cellular components of its hae-
molymph are concerned. Among the three main Drosophila haemocyte types men-
tioned in the literature, two of them, lamellocytes and crystal cells (Meister and
Lagueux 2003; Ribeiro and Brehélin 2006), seem to be present only in Drosophila
species. Furthermore, the third Drosophila haemocyte type, called plasmatocyte,
seems to be very different from its homonym observed in species belonging to other
insect orders, especially in Lepidoptera.
For these reasons, and in an effort to facilitate the comparison of results obtained
on cellular immune reactions, a thorough comparison between the Drosophila hae-
mocyte types and those described in Lepidoptera is in order, and changes in nomen-
clature are long overdue (Ribeiro and Brehélin 2006). In fact, the lack of
normalization in nomenclature has long been a standing issue, noted by more than
one author (Strand 2008; Hillyer 2016). Notwithstanding difficulties in reaching a
general agreement, there is a growing consensus and consistency in the homology
and nomenclature uniformity between the species of Lepidoptera and those belong-
ing to Drosophila (Brehélin and Zachary 1986; Ribeiro and Brehélin 2006; Lemaitre
and Hoffman 2007; Tsuzuki et al. 2014). In fact, there is actually a clear correspon-
dence between the morphology and physiology of the above mentioned insect hae-
mocytes and comparisons are possible with lepidopteran plasmatocytes, granulocytes
and oenocytoids, being homologous to Drosophila lamellocytes, plasmatocytes and
crystal cells, respectively (Ribeiro and Brehélin 2006).
8.3.2 tructural and Functional Analysis of Typical

S
Haemocyte types in Lepidoptera and their Corresponding
types in Drosophila
Insect haemocytes originate from mesodermal tissues, namely the hematopoietic

organs and the lymph glands, from where stem cells are derived. These differentiate
into specific haemocyte lineages identified by morphology, function, and molecular
markers (Lavine and Strand 2002). A summary of the accepted insect haemocyte
types and their respective functions is provided in Table 8.1.
Table 8.1 Most accepted insect haemocyte types for Lepidoptera and D. melanogaster, and
related specific functions
Haemocyte Type Main functions
Lepidoptera Prohaemocyte Haematopoietic stem cell
Plasmatocyte Spreading and adhesion, nodulation and encapsulation
Granular cell Adhesion, phagocytosis, coagulation, nodulation and
Granulocyte encapsulation
Oenocytoid Prophenoloxidase reservoir; wound cicatrisation,
cuticular melanization and sclerotization
Spherule cell Reservoir and transporter of components for cuticular
Spherulocyte renovation, pupae/chrysalis formation and silk
production for cocoons
Drosophila Prohaemocyte Haematopoietic stem cell
melanogaster Lamellocyte Spreading and adhesion; encapsulation
Plasmatocyte Adhesion, phagocytosis, coagulation, nodulation and
encapsulation
Crystal cell Prophenoloxidase reservoir; wound cicatrisation
8.3.2.1 Lepidopteran Haemocytes
Five main circulating haemocyte types have been recovered in all the lepidopteran
species studied to date: (i) prohaemocytes, (ii) plasmatocytes, (iii) granulocytes or
granular cells, (iv) oenocytoids and (v) spherulocytes or spherule cells.
(i) Prohaemocytes
Maintenance of circulating haemocytes in larvae of Lepidoptera has been attrib-
uted to both mitosis of haemocytes already in circulation and release from haema-
topoietic organs (Gardiner and Strand 2000). In all studied species, rare small
haemocytes, regular in shape and with a high nucleus-to-cytoplasmic ratio have
been reported. They are described as prohaemocytes, hypothesized to serve as stem
cells or immature haemocyte progenitors for one or more of the remaining types
(Grigorian and Hartenstein 2013). They are believed to be precursors of differenti-
ated haemocyte types. This interpretation, however, is supported mostly by cyto-
logical features and is controversial. Further work is needed before this hypothesis
can be confirmed. Additionally, different species of insects will likely show differ-
ences in haematopoietic organs and even in haematopoiesis as well (Liu et al. 2013).
(ii) Plasmatocytes
In vivo, plasmatocytes are spherical or oval cells (up to 20 μm long) with a regu-
lar shape, although they can sometimes appear spindle-shaped. In transmission
electron microscopy (TEM), they show few pseudopods, small clear vacuoles,
numerous polyribosomes and a moderate amount of rough endoplasmic reticulum
(RER). The Golgi apparatus is present, but is often little developed, and is devoid of
granules in most lepidopteran species. These cells always present pinocytotic vesi-
cles largely distributed in the cell membrane (Ribeiro et al. 1996; Ribeiro and
Brehélin 2006). In monolayers, after a few minutes incubation, plasmatocytes are
easily recognizable as they spread rapidly on contact with the glass slides, present-
ing a characteristic fibroblast-like morphology. They develop numerous pseudopo-
dia and long, wide lamellipodia. They show a large rounded nucleus, with small
scattered chromatin masses. It is widely recognized that plasmatocytes form the
bulk of capsules around foreign bodies too large to be phagocytised, or nodules
around masses of bacteria and necrotic melanised material, in vivo. Capsule and
nodule formations look identical at the cytological level (Ratcliffe and Gagen 1976,
1977; Lavine and Strand 2002), and in these formations plasmatocytes synthesize
numerous desmosomes and contain large amounts of microtubules in their cyto-
plasm (Ribeiro and Brehélin 2006). The role of plasmatocytes in phagocytosis is,
however, disputed. For some authors, they are phagocytes (Ratcliffe and Rowley
1975; Tojo et al. 2000; Ling and Yu 2006), but for others they are clearly not phago-
cytic cells (Akai and Sato 1973; Neuwirth 1974; Ribeiro et al. 1996; Beaulaton
1979; Ribeiro 2002; Ribeiro and Brehélin 2006).
(iii) Granulocytes or Granular Cells

After fixation upon haemolymph removal, lepidopteran granulocytes appear as
spherical (diameter from 5 to 8 μm), very refractive cells in phase contrast. In TEM,
they show a developed RER with enlarged cisternae filled with flocculent material,
numerous Golgi complexes and mitochondria, and sparse glycogen particles dis-
persed in the cytoplasm. Several thin and long pseudopodia/filopodia were often
visible. Similarly to plasmatocytes, granulocytes also possess numerous pinocytotic
vesicles in their cell membrane.
Three different kinds of membrane-bound inclusions have been described in
these cells (see Brehélin and Zachary 1986), namely vesicles containing dense gran-
ules or structured granules, both having a rather regular, rounded shape, and being
exclusively filled with materials synthesized by the Golgi apparatus. A third type of
vesicles resemble phagolysosomes, highly irregular in shape and filled with hetero-
geneous material, either originated from cytoplasmic areas in autolysis or from a
fusion between phagosomes and lysosomes containing hydrolases, synthesized by
the Golgi complex (Ribeiro et al. 1996). Inclusions of this type can become very
numerous and of large size (Raina 1976), for instance, at the end of the larval devel-
opment (Essawy et al. 1985; Ribeiro et al. 1996; Nardi et al. 2001, 2003).
Granulocytes, overloaded with large phagolysosomes, could be mistaken for spher-
ule cells on light microscopy.
One of the main functions of granulocytes is phagocytosis (Costa et al. 2005;
Ribeiro and Brehélin 2006). Granulocytes also represent the first cells to come into
contact, in small numbers, with a foreign body at the beginning of the capsule/nod-
ule formation. When in contact with a foreign body, they release their granular
content, another of their critical functions (Akai and Sato 1973; Ratcliffe and Gagen
1977; Schmit and Ratcliffe 1977; Ribeiro et al. 1996). According to most authors,
this exocytosis of typical opsonin-like material serves to attract plasmatocytes
(Gillespie et al. 1997), or at least to help plasmatocytes to build the capsule or nod-
ule (Pech and Strand 1996).
Granulocytes and plasmatocytes are recognized as defensive haemocytes in most
lepidopterans (Ribeiro et al. 1996; Brillard et al. 2001; Ribeiro and Brehélin 2006;
Costa et al. 2009). The former are involved in mediating phagocytosis and nodule
formation, whereas the latter act in the encapsulation process.
The pinocytic vesicles, clearly visible in the cell membranes of granulocytes but
also found on plasmatocytes, cannot go unnoticed, and studies on pinocytosis in
insect immunocompetent cells are needed. Pinocytosis is usually considered to be
constitutive, but it can also be a highly regulated, receptor-mediated process.
Depending on the molecular mechanism involved, pinocytosis can be divided into
four categories: caveolae-mediated, clathrin-dependent, macropinocytosis, and
dynamin- and clathrin-independent (Seto et al. 2002).
All of the abovementioned categories are involved in key signalling phenomena
in eukaryotic cells. It is reasonable to assume that membrane trafficking and signal
transduction are as important in insects as they are in mammals. Signalling path-
ways, namely those related to the presence of lipid rafts associated with c aveolae/
caveolin-dependent pinocytic vesicles, may interact and regulate components of the

membrane trafficking machinery in functions pertaining to the immune system,
such as recognition of foreign bodies and cellular and humoral responses in insects.
For instance, a large fraction of the cholera toxin can be endocytosed by clathrin-
dependent mechanisms, as well as by caveolae-mediated and clathrin-independent
endocytosis, in different cell types (Torgersen et al. 2001). Some bacterial patho-
gens have also been shown to exploit caveolin-1-rich membranes for infectious
entry into cells (Parton and Richards 2003).
Pinocytic vesicles seen in lepidopteran haemocytes (Ribeiro 1994; Ribeiro et al.
1996; Ribeiro 2002; Ribeiro and Brehélin 2006) resemble the caveolae that are
generally associated with lipid rafts and are described as being flask-shaped invagi-
nations present in the plasma membrane of many cell types. Caveolae have long
been implicated in endocytosis, transcytosis, and cell signalling. They have been
confirmed as directly involved in the internalization of membrane components such
as glycosphingolipids, extracellular ligands (e.g. folic acid or albumin), bacterial
toxins (e.g. cholera and tetanus toxins), and non-enveloped viruses, namely poly-
omaviruses. Unlike clathrin-mediated endocytosis, internalization through caveolae
is a triggered event that involves complex signalling (Pelkmans and Helenius 2002).
(iv) Oenocytoids
After fixation, oenocytoids appear as large cells (diameter up to 25 μm) rather
regular in shape, with variable refractivity in phase contrast microscopy, a low
nuclear to cytoplasmic ratio and an often eccentric nucleus. In TEM, the cytoplasm
is filled with numerous free ribosomes. Other typical cytoplasmic organelles are
poorly developed, especially the Golgi apparatus and the RER cisternae, which are
rare and very short, but often enlarged (Ribeiro and Brehélin 2006).
In Lepidoptera, prophenoloxidase, the precursor of the cascade responsible for
the melanisation reaction, is synthesized and stored within these haemocytes, and is
released into the haemolymph when these cells lyse (Neuwirth 1973; Essawy et al.
1985; Iwama and Ashida 1986; Ashida et al. 1988; Ribeiro 1994, 2002; Ribeiro
et al. 1996). This causes the characteristic darkening, due to the oxidation of phe-
nols to quinones, leading to spontaneous polymerization and to the formation of
insoluble melanin (Söderhäll and Cerenius 1998; Nappi and Ottaviani 2000;
Cerenius and Söderhäll 2004; Christensen et al. 2005). Activated prophenoloxidase
also plays an important role in cuticular melanization and sclerotization, during
larvae development (Marmaras et al. 1996).
(v) Spherulocytes or Spherule Cells
These are round cells, relatively stable in monolayers, containing a small number
of large inclusions (the spherules) that cause the cell to adopt an irregular shape. In
TEM, these inclusions show an internal structure of either lamellate concentric lay-
ers or a crystal-like lattice of dense particles, depending on the section (Ribeiro
et al. 1996). The exact functions of these cells aren’t currently known, but they are
considered as reservoirs and transporters of cuticular components. In G. mellonella,
spherule cells appear full of globular contents, becoming conspicuously empty of
their initial granules when the insects initiate silk production and pupation (Ribeiro
1994). In armyworm, Mithymna unipuncta, differential haemocyte counts during
the last instar larval stage showed a dramatic reduction of their numbers, with the
few present also devoid of sphere content (Ribeiro et al. 1996; 2002). Additionally,
cytochemical assays (Thiéry 1967) using the Periodic Acid-Thiosemicarbazide-
Silver Proteinate technique (PATAg) showed presence of polysaccharides in the
spherule inclusions (Ribeiro 1994). These results were in conformity with those
reviewed in Gupta (1991), Gupta and Sutherland (1967) and Ashhurst (1982), which
also pointed at the presence of neutral and acidic mucopolysaccharides, glycomuco-
proteins and glycosaminoglycan in the spherule content. Despite their eventually
different ontogeny, spherule cells participate in important physiological tasks,
namely those related to cuticle renovation and pupae/chrysalis formation and
cocoon silk production (Nittono 1960; Gupta 1991).
In insects, the cuticle, which is secreted by the epidermis, is a complex structure,
consisting primarily of an unbranched polymer of high molecular weight – an
amino-sugar polysaccharide composed of β(1-4) linked units of N-acetyl-D-
glucosamine – combined with lesser amounts of phenolics, lipids, waxes and miner-
als. The cuticle sclerotization is an irreversible process that darkens insect’s
exoskeleton and transforms the cuticular proteins in a water-insoluble matrix, via
the insertion of phenolic compounds and quinones and the deposition of melanin, in
a phenomenon known as tanning/darkening (Gullan and Cranston 1994).
Considering the underlying role of oenocytoids in the melanisation process
involved in sclerotization and the fact that spherule cells are transporters of cuticular
components, a close physiological and biochemical collaboration between these
two types of cells during the development, moulting and ecdysis of the insect larvae,
seem likely.
8.3.2.2 Drosophila Haemocytes
Drosophila has long been established as one of the best invertebrate models to study
haematopoiesis. As a result, the process of haemocyte formation in its embryonic
and larval stages is well documented (Mandal et al. 2007; Krzemien et al. 2007,
2010a, 2010b; Grigorian et al. 2011, 2013; Grigorian and Hartenstein 2013; Yu
et al. 2018). During the embryonic stage, haemocytes are produced by the head
mesoderm, whereas in the larval stage the lymph gland takes over this task (Lanot
et al. 2001; Holz et al. 2003). Since the circulating haemocytes in the adult flies have
been demonstrated to be either of embryonic or larval origin, adults were, until
recently, perceived as being incapable of haematopoiesis. However, Ghosh et al.
(2015) presented evidence that there are active haematopoietic centres in adult flies.
They house haemocytes from the two previously known lineages, in a network of
laminin A and pericardin. Moreover, they also harbour stem cells derived from the
larval lymph gland, capable of originating both crystal cells and fully functional
plasmatocytes. These clusters are akin to simplified versions of the vertebrate bone
marrow. This further reinforces the importance of Drosophila as a model for the
study of vertebrate haematopoiesis. There are in fact significant similarities between

the two processes, with conservation of signalling molecules, namely the phos-
phoinositol 3 kinase (PI3K) and GTPase Rac1 (Stramer et al. 2005; Wood et al.
2006), and the components of Toll, Imd, Jak/Stat and JNK pathways (Irving et al.
2005; Wertheim et al. 2005; Strand 2008).
Three main types of circulating haemocytes have been reported in Drosophila,
with a fourth being mainly described in the embryonic stage: (i) prohaemocytes, (ii)
lamellocytes, (iii) plasmatocytes and (iv) crystal cells.
(i) Prohaemocytes
Circulating cells, resembling lepidopteran prohaemocytes (and presumed to have
similar functions), are rare but present in Drosophila larvae, in particular during the
embryonic stage, likely as stem cells of the haematopoietic tissues (Brehélin 1982;
Lanot et al. 2001). Prohaemocytes rapidly differentiate to lamellocytes when insects
are immune-challenged by intruders, such as parasitoid wasps, and during meta-
morphosis (Lanot et al. 2001). Furthermore, invertebrate lymph glands contain
dividing stem cells or haemocyte progenitors called prohaemocytes (Grigorian et al.
2011, 2013; Grigorian and Hartenstein 2013).
(ii) Lamellocytes
These are large (25-40 μm), flat cells, that differentiate upon immune induction
and are rarely seen in healthy larvae (Brehélin 1982; Lanot et al. 2001). These hae-
mocytes exhibit the same characteristics as the lepidopteran plasmatocytes (Ribeiro
and Brehélin 2006). They have a regular shape, with very rare pseudopodia, lacking
cytoplasmic inclusions, and appearing as flat, thin and adhesive cells in monolayers.
They are recognized by reporters related to the Jun kinase signalling and L1 antigen
(Lanot et al. 2001; Asha et al. 2003). In their cytoplasm there are numerous polyri-
bosomes, whereas the Golgi apparatus and RER are poorly developed, with narrow
cisternae.
Like lepidopteran plasmatocytes, Drosophila lamellocytes form the bulk of cap-
sules and nodules, showing the formation of numerous desmosome-like junctions
(Russo et al. 1996) and microtubules (Rizki and Rizki 1979; Ribeiro and Brehélin
2006). Similarly, injected particulate material is phagocytosed in very low amounts,
if at all, by Drosophila lamellocytes (Brehélin 1982; Lanot et al. 2001), as is the
case with lepidopteran plasmatocytes (Costa et al. 2005).
(iii) Plasmatocytes
To add to the confusion in the nomenclature, the haemocytes that are called plas-
matocytes in Drosophila are not equivalent to the lepidopteran plasmatocytes, as
stated above. They have different histological and cytological features, different
behaviour in monolayers and different functions (Ribeiro and Brehélin 2006).
The Drosophila plasmatocytes are round cells that make up > 90% of circulating
haemocytes. They are strongly adhesive in vitro, and function as phagocytes that
engulf pathogens, dead cells and other entities, also participating in encapsulation
and in the production of AMPs (Brehélin 1982). Furthermore, they possess cell
membrane molecular markers that include the extracellular matrix protein peroxida-
sin and an uncharacterized surface marker called the P1 antigen (Nelson et al. 1994;
Asha et al. 2003). Although there is no true equivalent of lepidopteran granular
haemocytes in the haemolymph of Drosophila species, we must emphasize that the
cells called plasmatocytes in Drosophila look more similar to lepidopteran granular
haemocytes than to lepidopteran plasmatocytes. Like lepidopteran granular haemo-
cytes, circulating Drosophila plasmatocytes are spherical cells (diameter 5-8 μm),
with a developed Golgi apparatus and RER, enlarged cisternae filled with flocculent
material, numerous pinocytotic vesicles, thin pseudopodia and presence of more or
less numerous phagolysosome-like inclusions.
(iv) Crystal Cells
Crystal cells are non-adhesive haemocytes that comprise approximately 5% of
total circulating cells. They are considered equivalent to oenocytoids, due to high
similarities both in structure and function, but are distinguishable by the presence of
crystal-like cytoplasmic inclusions (Rizki and Rizki 1959; Nappi et al. 1995).
In phase contrast microscopy, D. melanogaster crystal cells are rather large with
a regular shape, an eccentric nucleus and irregular, sharp contour crystalline cyto-
plasmic inclusions. In other studied species of this genus, the above mentioned
inclusions are rounded and do not look like crystals, resembling the lepidopteran
oenocytoids instead (Rizki and Rizki 1980; Brehélin 1982). Crystal cells, like oeno-
cytoids express components of the prophenoloxidase cascade, the activation of
which leads to the formation of melanin (Rizki and Rizki 1959; Nappi et al. 1995).
8.4 The Bacterial Symbiont Arsenal
Xenorhabdus and Photorhabdus produce numerous secondary metabolites that play

different roles in their life cycles. These include not only several insecticidal and
antimicrobial compounds, but also other pathogenicity factors, namely cytotoxins
and cytolysins/haemolysins, as well as exoenzymes such as proteases and lipases.
These metabolites may be produced upon release into the host haemocoel in order
to suppress its immune response or actively circumvent it. This allows them to
quickly kill the insect during their growth phase, as well as to protect the insect
cadaver from competitors, afterwards. These may be saprotrophic organisms pres-
ent in soil or the insect own gut microbiota. Moreover, the metabolites convert the
host tissues into readily available nutrients to sustain the growth of both the bacteria
and the EPN.
8.4.1 Interacting with the Insect Host Immune System
There are several mechanisms by which both Xenorhabdus and Photorhabdus inter-
act with the insect host immune system. Although recognized by the insect immune
system, they suppress its humoral response by inhibiting the activation of its major
signalling pathways and response cascades, directly engaging and destroying the
cellular arm of the immune system (ffrench-Constant et al. 2007; Eleftherianos
et al. 2010a; Costa et al. 2010; Nielsen-LeRoux et al. 2012).
8.4.1.1 Recognition and Triggering of the Immune Reaction
The insect immune system is triggered when microbes are recognized by the host
cells, in a process mediated by pattern recognition receptors (PRRs), namely hemo-
lin, immunolectin-2 and peptidoglycan-recognition protein. These detect invariant
features of microorganisms called pathogen-associated molecular patterns (PAMPs)
(Seufi et al. 2012). PAMPs are, essentially, structural components of the bacteria
cell wall, such lipopolysaccharides (LPS), peptidoglycan, lipoteichoic acid or lipo-
proteins. Specific recognition of a wide array of PAMPs by the PRRs allows the host
to react to the pathogen triggering signalling responses (Medzhitov and Janeway
2002). Such a mechanism constitutes a very effective defence network against any
possible invading microorganisms.
Hemolins are bacteria-inducible immunoglobulin-like proteins acting as multi-
functional molecules involved in a diverse range of cell interactions. They are able
to: (i) bind lipopolysaccharides (Ladendorff and Kanost 1991; Daffre and Faye
1997; Yu and Kanost 2002); (ii) be upregulated during metamorphosis (Yu and
Kanost 1999; Lindquist et al. 2005); (iii) agglutinate bacterial cells (Schmidt et al.
2010); (iv) promote opsonin-like effects, such as increased cell adhesion and phago-
cytosis (Lanz-Mendoza et al. 1996; Bettencourt et al. 1997); (v) bind to haemocytes
and inhibit their aggregation (Kanost et al. 1994), and (vi) regulate embryonic
development (Bettencourt et al. 2000, 2002).
Lectins are carbohydrate-binding proteins, and imunolectin-2 is a C-type lectin
that is present at a constitutively low level in the larval haemolymph, recognized as
a potent protector from bacterial infection. Its synthesis was experimentally induced
after injection of Gram-negative bacteria or LPS, stimulating prophenoloxidase in
haemolymph (Yu and Kanost 2003).
After pathogen recognition, insects produce a plethora of AMPs that include
attacin, cecropin and moricin in Lepidoptera and metchnikowin, diptericin, droso-
mycin and attacin in Drosophila. The recognition of pathogenic Photorhabdus and
Xenorhabdus bacteria by PRRs, and the subsequently produced antimicrobials,
have been shown to slow down the otherwise rapid killing of the infected insects.
The genes encoding PRRs and AMPs are known to be transcriptionally induced at
higher levels after infection by EB. Accordingly, silencing by RNA interference of
the same genes results in an increase of the larval sensitivity to the pathogens
(Eleftherianos et al. 2006a, 2006b, 2010b). Despite the fact that the insects are
clearly able to deploy various immune responses towards pathogenic bacteria, these
systems are, in most cases, simply not effective enough. The bacteria, in the end,
almost always prevail as they have the necessary genetic and molecular strategies to
deal with, and dismantle the host response and escaping death (Costa, 2008).
8.4.1.2 Suppression of the Humoral Response
As far as the suppression of the insect immune system is concerned, the first step
appears to be the inhibition of the biosynthesis of eicosanoids (Eom et al. 2014;
Sadekuzzaman and Kim 2017; Sadekuzzaman et al. 2017a, b). Eicosanoids are a
group of oxygenated C20 polyunsaturated fatty acids involved in the activation of
various cellular and humoral immune responses, both in vertebrates and inverte-
brates (Park and Kim 2000; Park et al. 2004a, b, 2005; Kim et al. 2005; Shrestha and
Kim 2007; Stanley and Kim 2014). They are extremely important in mediating the
insect immune responses, being responsible for: (i) the increase of circulatory hae-
mocytes and their migration, (ii) the expression of AMPs and (iii) the release of
prophenoloxidase, the zymogen of phenoloxidase involved in the melanisation cas-
cade (Kim et al. 2018). These polyunsaturated fatty acids are synthetized from ara-
chidonic acid, which is in turn produced as the result of membrane lipid hydrolysis
by phospholipase A2 (Burke and Dennis 2009; Cao et al. 2013). By inhibiting the
production of arachidonic acid, several immune response pathways are directly and
indirectly impacted. The host immune system hence becomes severely compro-
mised, making phospholipase A2 an obvious target for any pathogen. This inhibition
is achieved, in the case of X. nematophila, by producing, at least, eight different
metabolites, that are synthetized at different stages of bacterial growth and provide
differential immunosuppressive activity (Eom et al. 2014). All of the tested com-
pounds produced significant results to varying degrees, but benzylideneacetone
(BZA), Pro-Tyr (PY) and oxindole stood out as the most effective. Oxindole pre-
vented phenoloxidase activity and BZA, which had already been reported to inhibit
aggregation, spreading and nodulation of haemocytes (Kwon and Kim 2008), was
shown to significantly inhibit haemocyte nodulation and, along with PY, exhibited
phospholipase A2 inhibitory activity and cytotoxicity against insect cells.
Another target for immunosuppression is phenoloxidase, a metalloenzyme that
has a crucial role in the invertebrate immune response, triggering the deposition of
melanin onto the cell surface of the invading pathogens, opsonizing them in order to
stimulate encapsulation, effectively isolating the intruder from the surrounding hae-
molymph (Gillespie et al. 1997). Xenorhabdus and Photorhabdus are able to cir-
cumvent this melanisation reaction by targeting different steps of its cascade,
through different mechanisms. For instance, Photorhabdus produces a hydroxystil-
bene compound that serves a dual function as antibiotic, inhibiting the growth of
microbial competitors in the insect cadaver, and as a potent inhibitor of activated

phenoloxidase (Eleftherianos et al. 2007). Rhabduscin, an isocyanide-containing
tyrosine derivative produced by both bacterial genera, is a virulence factor located
in the bacterium outermost layer. It acts as an inhibitor for phenoloxidase, mimick-
ing its substrate (Crawford et al. 2012). Another example are the rhabdopeptide/
xenortide peptides, a large family of compounds that are commonly produced by
Xenorhabdus and Photorhabdus. Given their high similarity with protease inhibi-
tors are likely responsible for disrupting the serine cascade that results in the activa-
tion of prophenoloxidase into phenoloxidase (Tobias et al. 2018).
Other compounds target the proteasome, whose main task is to degrade proteins
that are no longer needed for the normal cellular function, and may be implicated in
the removal and disabling of toxins and inhibitors produced by the pathogens during
infection.
Photorhabdus produces glidobactin and cepafungin, some of the more potent
proteasome inhibitors currently known (Stein et al. 2012). These compounds are
included, alongside syringolins, in the syrbactin natural product class (Groll et al.
2008). These bear a 12-membered dipeptide-macrolactam resulting from linkage of
a vinylogous amino acid and a modified lysine residue, a unique structure that con-
fers them particular chemical and biological properties (Krahn et al. 2011).
Xenorhabdins are dithiolopyrrolone derivatives, first isolated from Xenorhabdus,
that were described as having a significant antibacterial activity against Gram-
positive bacteria, but little effect against Gram-negative bacteria (McInerney et al.
1991a). These compounds are members of the dithiolopyrrolone group of bicyclic
antibiotics that have been isolated from Actinomycetes and Proteobacteria (Li et al.
2014). Thiolutin, one of the most well studied members of this group, has recently
been reported as a zinc-chelator, capable of inhibiting several metalloproteases,
including one involved in the proteasome (Lauinger et al. 2017).
8.4.1.3 Directly Engaging the Cell-Mediated Response Effectors
In their interactions with the cellular arm of the insect immune system, both
Xenorhabdus and Photorhabdus produce several different types of compounds that
directly target haemocytes, namely cytotoxins, cytolysins, haemolysins and prote-
ases, as well as other pathogenicity factors. For instance, Xenorhabdus also employ
specific morphological features that have been previously characterized and include:
i) a cytotoxic fimbrial structural subunit (MrxA), a pore-forming toxin that lyses
haemocytes (Banerjee et al. 2006); ii) a pilin subunit, secreted through outer mem-
brane vesicles (OMVs) with binding cytotoxicity (Khandelwal et al. 2004a) and
agglutinating properties against haemocytes (Khandelwal et al. 2004b); iii) lipo-
polysaccharides, which confer adverse effects on haemocyte functions (Giannoulis
et al. 2008), and iv) type 1 fimbriae, that exhibit agglutinating activity with sheep,
rabbit, and human erythrocytes and with haemocytes of G. mellonella (Moureaux
et al. 1995).
Type 1 fimbriae, filamentous appendages that are expressed by a variety of

Enterobacteriaceae, are mannose-sensitive haemagglutination (MSHA) factors
comprised of filamentous surface proteins that have been shown to facilitate bacte-
rial adherence to specific host tissues by carbohydrate binding to surface receptors
and to mannose-containing residues. They mediate the agglutination of erythrocytes
of many vertebrate species and contribute to the adherence of pathogenic bacteria to
eukaryotic cells (Sharon and Ofek 1986; Clegg and Gerlach 1987; Paranchych and
Frost 1988; Gerlach et al. 1989; Johnson 1991; Krogfelt 1991; Forst et al. 1997;
Duncan et al. 2005; Pizarro-Cerdá and Cossart 2006; Chandra et al. 2008). Several
other types of enterobacterial fimbriae exhibit mannose-resistant haemagglutination
(MRHA) and cell adherence, by recognizing either a carbohydrate moiety of a
membrane glycoprotein (or glycolipid) or a carbohydrate-conjugated mucosal pro-
tein (Graaf and Mooi 1986; Johnson 1991; Hultgren et al. 1993). Fimbriae are an
important virulence factor in enterotoxigenic Escherichia coli and uropathogenic
strains of E. coli and Proteus mirabilis, enhancing colonization of epithelial cells
(Gaastra and Graaf 1982; Johnson 1991; Massad et al. 1994; Li et al. 1999).
The global regulator Lrp has long been implicated in the regulation of fimbriae
production, being reported as both a positive and a negative regulator of the pap
operon in E. coli (Woude et al. 1996) and the fim gene cluster responsible for type 1
fimbriae production in Salmonella enterica (Baek et al. 2011). Lrp is a positive
regulator of the mrx operon in X. nematophila (He et al. 2004), further emphasising
the importance of this regulator in its motility and pathogenicity.
It has been demonstrated that X. nematophila, during growth in artificial medium,
produces different factors, assessed by chromatographic separation that, when incu-
bated, exhibited protease, cytolytic and/or haemolytic activities in vitro towards
midgut epithelium and haemocytes from: G. mellonella (Pyralidae), Agrius convol-
vuli (Sphingidae), Mythimna unipuncta (=Pseudaletia unipuncta) (Noctuidae),
Spodoptera litorallis (Noctuidae), Sl2b cell line from S. litorallis (INRA-France),
and sheep and rabbit erythrocytes (Ribeiro et al. 1999; Ribeiro 1994, 2002). The
same studies showed that FITC-labelled LPS from X. nematophila, E. coli and
Serratia marcescens did not exhibit any direct cytolytic or haemolytic effects
against any of the aforementioned types of cells and gut tissues. In fact, only plas-
matocytes showed slight adherence to the FITC-labelled cell membrane of E. coli,
with no induced mortality. Although LPS extracted from X. nematophila induce
lysozyme expression in the fat body of S. exigua, infection with live bacteria in the
same host suppresses expression of cecropin and other AMPs, probably through
inhibition of the eicosanoid pathway (Bae and Kim 2003; Ji and Kim 2004; Hwang
et al. 2013).
The fractions that exhibited protease activity targeted and degraded the midgut
epithelium and caused monolayers of haemocytes adherent to glass slides to become
unstuck, impairing their ability to recognize and adhere to foreign substrates,
whereas the fractions exhibiting cytolytic/haemolytic activities lysed all the afore-
mentioned cells types, with the exception of rabbit erythrocytes (Ribeiro et al.
1999). Massaoud et al. (2010) did not observe any correlation between the pathoge-
nicity of Xenorhabdus strains and their protease production, but there are indications
that they may indeed play a role in pathogenicity, being involved in the destruction
of AMPs (Eleftherianos et al. 2018).
Photorhabdus produces a serralysin-type metalloprotease, PrtA that targets and
selectively cleaves 16 haemolymph immune proteins in vitro, collectively named
PAT (PrtA target). Included in this group are the coagulation cascade effectors sco-
lexins A and B, proteins involved in recognition processes such as β-1,3 glucan
recognition protein 2, and proteins involved in immune signalling and regulation,
such as the haemocyte aggregation inhibitor protein (HAIP), six serpin-1 variants,
including serpin-1I, and serine proteinase homolog 3 (Felföldi et al. 2009).
The inactivation of serine proteinase homolog 3 has been demonstrated to sig-
nificantly reduce the ability of Manduca sexta larvae to overcome Photorhabdus
infection, due to the strong downregulation of multiple antimicrobial effector genes
and of the gene encoding prophenoloxidase (Felföldi et al. 2011).
Brillard et al. (2001) evaluated the haemolytic activity of several strains of
Xenorhabdus and Photorhabdus, through assays in sheep blood agar plates, liquid
haemolytic assays using culture supernatants and sheep and rabbit erythrocytes in
suspension, and lepidopteran haemocytes in monolayers. The P. luminescens strains
evaluated displayed different haemolysis patterns in the blood plates and, in the
liquid assays showed either no extracellular cytolytic activity against any of the cell
types, or only a weak activity against rabbit erythrocytes. Given the presence of
several putative haemolysin sequences in the P. luminescens genome, the authors
hypothesized that the lack of extracellular haemolytic activity observed could be
due to factors such as the need for these compounds to be processed like toxin com-
plexes or the fact that cytolysis may be contingent on contact between the bacteria
and the target cells. The X. nematophila F1 strain tested produced a halo of total
discoloration in the blood agar plates, and showed extracellular cytolytic activity
against all three cell types used in the liquid assays.
Two distinct and successive bursts of extracellular cytolytic activity were detected
during the in vitro growth of X. nematophila F1, corresponding to two growth stage-
specific extracts termed C1 and C2. These were predicted to represent two discrete
haemolysins regulated according to the growth phase. The C1 cytolytic activity
occurred when bacterial cells reached the stationary phase and the corresponding
extract exhibited haemolytic activity against sheep, but not rabbit, erythrocytes.
Both lepidopteran immunocompetent cells, known for their major role in cellular
immunity (Givaudan and Lanois 2000), were susceptible to C1 activity, with granu-
locytes being much more sensitive than plasmatocytes. Lysis by necrosis of the
haemocytes was preceded by a dramatic vacuolization of the cells, that appeared
swollen and with dilated endoplasmic reticulum vesicles. In contrast, the second
burst of cytolytic activity (C2) occurred late during the stationary phase and caused
haemolysis of rabbit, but not sheep, erythrocytes. C2 activity was effective against
granulocytes and plasmatocytes, with plasmatocytes being much more sensitive
than granulocytes. The affected cells appeared shrunken and devoid of lamellipodia,
with condensed chromatin evident in the cytoplasm. The C1 and C2 extracts were
predicted to represent two discrete haemolysins, regulated according to the
b acterium growth phase, with the former being flhD dependent and heat labile and
the latter being flhD independent and heat resistant (Brillard et al. 2001).
Ribeiro et al. (2003) reported the purification of a flhDC-dependent, heat-labile
cytotoxin responsible for the C1 cytolytic activity, a cation-selective Ca-independent
porin-like peptide called α-Xenorhabdolysin (αX), and demonstrated that the
plasma membrane of insect haemocytes and sheep erythrocytes were the primary
targets of this toxin. Electrophysiological observations indicated that the initial
effect of αX on macrophage/granulocytes plasma membranes was an increase of
monovalent cation permeability, sensitive to potassium channel blockers. As a result
of this increase in permeability, several events occurred intracellularly, such as
selective vacuolization of the endoplasmic reticulum, cell swelling, and cell death
by colloid-osmotic lysis. These effects, inhibited by potassium channel blockers,
were totally independent of Ca2+ cations. The size of the pores created by αX on the
plasma membranes of insect granulocytes and sheep erythrocytes increased with the
toxin concentration, leading to rapid cell lysis.
Later, Vigneux et al. (2007) reported the molecular characterization of αX as the
XaxAB binary cytotoxin, an α-pore-forming toxin (α-PFT) encoded by the xaxAB
genes. This was the prototype of a new family of hemolysins with both necrotic and
apoptotic activities in insect haemocytes and mammalian cells, and indicated that
homologues of the xax operon were present in the insect pathogenic bacteria P.
luminescens and Pseudomonas entomophila, as well as in the human pathogens
Yersinia enterocolitica and Proteus mirabilis. Waterfield et al. (2008) also identified
a XaxAB homolog as a potential virulence factor of P. asymbiotica in a rapid viru-
lence annotation screening. More recently, homologous cytotoxins were found in Y.
enterocolitica and named YaxAB (Wagner et al. 2013). Zhang et al. (2014) reported
on the cytotoxic and insecticidal activity of the XaxAB-like cytotoxin Plu1961/
Plu1962 from P. luminescens, termed PaxAB (ffrench-Constant and Dowling 2014).
Recent works describing the structures of YaxAB (Bräuning et al. 2018) and XaxAB
(Schubert et al. 2018) provided further insight into the mechanisms involved in the
formation of the pore complexes and the dynamics of the aggregation of the two
subunits.
Schubert et al. (2018) compared the available data and pointed out that, although
the protomer structures of YaxAB and XaxAB are very similar, there may be a
species-dependent size variability, since the YaxAB pore is comprised of 8 to 12
heterodimers, as opposed to the 12 to 15 heterodimers found in the XaxAB pore.
The proposed sequence of events leading to pore formation also differs between the
two cytotoxins. YaxA is hypothesized to enter the membrane first and then recruit
YaxB (Bräuning et al. 2018), whereas XaxA and XaxB first heterodimerize/oligo-
merize in solution, and then associate with the membrane as heterodimers or oligo-
mers (Schubert et al. 2018).
XaxAB is one of the two Xenorhabdus haemolysins directly regulated by FliZ, a
global regulatory protein that controls the expression of the master flagellar regulator
operon flhDC (Lanois et al. 2008; Jubelin et al. 2013), the other being the cell sur-
face-associated XhlA, encoded by the xhlBA operon and belonging to the two-
partner secretion system family (Cowles and Goodrich-Blair 2005). The first
member of the two-partner secretion system family to be characterized was the
Serratia marcescens haemolysin ShlA and its transporter ShlB, encoded by the
shlBA operon. ShlB belongs to the TpsB protein family and was shown to be essen-
tial to the secretion and activation of ShlA, which belongs to the TpsA protein fam-
ily. Homologues of the shlBA operon were also found in other Enterobacteriaceae,
such as P. mirabilis (hpmBA), Edwardsiella tarda (ethBA) and P. luminescens
(phlBA) (Brillard et al. 2002) and, more recently, X. nematophila (xhlBA) (Cowles
and Goodrich-Blair 2005). XhlA was reported to be cell surface-associated and
exhibited cytotoxic activity against rabbit and horse erythrocytes, and against both
immunocompetent lepidopteran haemocytes, with plasmatocytes being far more
sensitive than granulocytes. It was hypothesized that XhlA might be responsible for
the C2 activity reported by Brillard et al. (2001), since the observed biological
effects of XhlA were very similar to those reported for the C2 extract, in all the
comparable activities (Cowles and Goodrich-Blair 2005). However, the fact that
XhlA appears to be outer-membrane bound and FlhDC-dependent (Cowles and
Goodrich-Blair 2005), whereas C2 was clearly secreted to the extracellular medium
and was reported as being flhD independent (Brillard et al. 2001) makes it improb-
able that XhlA is the cytotoxin responsible for the C2 activity, hinting that there is a
still uncharacterized haemolytic agent being produced by X. nematophila.
The haemolytic activity of P. luminescens TT01 was evaluated and PhlA, a
homologue of XhlA, was identified and characterized (Brillard et al. 2002).
Haemolysis was reported in sheep and horse blood agar plates, but couldn’t be
attributed to PhlA since a phlA-null mutant still produced the same activity suggest-
ing a second, distinct haemolysin being produced by this strain. Haemolytic activity
was also reported in liquid haemolytic assays using exponentially growing bacteria,
incubated with horse erythrocytes. This activity was attributed to PhlA, since no
haemolysis was produced by the phlA-null mutant. The authors also reported that,
when filter-sterilized bacterial supernatants of the strains tested were used instead of
the cultures, no haemolytic activity was observed. These results are in accordance
with those obtained by Brillard et al. (2001) that reported that either no extracellular
cytolytic activity or only a weak activity against rabbit erythrocytes was observed
when using culture supernatants of Photorhabdus strains. This lack of activity in the
absence of bacterial cells could point to PhlA being cell membrane-associated, as
XhlA was reported to be (Cowles and Goodrich-Blair 2005), but a more exhaustive
study is needed before that conclusion can be drawn. Brillard et al. (2002) also
reported that PhlA was not a major virulence factor in the infection of S. littoralis
by P. luminescens, whereas XhlA was reported to be essential for full virulence of
X. nematophila against M. sexta (Cowles and Goodrich-Blair 2005).
8.4.2 Killing the Insect
Once the immune system has been neutralized, the pathogens start multiplying and
producing a plethora of virulence agents and insecticidal toxins, in order to quickly
kill the host. These toxins can be separated into four main groups (Rodou et al.
2010): i) toxin complexes (TCs), ii) “makes caterpillars floppy” toxins (Mcf), iii)
Photorhabdus insect-related proteins (PirAB) and iv) Photorhabdus virulence cas-
settes (PVC).
TCs are large multimeric protein complexes, some of which are highly toxic to
insects and destroy the larvae midgut both when injected and per os (Bowen et al.
1998; Waterfield et al. 2001; Silva et al. 2002). TCs were first described in
Photorhabdus and Xenorhabdus, being encoded by tc (Waterfield et al. 2001) and
xpt genes (Morgan et al. 2001), respectively. However, homologs are present in a
wide range of other pathogens (ffrench-Constant and Waterfield 2005). TCs com-
prise three subunits, each belonging to a different class of proteins, termed A, B and
C and grouped based on their size and sequence similarity (ffrench-Constant et al.
2007); biological activity depends on the presence of the three subunits (Lang et al.
2010; Sheets et al. 2011). The C subunit, consisting of a core domain, highly homo-
logue between all members of the C class, and a hyper variable region (Lang et al.
2011), was identified as the actual functional component of the toxin. It has an
ADP-ribosyltransferase activity and induces major changes on the actin cytoskele-
ton, which most likely cause the inhibition of haemocyte phagocytosis. Different
variants of the C subunit target different proteins, with TccC3 and TccC5 being well
document examples. The former modifies actin, largely disabling the monomeric
actin sequestering mechanisms that prevent its polymerization under normal cir-
cumstances, leading to the extensive clustering of actin, while the latter may cause
persistent activation of Rho GTPases, leading to the massive formation of stress
fibers. The effect of these two molecules is synergistic, as application of both caused
complete aggregation of the actin cytoskeleton, forming star-like clusters all over
the cells (Lang et al. 2010, 2011; Aktories et al. 2011).
TCs function differently from any other known toxins, forming discrete compart-
ments for protein unfolding and processing, and translocating the cytotoxic payload
in a novel way (Gatsogiannis et al. 2013; reviewed in Meusch et al. 2014). The A
subunit forms a translocation channel to which a cocoon formed by the B and C
subunits then attaches. The cytotoxic domain of C is then cleaved and translocated
through the pore formed by the A subunit into the cytoplasm of the host cell.
Mcf toxins, when injected, induce apoptosis in the insect’s midgut epithelium,
disrupting osmoregulation and leading to the characteristic loss of body turgor that
gives them their name (Daborn et al. 2002; Waterfield et al. 2003; Dowling et al.
2004, 2007). The Mcf1 toxin, a gut-active multidomain protein is taken up into
target cells by endocytosis. It induces apoptosis by means of a BH3-like domain.
BH3 is one of four specific regions of homology (BH1, BH2, BH3 and BH4) found
in the Bcl-2 family of proteins. These domains are critical in the interactions with
other apoptotic family members and regulatory pro- and anti-apoptotic proteins, and
play a pivotal role in their function as cell death regulators. BH3 is a potent cell
death mediator that triggers the mitochondrial pathway, releasing cytochrome c and
altering the membrane potential (Lutz 2000). Mcf1 also affects insect haemocytes
and appears to have an anti-phagocytic activity, causing unexpected alterations in
the actin cytoskeleton of host phagocytes, quickly paralysing them upon internaliza-
tion, effectively preventing cell migration and any further phagocytosis – “freezing”
phenotype. The mechanism by which Mcf1 affects the actin cytoskeleton may be
related to the inactivation of Rho GTPases, namely Rac (Vlisidou et al. 2009).
Binary proteins, that include PirAB, are toxic to some insects and destroy the
midgut epithelium of the larvae upon ingestion, causing the swelling and shedding
of the apical membranes normally seen in presence of other gut active toxins
(Duchaud et al. 2003; Waterfield et al. 2005; Blackburn et al. 2006). Also included
in this group are the Plu1961/Plu1962 cytotoxins (Zhang et al. 2014), that are highly
toxic to larvae when injected, causing necrosis of the midgut epithelium and are
quite similar to the XaxAB haemolysin from X. nematophila (Vigneux et al. 2007),
a pore-forming cytolysin (Ribeiro 2002; Ribeiro et al. 2003) causing necrosis and
apoptosis of insect haemocytes.
PVC are prophage-like loci predicted to encode 15-20 proteins, showing a differ-
ent putative effector sequence each. Despite the fact that the structure of PVC prod-
ucts is highly similar to that of known bacteriocins, they show no antibacterial
activity. Instead, the PVC-encoded effectors are toxic when injected into insect lar-
vae, and are responsible for a dramatic condensation of the actin cytoskeleton of
insect haemocytes, leading to cell death (Yang et al. 2006).
8.4.3 Settling in and Getting Rid of Competition
After successfully evading the insect’s immune system and producing enough tox-
ins to kill it, the next step is the production of hydrolytic enzymes to start converting
the insect biomass into a nutrient-rich soup, with metabolites that facilitate the
growth of both symbionts (Vizcaino et al. 2014). Antimicrobial compounds are
needed to keep other microorganisms (both present in the soil environment or living
in the insect gut or on the nematode cuticle), from proliferating in the cadaver and
competing with them for the readily available resources. To this end, Xenorhabdus
produces a veritable arsenal (Bode 2009), that fall into one of two categories
(Boemare et al. 1992): i) broad spectrum antibiotics (BSA) and ii) narrow-spectrum
bacteriocins (NSB). BSA are active against a wide range of microorganisms.
Foremost among them are xenoamicins, large hydrophobic depsipeptides active
against parasitic protozoa, without antibacterial activity (Zhou et al. 2013), xeno-
coumacins, benzopyranone derivatives that are highly active against Gram-positive
bacteria and also exhibit antimycotic activity against several fungal species
(McInerney et al. 1991b; Reimer et al. 2009), and PAX-peptides, that show antifun-
gal and antibacterial activity (Gualtieri et al. 2009; Fuchs et al. 2011).
NSB include the phage-derived xenorhabdicin (Thaler et al. 1995), and the coli-
cin E3-type killer protein xenocin (proteins involved in signalling, regulation,
pathogenicity, as well as in metabolism) produced by Xenorhabdus. NSB are active
mainly against bacteria from the same genus or closely related genera, likely pro-
duced not only to eliminate direct competition for nutrients (Singh and Banerjee
2008), but also to ensure the bacteria vertical transmission to the nematode progeny,
upon recolonization of the IJs, before the nematodes emerge from the insect cadaver
to search for a new host (Thaler et al. 1995).
Since Photorhabdus and Xenorhabdus have never been isolated from environ-
mental samples, it is presumed that their entire life cycles unfold within a biological
reservoir. Concomitantly, in their tripartite way of life (nematode gut - insect gut -
insect haemocoel) the EB display two different, perhaps opposed, life styles as
mutualistic symbionts and as parasite/pathogenic symbionts.
Insect guts present distinctive environments for microbial colonization, and the
bacteria naturally present in the gut potentially provide many beneficial services to
their hosts. They have also been shown to positively contribute to nutrition, protec-
tion from parasites and pathogens, modulation of immune responses, and commu-
nication (Engel and Moran 2013).
After leaving the nematode, the insect gut represents the first challenging sector
of their dual lifestyles. In fact, Xenorhabdus and Photorhabdus face a new task,
because the indigenous microbiota poses an obstacle to the colonization by non-
indigenous species, including pathogens, while also playing an important role in
resisting infection. Gut bacterial consortia adapt by the transfer of plasmids and
transconjugation between bacterial strains, and some insect species provide ideal
conditions for bacterial conjugation, suggesting that the gut is a “hot spot” for gene
transfer (Dillon and Dillon 2004). It is in this adverse environment that the EB will
be introduced, to be challenged by various detrimental conditions, which they have
to sense and adapt to, in order to prevail. To achieve this goal, several complex
mechanisms of sensing, signalling, and regulation have evolved in both genera
(Heermann and Fuchs 2008).
A comparative analysis between P. luminescens and Y. enterocolitica uncovering
candidate genes encoding proteins involved in signalling, regulation and pathoge-
nicity, as well as in metabolism, employed in the gut invasion and subsequent insect
exploitation of nutrients identified a set of factors shared by the two pathogens
involved in the host infection process, in persistence within the insect, or in host
exploitation. These results not only improved the understanding of the biology of
both pathogens, but also revealed some implications on the evolution of invertebrate
and vertebrate virulence factors.
Carneiro et al. (2008) reported a dramatic decrease in the numbers and viability
of natural enteric microbiota in the intestinal lumen of the lepidopteran Diatrea sac-
chralis, upon infection by Photorhabdus. Oral infection by Photorhabdus is known
to trigger gut immune responses in the host, namely the expression of nitric oxide
synthesis specifically in the gut, which prevents the pathogens from crossing into
the haemolymph via the gut wall (Eleftherianos et al. 2009). However, the efficacy
of these immune responses can vary dramatically and in a predictable way depending
on the stage of development and/or age of the insect. Age-related variation in immu-
nity in the adult stage has been frequently reported (DeVeale et al. 2004) and, in
fact, older insect larvae have a reduced immune capacity, which is marked by an
increased colonization of their gut (Eleftherianos et al. 2008).
Joshi et al. (2008) reported that X. nematophila secretes, through OMVs, an
insecticidal protein, a GroEl homolog that confers oral toxicity to larvae of
Helicoverpa armigera through gut membrane binding. This protein is made up of
three different domains exhibiting various levels of binding and insecticidal activity.
Similarly, Singh et al. (2013) reported the identification of the xenocin operon of X.
nematophila that includes the ximB and xciA genes encoding, respectively, the
42-kDa immunity protein (IP) and a 64-kDa xenocin, possessing an RNase catalytic
domain, with a specific intramolecular target in 16S rRNA (Zarivach et al. 2002),
responsible for its strong lethal effect on the competing microbes present in the
larval gut.
These antibacterial xenocins are coexpressed with periplasmic IP, which protects
Xenorhabdus from the lethal effects of their own xenocins. For this purpose,
McQuade and Stock (2018) reported that the xenocins may be exported from the
cytoplasm, through the cellular membrane, by the Sec translocase or Twin-arginine
translocation (Tat) pathways into the periplasmic space. In certain physiological
conditions, the external delivery of xenocins is processed by the type VI secretion
system (Singh et al. 2013; McQuade and Stock 2018).
Gram-negative bacteria use a well-known pathway to export products through
their OMVs (Beveridge 1999). Khandelwal and Banerjee (2003) demonstrated the
insecticidal potential of the outer membrane-associated proteins secreted by X.
nematophilus. These proteins exhibited in vivo oral cytotoxicity, targeting the gut of
neonate larvae and in vitro cytotoxicity to Sf-21 cells. The presence of chitinase
activity together with bacteriocins, adhesins, and pore-forming proteins in the
insecticidal multiprotein complex found in OMVs supports the role of this complex
in pathogenicity, as these proteins are known to mediate host-pathogen interactions
in other pathogenic bacteria. OMVs provide an efficient mechanism of protection,
transport and delivery of effector molecules within the larval host, not only in the
insect gut, but also in the haemocoel, where they target the haemocytes.
8.5 Conclusion
EB and their associated nematodes, acting as biological reservoirs and delivery vec-
tors to their insect pest targets, have great ecological and socioeconomic impor-
tance. Their genetic, molecular, cellular, metabolic, adaptive and co-evolutionary
features make these nematode-bacterial symbionts excellent models for understand-
ing the genes and molecular regulators that govern the fundamental biological pro-
cesses in hosts and pathogens alike.
Regardless of the end result, pathogenicity is a complex equation that balances
the strategies of both pathogens and hosts, the first striving to overcome, the other
Fig. 8.1 Schematic overview of the interaction between the symbiotic pairs Steinernema:
Xenorhabdus and Heterorhabditis: Photorhabdus and their hosts, with emphasis on the different
means by which the bacteria overcome and circumvent the insect immune system (Illustration cre-
ated with BioRender)
to defend, and both trying to maximize their chances of survival, managing their
mutual interactions and those with the surrounding environment. From the insect
standpoint, we addressed the immune response to the pathogens challenge, and elu-
cidated the structure and function of their immunocompetent cells, the haemocytes.
We dissected the different ways by which the EPN bacterial symbiont circumvents
and disables the insect immune system, rendering the various defence mechanisms
at work during infection all but useless (Fig. 8.1).
On the other side, looking at the life cycle of the EPN symbionts, we focused on
their tripartite life, in which the bacterium shows mutualistic and pathogenic facets,
modulating basic functions of the nematode host, facilitating its survival and, there-
fore, ensuring its own, ultimately influencing the insect response, only to over-
whelm it.
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Chapter 9
Effects of Entomopathogenic Nematodes
and Symbiotic Bacteria on Non-target
Arthropods
Ramandeep Kaur Sandhi and Gadi V. P. Reddy
Abstract The concerns over environmental risks of chemical pesticides and their
consequences for insect pest control have stimulated the search for alternative con-
trol measures such as biological control by entomopathogens. Nematodes of the
families Steinernematidae and Heterorhabditidae are biological control agents that
are non-toxic to humans, safer to environment than chemical pesticides, and can
easily combined with other control tactics. However, there has always been concern
regarding potential non-target effects of these parasitic nematodes. Only a few stud-
ies have examined the action of entomopathogenic nematodes on non-target organ-
isms and these cases have shown effects ranging from negligible to harmful. In
some cases, these nematodes can cause mortality to non-target organisms, usually
on a temporary and restricted area basis. We review herein the direct and indirect
effects of entomopathogenic nematodes on non-target organisms, from both labora-
tory and field studies.
Keywords Biological control · Entomopathogenic nematode · Steinernematidae ·

Heterorhabditidae · Non-target organism
9.1 Introduction
The continuous use of chemical pesticides for insect pest control has led to various
environmental problems including human illness, soil and water pollution, and
insecticide resistance in insect pests. Frequent use of some insecticides may increase
pesticide resistance in crop pests, increase the cost of crop production, and harm
non-target organisms, including the natural enemies of pest insects. The negative
effects of organophosphate and carbamate insecticides, for example, both on non-
target organisms in soil (Cockfield and Potter 1983, Floate et al. 1989) and aquatic
R. K. Sandhi · G. V. P. Reddy (*)

Department of Research Centers, Western Triangle Agricultural Research Center, Montana
State University-Bozeman, Conrad, MT, USA

https://doi.org/10.1007/978-3-030-23045-6_9
248 R. K. Sandhi and G. V. P. Reddy
environments (Hurlbert et al. 1972) have been well documented. Development of

integrated pest management (IPM) strategies with little or no reliance on chemical
pesticides has become an important goal for modern agriculture. One of the impor-
tant IPM strategies is to maximize the impact of biological control agents, such as
predators, parasitoids, and pathogens (bacteria, fungi, viruses, and nematodes).
Entomopathogenic nematodes (EPNs) (families Steinernematidae and
Heterorhabditidae) are the insect parasites that kill their hosts with the help of mutu-
alistic symbiotic bacteria (Xenorhabdus spp. and Photorhabdus spp. for steinerne-
matids and heterorhabditids, respectively). These symbionts are motile
Gram-negative bacteria in the family Enterobacteriaceae (Poinar 1990; Adams and
Nguyen 2002; Lewis and Clarke 2012). Steinernematidae and Heterorhabditidae
nematodes have the only one free-living stage, being the third juvenile stage, also
called as infective juveniles (IJs). This stage enters the host through natural open-
ings (mouth, anus, and spiracles), or in some cases, through the cuticle (Campbell
and Gaugler 1991). These infective juveniles carry their symbiotic bacterium in a
bi-lobed vesicle in the intestine (Martens et al. 2003). After entering the host’s hae-
mocoel, nematodes release bacteria into the host hemolymph, causing septicemia,
which kills the host in 24–48 h. Nematodes molt and complete up to three genera-
tions within the host body. Once the nutrient supply is depleted, IJs burst out of the
cadaver and search to find new hosts (Poinar 1990; Lewis and Clarke 2012). For
more details on EPN biochemistry see Chap. 8 of this Volume.
EPN’s have been investigated worldwide for the management of a wide range of
economically important pest insects because of their suitability for mass produc-
tion, ease of application, host-seeking ability, ability to rapidly kill their hosts, and
safety to mammals and other non-target organisms. They are also exempt from the
U.S. federal pesticide regulation, making commercialization feasible. However, due
to their sensitivity to UV radiation and unfavorable temperatures, EPNs have been
successful primarily for the management of soil-inhabiting pests and pests in green-
houses or other forms of protected cultivation (Georgis and Gaugler 1991).
However, as for any pest control tactic, concerns for potential non-target effects
need to be understood. The ecological impacts of field application of EPNs on non-
target arthropods is an important but relatively neglected topic. EPNs, which as a
group have wide host ranges, are more likely to affect non-target arthropods (insect
biocontrol agents such as parasitoids and predators). On the other hand some EPN
species have limited host ranges. However, the risk of EPNs to non-target species
under field conditions may be less than those implied by laboratory tests because of
the limited dispersal and lack of persistence of these nematodes (van Lenteren et al.
2003; Lynch and Thomas 2000). Here we discuss the potential of EPNs to cause
either direct or indirect harm to non-target organisms under laboratory or field con-
ditions. We also discuss future prospects for further work needed to avoid important
non-target impacts of EPNs.
9 Effects of Entomopathogenic Nematodes and Symbiotic Bacteria on Non-target… 249
9.2 Symbiotic Relationships of EPNs with Bacteria
Different species of EPNs (of Steinernema or Heterorhabditis) host different spe-

cies of symbiotic bacteria (Table 9.1) (Boemare (2002); Ganguly (2006); Stock and
Goodrich-Blair (2008); Plichta et al. (2009); Askary (2010); Tailliez et al. 2012;
Kuwata et al. 2013; Ferreira et al. (2014). The EPN life cycle consists of two stages
(Fig. 9.1): i) a free-living stage in the soil that carries the symbiotic bacteria in their
gut and searches for new insect hosts and ii) a parasitic stage in which infective
juveniles penetrate the host, release their bacterial symbionts, and reproduce
(Emelianoff et al. 2007). The symbiotic bacteria require the nematode species for
dissemination from one host to another (Akhurst and Boemare 1990; Boemare
2002), and in return nematodes rely on nutrients and metabolites produced by their
symbionts for growth and reproduction (Goodrich-Blair 2007). Also, nematodes
shelter the bacteria from unfavorable environmental conditions, and bacteria pro-
duces antibiotic and hydrolytic enzymes that degrade the insect body into a nutrient
soup that provides nutrition to the nematodes. The bacteria also release toxins that
prevent other pathogens or microorganisms from attacking tissues of the moribund
host. These toxins can be insecticidal, antifungal, or antibacterial that inhibit the
other pathogens to infect or colonize the hosts, thus making the host specific to the
EPNs (Khandelwal and Banerjee-Bhatnagar 2003). This association characterizes
the symbiotic relationship between nematodes and bacteria (Burnell and Stock
2000; Dowds and Peters 2002; Hazir et al. 2003).
Other than steinernematids and heterorhabditids, nematode species in several
other families (Phaenositylenchidae, Merminthidae, Sphaeririidae,
Tetradonematidae and Allantonematidae) are parasitic on insects (Stock and Hunt
2005). Members of these families have been recovered from insects belonging to
different orders (Coleoptera, Diptera, Thysanoptera, Lepidoptera, and
Hymenoptera). However, nematodes in these families do not have the mutualistic
bacteria found in steinernematids and heterorhabditids, and their inability to repro-
duce in artificial media make them of little use for pest control (Arthurs et al. 2004).
9.3 Bacterial Symbionts as Biocontrol Agents
Xenorhabdus and Photorhabdus, the bacterial symbionts of steinernematid and het-

erorhabditid nematodes, are gram-negative bacteria belonging to family
Enterobacteriaceae. They produce chemical compounds with antimicrobial, nema-
ticidal, insecticidal, and even anti-cancer activity (Webster et al. 2002). Xenorhabdus
species can infect species of both Lepidoptera (Ji and Kim 2004; Khandelwal et al.
2004; Banerjee et al. 2006; Kumari et al. 2013; Kalia et al. 2014) and Hymenoptera
(Dudney 1997) when injected artificially into their bodies in the laboratory.
Table 9.1 Bacterial species mutualistically associated with different EPN species
Entomopathogenic nematodes Bacteria
Steinernematidae
Steinernema affine Bovien; S. anatoliense Hazir, Stock and Xenorhabdus bovienii
Keskin; S. feltiae Filipjev; S. intermedium Poinar; S. jollieti Akhurst
Spriridonov, Krasomil-Osterfeld and Moens; S. kraussei Steiner; S.
litorale Yoshida; S. oregonense Liu and Berry; S. puntauvense
Stock, Uribe-Lorio and Mora; S. silvaticum Sturhan, Spiridonov
and Mrácek; S. weiseri Mrácek, Sturhan and Reid
S. bicornutum Tallosi, Peters and Ehlers X. budapestensis Lengyel,
Lang, Fodor, Szállás,
Schumann, and
Stackebrandt
S. riobrave Cabanillas, Poinar and Raulston X. cabanillasii Tailliez,
Pages, Ginibre, and
Boemare
S. diaprepesi Nguyen and Duncan X. doucetiae Tailliez,
Pages, Ginibre, and
Boemare
S. serratum Li X. ehlersii Lengyel, Lang,
Fodor, Szállás, Schumann,
and Stackebrandt
S. hermaphroditum Stock, Griffin and Chaenari X. griffiniae Tailliez, Pages,
Ginibre, and Boemare
S. karii Waturu, Hunt and Reid; S. monticolum Stock, Choo and X. hominickii Tailliez,
Kaya Pages, Ginibre, and
Boemare
S. abbasi Elawad, Ahmad and Reid; S. thermophilum Ganguly and X. indica Somvanshi, Lang,
Singh Ganguly, Swiderski,
Saxena, and Stackebrandt
S. scapterisci Nguyen and Smart X. innexi Lengyel, Lang,
Fodor, Szállás, Schumann,
and Stackebrandt
S. aciari Qui, Yan, Zhou, Nguyen and Pang X. ishibashii Kuwata, Qiu,
Wang, Harada, Yoshida,
Kondo, and Yoshiga
S. kushidai Mamiya X. japonica Nishimura,
Hagiwara, Suzuki, and
Yamanaka
S. scarabaei Stock and Koppenhöfer X. koppenhoeferii Tailliez,
Pages, Ginibre, and
Boemare
S. arenarium Artyukhovsky X. kozodoii Tailliez, Pages,
S. australe Edgington, Buddie, Tymo, Hunt, Nguyen, France, X. magdalenensis Tailliez,
Merino and Moore Pages, Edginton, Tymo,
and Buddie
S. carpocapsae Weiser; S. websteri Cutler and Stock X. nematophila corrig.
Poinar and Thomas
(continued)

S. cubanum Mrácek, Hernandez and Boemare; S. glaseri Steiner X. poinarii Akhurst
S. puertoricense Román and Figueroa X. romanii Tailliez, Pages,
S. siamkayai Stock, Somsook and Kaya X. stockiae Tailliez, Pages,
S. costaricense Stock, Uribe-Lorio and Mora; S. rarum de Doucet X. szentirmaii ehlersii
Lengyel, Lang, Fodor,
Szállás, Schumann, and
Stackebrandt
S. akhursti Qui, Hu, Zhou, Mei, Nguyen and Pang; S. apuliae Not known
Triggiani, Mrácek and Reid; S. ashiunense Phan, Takemoto and
Futai; S. asiaticum Anis, Shahina, Reid, and Rowe; S. longicaudum
Shen and Wang; S. leizhouense Nguyen, Qui, Zhou, and Pang; S.
loci Phan, Nguyen, and Moens; S. neocurtillae Nguyen and Smart;
S. pakistanense Shahina, Anis, Reid, Rowe and Maqbool; S. ritteri
de Doucet and Doucet; S. robustispiculum Phan, Subbotin,
Waeyenberge, and Moens; S. sangi Phan, Nguyen, and Moens; S.
sasonense Phan, Spiridonov, Subbotin, and Moens; S. sichuanense
Mrácek, Nguyen, Tailliez, Boemare, and Chen; S. tami Luc,
Nguyen, Spiridonov, and Reid; S. thannhi Phan, Nguyen and
Moens; S. yirgalemense Nguyen, Tesfamariam, Gozel, Gaugler,
and Adams
Heterorhabditidae
Heterorhabditis gerrardi Plichta, Joyce, Clarke, Waterfield, and Photorhabdus asymbiotica
Stock Fischer-Le Saux, Viallard,
Brunel, Normand, and
Boemare subsp. nov.
H. zealandica Poinar (South Africa strains) P. heterorhabditis Ferreira,
van Reenen, Endo, Tailliez,
Pages, Sproer, Malan, and
Dicks (strains SF41 and
SF783)
H. indica Poinar, Karunakar and David; H. marelata Liu and Berry P. luminescens subsp.
Akhurstii Fischer-Le Saux,
Viallard, Brunel, Normand
and Boemare subsp. nov.
H. bacteriophora Poinar (HP88) P. luminescens subsp.
Laumondii Fischer-Le
Saux, Viallard, Brunel,
Normand and Boemare
H. bacteriophora (Brecon) P. luminescens subsp.
Luminescens Thomas and
Poinar
H. bacteriphora (NC1); H. downesi Stock, Burnell, and Griffin; H. P. temperata Fischer-Le
megidis Poinar, Jackson, and Klein (Nearctic group); H. zealandica Saux, Viallard, Brunel,
Poinar Normand, and Boemare
(continued)

H. megidis (Palaearctic group) P. temperata temperata
Fischer-Le Saux, Viallard,
Brunel, Normand, and
Boemare
H. amazonensis Andaló, Nguyen, and Moino; H. baujardi Phan, Not known
Subbotin, Nguyen, and Moens; H. brevicaudis Liu; H. floridensis
Nguyen, Gozel, Koppenhofer, and Adams; H. Mexicana Nguyen,
Shapiro-Ilan, Stuart, McCoy, James, and Adams; H. poinari
Kakulia and Mikaia; H. taysearae Shamseldean, Abou El-Sooud,
Abd-Elgawad, and Saleh
Fig. 9.1 Life cycle of entomopathogenic nematodes
Similarly, species of Photorhabdus are virulent against a wide range of insects,

including species of Lepidoptera (Mohan et al. 2003; Mahar et al. 2005; Jallouli
et al. 2013); Coleoptera (Blackburn et al. 2005; Shrestha and Kim 2010),
Thysanoptera (Gerritsen et al. 2005; Uma et al. 2010), Hemiptera (Blackburn et al.
2005; Fand et al. 2012; Kumar et al. 2014), Orthoptera (Mahar et al. 2004),
Hymenoptera (Bowen and Ensign 1998), and Diptera (Ahn et al. 2013) in the labo-
ratory when hosts are infected orally or by injection. Bussaman et al. (2009) reported
more than 80% mortality in female mushroom mites, Luciaphorus perniciosus
Rack (Pygmephoridae) within 3 days of application of P. luminescens. Some field
studies reported the insecticidal virulence of P. luminescens in a variety of hosts: the
cabbage white butterfly, Pieris brassicae L. (Lepidoptera: Pieridae) (Mohan et al.
2003), Drosicha mangiferae Stebbins (Hemiptera: Margarodidae) (Mohan et al.
2004), and pupae of diamondback moth, Plutella xylostella L. (Lepidoptera:
Plutellidae) (Razek-Abdel 2003). Jallouli et al. (2013) reported near 100% mortality
in a stored grain pest, Ephestia kuehniella Zeller (Pyralidae), when treated with P.
temperata K122 used at the rate of 12 × 108 cells/ml.
Few researchers have examined the effect of the symbiotic bacteria or their sec-
ondary metabolites on non-target arthropods, independent of their EPNs. Lalitha
et al. (2012) studied the effect of P. luminescens (the symbiotic bacteria of H. bac-
teriophora strain PDBC Hbb1) on pupae and adults of the egg parasitoid
Trichogramma chilonis Ishii (Hymenoptera: Trichogrammatidae) and eggs and lar-
vae of the predator Chrysoperla zastrowi sillemi Esben-Peterson (Neuroptera:
Chrysopidae). There was no physical changes in the eggs, larvae, and adults of T.
chilonis and C. zastrowi sillemi, and no significant reduction was observed in egg
hatch, adult emergence, or parasitism from the bacteria. Mohan and Sabir (2005)
tested the virulence of P. luminescens (isolated from H. indica) against T. chilonis
and Trichogramma japonicum Ashmead (Hymenoptera: Trichogrammatidae) inside
eggs of their host Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) in the
laboratory at the rate of 1 × 105 cells per ml, and reported 84% reduction in emer-
gence of Trichogramma adults from treated host eggs.
However, despite biological activity of EPN symbiotic bacteria when applied
artificially, to date, free-living forms of Xenorhabdus and Photorhabdus have not
been found in the soil. Rajagopal and Bhatnagar (2002) and Mitani et al. (2004)
reported that the nematodes alone can cause host mortality but cannot reproduce.
Similarly, bacteria can cause mortality on their own but need nematodes for their
reproduction and, apparently, cannot live alone in soil without nematodes (Burnell
and Stock 2000). These observations suggest that there is little likelihood that either
EPNs or their symbiotic bacteria can be effective for pest control, when used alone.
9.4 on-target Effects of EPNs and Bacterial Symbionts

N
on Arthropods
EPNs typically have broad host ranges, suppressing a wide range of insects, includ-
ing species of Coleoptera, Lepidoptera, Diptera, Thysanoptera, Orthoptera and
Isoptera (Grewal et al. 2005; Půža and Mráček 2005; Arthurs and Heinz 2006;
Barbara and Buss 2006; Malan and Manrakhan 2009; Khan et al. 2016). The
numbers of infective juveniles (IJs) applied in inundative programs are high, rang-
ing from 2.5 to 7.5 × 109 IJs per hectare (Georgis et al. 2006).
Despite being safer than conventional chemical pesticides to the environment,
EPNs may have some potential to affect some non-target arthropods due to broad
host ranges and the high doses used in the field. They have successfully controlled
insect pests in several habitats, including soil, holes and galleries of borers, leaf
mines, curled leaves, flowers, and buds, as well as, in some cases, on simple leaf
surfaces. These habitats provide protection to EPNs from dryness and other unfa-
vorable environmental conditions. In such habitats, non-target organisms may come
in contact with EPNs and get affected. Also, EPNs may also have indirect effects on
non-target organisms, as for example through the ingestion by predators of insects
that are infected by EPNs, or the death of parasitoids inside infected hosts. It is
known that predation of EPN-infected insects also impairs the development of
EPNs themselves within the insect because, in addition to destroying the host, EPN
development can be harmed by more rapid host dessication (Baur et al. 1998; Foltan
and Puza 2009). Previous reviews on non-target effects due to EPNs include Poinar
(1989), Bathon (1996), and Piedra-Buena et al. (2015), as well as various individual
studies, most based on laboratory assays (Table 9.2), discussed here by taxonomic
groupings of non-target species.
9.4.1 Coleoptera
Most studies of EPN effects on non-target organisms assessed levels of mortality

under laboratory conditions. Georgis et al. (1991) found that adult predators were
not susceptible to EPN nematodes under laboratory conditions, but their immature
stages were more prone to infection. In the field, Georgis et al. (1991) did not
observe any effects of EPNs on non-target predatory beetles in the Carabidae,
Staphylinidae, or Histeridae in four crops (turfgrass, corn, cabbage, and cranberries)
treated with S. carpocapsae, S. feltiae, or H. bacteriophora.
Farag (2002) reported high mortality of larvae of Coccinella undecimpunctata L.
(Coccinellidae) due to H. taysearae and S. carpocapsae strain S2 under laboratory
conditions. Similarly, Rojht et al. (2009) found 93% mortality of twospotted lady
beetle, Adalia bipunctata (L.) (Coccinellidae) in laboratory due to S. feltiae, S. car-
pocapsae, or H. bacteriophora. However, Shapiro and Cottrell (2005) found lower
mortality (< 30%) to lady bird beetles, C. septempunctata, C. maculata, H. axyridis,
and Olla v-nigrum as compared to 90% mortality to their host, Agrotis ipsilon
(Hufnagel) (Lepidoptera: Noctuidae) due to H. bacteriophora. However, S. carpo-
capsae caused 80–85% mortality in C. septempunctata, C. maculata, and O. v-
nigrum which was still lower than the A. ipsilon mortality (100%). Shapiro and
Cottrell (2005) predicted that EPNs applications have significantly less impact on
lady beetle populations (relative to impacts on the target pest) in crop fields than
suggested by studies in the laboratory or in greenhouses. Their reason for this pre-
diction was that contact between nematodes and lady beetles would be limited
9
Table 9.2 Examples of virulence of EPNs towards non-target arthropods

Laboratory
(L)/Field (F)/
Insect life Greenhouse
EPNs species Insect Order Non-target insects stage (G) Dose Comments References
H. bacteriphora NC Coleoptera Ground beetles, Last larval or L 20 nematodes Adults possessed low Georgis
strain and S. Harpalus sp. and nymphal per cm2 or 400 susceptibility to nematodes; et al.
carpocapsae All strain Pterostaticus sp.; stages and IJs per larva/ Mortality rate was 25–44% in (1991)
Cicindela sp., adults adult late instars
Tetracha sp., and
Philonthus sp.
S. carpocapsae Coleoptera Carabid beetles, Adults F 0; 1.5 × 104; No effect found Ropek and
Bembidion 3 × 104; Jaworska
properans Steph. 6 × 104 IJs (1994)
and Pterostichus
cupreus L.
S. carpocapsae (Cxrd Coleoptera Coleomegilla Beetles L 250 IJs/beetle Mortality was higher in Shapiro and
strain) and H. maculate, Olla lepidopterous host, Agrotis Cottrell
bacteriophora (VS v-nigrum, ipsilon than all 4 lady beetles; (2005)
strain), Harmonia axyridis,
and Coccinella
septempunctata
S. feltiae, S. Coleoptera Two spotted lady Larvae L 50, 250, and Mortality rate at 25 °C was Rojht et al.
carpocapsae, and H. beetle, Adalia 500 IJs/larva >93%; Infectivity of EPNs (2009)
bacteriophora bipunctata species increased with
increasing temperature; H.
bacteriophora was found to be
least effective and other
Effects of Entomopathogenic Nematodes and Symbiotic Bacteria on Non-target…
species and mixed suspension

of species showed almost the
same effects
255
(continued)
256
Laboratory
(L)/Field (F)/
H. amazonensis isolate Coleoptera Carabid beetle Larvae, L 75, 150, 300 Only 1st instar predator larvae Mertz et al.
RSC 5 and H. Calosoma pupae and and 600 IJs/ susceptible to EPNs at (2015)
amazonensis isolate JPM granulatum adults insect stage concentrations greater than
4 150 IJs; EPN isolates were
found to be safer for predator
S. carpocapsae (BA2), Coleoptera Seven spotted lady 2nd instar L 50, 25 and S. feltiae (SF), S. carpocapsae Metwally
S. carpocapsae (S2), H. beetle, Coccinella larvae 12.5 IJs/larva (all), S. riobravae (SR), H. et al.
sp. (D1), S. feltiae (SF), septempunctata bacteriophora (HP88), S. (2016)
S. carpocapsae (All), S. carpocapsae (BA2), and S.
riobravae (SR), S. scabtarsci (SS) caused
scabtarsci (SS), S. 50–100% mortality; H.
glaseri (SG), H. bacteriophora was least
bacteriophora (HP88), effective;
and H. marilatus (MAR)
S. carpocapsae Hymenoptera Eulophid parasitoid Larvae and L 0, 5, 25, 50, 100 per cent mortality of wasp Sher et al.
wasp, Diglyphus adults and 250 IJs per larvae after 48 h; Adults (2000)
begini larva and 0 to showed no infection
320
nematodes/
adult
S. carpocapsae Hymenoptera Cardiochiles C. L 200 IJs/ larva Only 0–10% immature Shannag
“Mexican” strain diaphaniae diaphaniae parasitoids parasitized by and
parasitized nematodes within the hosts. Capinera
melonworm Indirectly, affected the (2000)
larvae emergence rate of parasitoid in
the host
R. K. Sandhi and G. V. P. Reddy
Laboratory
(L)/Field (F)/
9
S. affinis, S. feltiae Hymenoptera Apis mellifera Worker and In-situ in 9–10 IJs 20–71.4% mortality in both Zoltowska
mellifera drone bees brood combs sexes; S. feltiae more invasive et al.
and L than S. affinis especially (2003)
against worker bees. Reduction
in protein content of worker
bees larvae
S. carpocapsae (Sal) Hymenoptera Ichneumonid, Larvae L 10 IJs/cm2 70.7 and 85.2% mortality in Lacey et al.
Mastrus ridibundus (152 IJs) Mastrus ridibundus and (2003)
and Liotryphon Liotryphon caudatus,
caudatus respectively
S. thermophilum, S. Hymenoptera Ants, Messor Adults L 100 IJs/ant S. thermophileum (68–100% Anes and
glaseri, and H. indica himalayans mortality) > H. indica Ganguly
(40–100% mortality) > S. (2015)
glaseri (24–72% mortality)
Commercial products Hymenoptera Bumblebee, Adult bees L 10, 25, and 50 Both products caused very Dutka et al.
containing mixture of Bombus terrestris IJs/cm2 of high mortality after only 72 h (2015)
Steinernema and treatment box exposure, with the first deaths
Heterorhabditis sp. and evident after 48 h
S. Kraussei
S. feltiae, S. carpocapsae Neuroptera Green lacewing Larva L 50 IJs, 250, At 15 °C, S. feltiae was most Rojht and
and H. bacteriophora (Chrysoperla and 500 IJs/ virulent; at 20 °C and 25 °C, S. Trdan
carnea) larva carpocapsae and two mixed (2007)
suspensions (S. carpocapsae ×
S. feltiae and S. carpocapsae ×
H. bacteriophora) showed
higher infectivity
(continued)
257
258
Laboratory
(L)/Field (F)/
S. carpocapsae (BA2), Neuroptera Green lacewing, 2nd instar L 50, 25 and S. carpocapsae (All) was the Metwally
S. carpocapsae (S2), H. Chrysoperla larvae 12.5 IJs/larva most effective with 55–88% et al.
sp. (D1), S. feltiae (SF), carnea mortality; Heterorhabditis sp. (2016)
S. carpocapsae (All), S. D1 was least effective
riobravae (SR), S. (38–73% mortality)
scabtarsci (SS), S.
glaseri (SG), H.
bacteriophora (HP88),
S. carpocapsae (BA2), Hemiptera Minute pirate bug, Immature L 50, 25 and S. carpocapsae caused 40% Metwally
S. carpocapsae (S2), H. Orius albidipennis and adults 12.5 IJs/ml mortality; all other species et al.
sp. (D1), S. feltiae (SF), were not virulent. (2016)
S. carpocapsae (All), S.
riobravae (SR), S.
scabtarsci (SS), S.
glaseri (SG), H.
bacteriophora (HP88),
H. bacteriphora NC Dermaptera Earwig, Labidura Last larval or L 20 nematodes No mortality observed Georgis
strain and S. riparia nymphal per cm2 or 400 et al.
carpocapsae all strain, stages and IJs per larva/ (1991)
and S. feltiae SN strain adults adult
R. K. Sandhi and G. V. P. Reddy
Laboratory
(L)/Field (F)/
9
S. carpocapsae, S. Diptera Aphid predator, 3rd instar L and G 10, 100, and Mortality caused by all 3 Powell and
feltiae, and H. Aphidoletes larvae 1000 IJs/ larva species but comparatively less Webster
bacteriophora aphidimyza in laboratory due to S. feltiae in laboratory; (2004)
and 100 and H. bacteriphora, not S.
1000 IJs per/ carpocapsae reduced pupal
larva in emergence; in greenhouse,
greenhouse adult emergence was reduced
by both S. carpocapsae and H.
bacteriophora; mortality
(5–81%) in greenhouse was
lower than laboratory mortality
(8–93%)
S. thermophilum, S. Arachnids Spider, Neoscona Adults L 100 and 500 Maximum of 30, 20, and 10% Anes and
glaseri, and H. indica (Araneae) theisi IJs/adult mortality caused by H. indica, Ganguly
S. thermophilum, and S. (2015)
glaseri, respectively
259
because lady beetles spend their time looking for hosts on the crop foliage, while
EPNs would be active only in the soil and were present briefly at most on foliage, at
the time of treatment.
Foltan and Puza (2009) also found that the carabid Pterostichus melanarius
Illiger (Carabidae) avoided feeding on prey infected with Phasmarhabditis her-
maphrodita Schneider (Rhabditidae) and Steinernema affine (Bovien)
(Steinernematidae). The presence of either of the two nematodes deterred the bee-
tles from consuming infected carabid cadavers. Mertz et al. (2015) reported that first
instar larvae of Calosoma granulatum Perty (Carabidae) were susceptible (70%
mortality) to two strains of H. amazonensis when this EPN was applied topically at
concentrations greater than 150 IJs/ml. Mertz et al. (2015) also found a repellent
effect of H. amazonensis infection (in the larvae of noctuid Spodoptera frugiperda
Smith (Lepidoptera: Noctuidae) on feeding by third instar larvae or adults of C.
granulatum. Larvae of C. granulatum that did feed on noctuids infected by H. ama-
zonensis, however, died, although H. amazonensis had been reported to be safe to C.
granulatum (Mertz et al. 2015). However, Metwally et al. (2016) observed that S.
carpocapsae (All) was highly virulent (causing 72–100% mortality) to the larvae of
the seven spotted lady beetle, C. septempunctata, under laboratory conditions.
There are very few studies reporting the possible effects of EPNs on non-target
beetles under field conditions. Ropek and Jaworska (1994) did not observe any mor-
tality under field conditions to the carabid beetles Bembidion properans Stephens or
Pterostichus cupreus L. (Carabidae) when fields of annual legumes were treated
with S. carpocapsae. Similarly, Dillon et al. (2012) found no effects of S. carpocap-
sae on the richness or abundance of 65 species of non-target beetles, including 11
saproxylic species in a forest ecosystem. In contrast, reports suggest that field appli-
cations of H. megidis can reduce the population densities of the weevil Barypeithes
sp. (Curculionidae) and that applications of S. feltiae reduced numbers of four non-
target chrysomelids or carabids (Buck and Bathon 1993; Koch and Bathon 1993).
9.4.2 Hymenoptera
EPNs have also been suggested as a potential threat to the pollinator bees. In prin-
ciple, adult bees might be infected by EPNs while foraging on flowers for nectar or
pollen (Morse and Flotum 1997). Some pollinator bees such as bumble bees
(Bombus spp.) are reported to be hibernating under rotten tree stumps, piles, leaf
litter, and soil (Alford 1969) which might be a route in EPN infection. Kaya et al.
(1982) observed that larvae and adults of honey bees, Apis mellifera L. (Apidae),
were susceptible to EPN nematodes, but EPNs did not adversely affect the whole
colonies. Nguyen and Smart (1991) reported low mortality in honey bees due to
Steinernema scapterisci. Zoltowska et al. (2003) observed sensitivity of honey bee
larvae to infection by S. affinis and S. feltiae in both combs and petri dishes.
Similarly, Dutka et al. (2015) found high mortality in Bombus terrestris L. (Apidae)
due to two commercially available EPN products containing mixture of Steinernema
and Heterorhabditis sp. and S. kraussei under laboratory conditions. Taha and
Abdelmegeed (2016) observed no mortality in honey bees, A. mellifera, when hives
were treated with H. bacteriophora to control wax worms, Galleria mellonella L.
(Lepidoptera: Pyralidae).
Shannag and Capinera (2000) reported the direct infection of hymenopteran
parasitoid, Cardiochiles diaphaniae Marsh (Braconidae) by S. carpocapsae after
they have emerged from infected hosts, melonworm, Diaphania hyalinata L., and
pickleworm, D. nitidalis Stoll (both Lepidoptera: Crambidae), but before comple-
tion of the pupal cocoon. Similarly, Lacey et al. (2003) reported infection of devel-
oping larvae of two ectoparasitic ichneumonids, Mastrus ridibundus Gravenhorst
and Liotryphon caudatus Ratzeburg (Ichneumonidae) by S. carpocapsae due to
direct application in laboratory experiments. The potential compatibility of applica-
tion of EPNs with parasitoid populations was observed because both parasitoids
were able to avoid nematode-infected larvae and, in contrast, focused their oviposi-
tion on healthy cocooned host larvae of the codling moth, Cydia pomonella L.
(Lepidoptera: Tortricidae) that escaped the nematode infection. Similarly, the eulo-
phid parasitoid Diglyphus begini Ashmead (Eulophidae) avoided ovipositing in lar-
vae of the agromyzid leafminer Liriomyza trifolii Burgess (Diptera: Agromyzidae)
infected with S. carpocapsae (Sher et al. 2000). Anes and Ganguly (2015) found
that the EPNs, S. thermophilum, S. glaseri, and H. indica were lethal to the benefi-
cial ant Messor himalayanus Forel (Formicidae), causing 72–100% mortality under
laboratory conditions, when applied topically.
9.4.3 Neuroptera
Green lacewings (Chrysopidae) are predators of mites, aphids, whiteflies, scales,

and other small insects. Some neuropterans spend some time in soil foraging for
hosts, potentially bringing them into contact with EPNs. Rojht and Trdan (2007)
studied the effects of three EPNs (S. feltiae, S. carpocapsae and H. bacteriophora)
individually and in mixed suspensions on larvae of green lacewing, Chrysoperla
carnea Stephens (Chrysopidae) at different temperatures in the laboratory with
three doses: 50, 250, and 500 IJs/ larva. Steinernema feltiae was found to be virulent
at 15 °C. At 20 and 25 °C, S. carpocapsae and two mixtures (S. carpocapsae × S.
feltiae and S. carpocapsae × H. bacteriophora) caused the highest mortality in C.
carnea. Metwally et al. (2016) reported high rates of mortality (30–100%) in 2nd
instar larvae of C. carnea by different EPN strains at 50, 25 and 12.5 IJs/larva doses,
under laboratory conditions.
9.4.4 Hemiptera
The minute pirate bug Orius albidipennis Rueter (Anthocoridae) is an insect preda-
tor in many agricultural crops. The nymphs and adults feed on a wide range of
arthropods including aphids, chinch bugs, springtails, plant bugs, thrips, eggs and
small larvae of corn earworms, whiteflies, spider mites. The adults of minute pirate
bugs overwinter in plant debris or leaf litter that may bring them into contact with
EPNs. Metwally et al. (2016) reported S. carpocapsae as the most virulent EPN
tested against O. albidipennis.
9.4.5 Diptera
The cecidomyiid fly Aphidoletes aphidimyza Rondani (Cecidomyiidae) is an aphid

predator that is a commercially available biological control agent, used in both
greenhouses and orchards. The larvae of this predator drop from the plant foliage to
the soil, where they pupate. The pupae will wiggle to the soil surface after emer-
gence from the cocoon and emerge as adults. This time in soil provides opportunity
for contact with EPNs. Powell and Webster (2004) observed mortality of A. aphidi-
myza following treatment with S. carpocapsae, S. feltiae, and H. bacteriophora.
However, the levels of mortality found in a greenhouse study was lower than that in
the laboratory test.
9.4.6 Other Arthropods
The susceptibility of some isopods, diplopods, symphylans, and chelicerates to

EPNs has been reviewed (Poinar 1989). Steinernema carpocapsae (DD 136) can
infect the symphylan Scutigerella immaculata Newport (Symphyla: Scutigerellidae)
under laboratory conditions (Swenson 1966). As symphylans live in the soil litter,
there might be possibility of contact between them and EPNs. Similarly, the terres-
trial isopods Porcellio scaber Latreille (Isopoda: Porcellionidae) and Armadillidium
vulgare Latreille (Isopoda: Armadillidiidae) were susceptible to S. feltiae infection
under laboratory conditions (Poinar and Paff 1985). The tick Boophilus annulatus
Say (Acari: Ixodidae) was susceptible to S. carpocapsae under laboratory condi-
tions. But no development of S. carpocapsae occurred in the cadavers of infected
females (Samish and Glazer 1992; Glazer and Samish 1993). The nematode S. fel-
tiae when applied at 80 nematodes/cm2 of soil reproduced successfully in the sow-
bug Porcellio scaber Latr. and in the millipede Blaniulus guttulatus Fabricius
(Julida: Blaniulidae), under greenhouse conditions (Jaworska 1994). Zhioua et al.

(1994) found penetration by S. carpocapsae and subsequent mortality of female
ticks, Ixodes scapularis Say (Acari: Ixodidae). However, most of these studies were
conducted under laboratory conditions in which high contact between nematodes
and the target organism was assured, under highly favorable physical conditions.
Fewer field studies on the above mentioned groups have been reported. Rahayu
(1983) found reduction in numbers of the Collembolan Onychiurus armatus
Tullberg (Collembola: Onychiuridae) in both field and laboratory studies from
infection by S. carpocapsae. Similarly, Edwards and Oswald (1981) reported sup-
pression of Collembola and mites by S. carpocapsae in sugar beet fields. In con-
trast, Georgis et al. (1991) did not found any effects on non-target arthropods,
including species of Gryllidae, Collembola, Gamasida, Actinedida, and Oribatida
from exposures to S. carpocapsae, S. feltiae, or H. bacteriophora. The immature
stages and adults of the earwig Labidura riparia Pallas (Dermaptera: Labiduridae)
were found to be resistant to H. bacteriophora NC strain and S. carpocapsae infec-
tion, when treated in the laboratory with 400 IJs per larva or adult (Georgis et al.
1991).
Wang et al. (2001) found the long term effect of H. bacteriophora, S. carpocap-
sae, and S. riobravis to be much less severe in Lycosidae, Staphylinidae, Araneae,
Formicidae, Scelionidae, and Sminthuridae than the effect of the insecticide chlor-
pyriphos in turf grass, with the degree of impact varying considerably among non-
target arthropod groups. Indeed, the population levels of oribatid mites were
significantly higher in EPN treated plots than in untreated controls (Wang et al.
2001). However, Peck (2009) found reduction in the abundance of insects and
Collembola in plots of turf grass treated with H. bacteriophora. However, the appar-
ent reduction of these aggregated groups was not detected when data were broken
apart into more specific taxonomic groups, due to complications related to the tre-
mendous amount of microarthropods collections produced by sampling. Anes and
Ganguly (2015) observed 10–30% mortality in the spider Neoscona theisi
Walckenaer (Araneae: Araneidae) following application of S thermophilum, S. gla-
seri, and H. indica under laboratory conditions. However, it is important to notice
the possible lack of exposure of spiders to EPNs due to the spider habitat character-
istics. Some other researchers have also found negative impacts of EPNs on terres-
trial isopods, diplopods, and ticks under laboratory conditions (Jaworska 1991;
Mauleon et al. 1993; Hill 1998).
However, an increase in the population of isotomid collembolans, anysitid mites,
and gnaphosid spiders under trees after nematode application was found. The
increase in the anysitid mite population was apparently related to the increased for-
aging activity by the mite in the nematode-treated areas. The anysitid mites are
important natural enemies in orchards, feeding on thrips, collembola, and nema-
todes (Goh and Lange 1989; Cuthbertson and Murchie 2004).
9.5 Intraguild Predation
Most of the above studies have focused on the direct effects of the nematode/bacte-
ria complex on non-target speciess. EPNs can directly reduce the populations of
some non-target organisms through significant non-target mortality. However, there
are some indirect effects such as Intraguild Predation (IGP) that can affect the popu-
lation densities of non-target organisms that ultimately minimize their input in bio-
logical control. Intraguild predation is defined as two species sharing a common
host and engaging in predation or parasitism of each other (Holt and Polis 1997).
IGP is relatively common among agents used for the biocontrol of arthropod pests,
as compared to agents used against weeds or plant pathogens (Rosenheim et al.
1995). The simultaneous application of EPNs with other types of biocontrol agents
such as predators and parasitoids place large populations of both agents together,
setting up conditions for IGP. Akhurst (1990) reported that when biocontrol agents
such as parasitoids and entomopathogenic nematodes co-infect an insect, the nema-
todes either can infect immature parasitoids or inhibit their development. Factors
such as the exact insect host, the parasitoid species, the host specificity of the nema-
tode, and the relative timing of co-infestation of the agents can all affect these inter-
actions. Predators can also be affected, as they can become infected with nematodes
by eating a nematode-infected prey.
IGP interactions have been described by Georgis and Hague (1982) and Battisti
(1994) for Steinernema spp. interacting with sawflies and their parasitoids (ichneu-
monids). In both studies, the survival rate of parasitoids was lower in nematode-
treated plots as compared to control. No infection of parasitoid larvae was found
inside hosts (Battisti 1994). The parasitoid larvae inside cocoons were less suscep-
tible to nematodes than the parasitoid parasitizing the sawfly in the laboratory tests
(Georgis and Hague 1982).
Sher et al. (2000) examined the interaction between S. carpocapsae and the eulo-
phid parasitoid wasp, D. begini for the control of leafminer, L. trifolii on chrysan-
themum. It was found that parasitoids were affected by nematodes either indirectly
through host mortality or directly through nematode infection of the parasitoid lar-
vae. The nematodes attacked paralyzed (by the parasitoid) leafminer larvae and
healthy (non-parasitized) leafminer larvae equally, which suggested that behavioral
avoidance by the nematode did not reduce infection of healthy leafminer larvae.
Sher et al. (2000) also found that the total mortality of leafminers was higher when
both agents were used together, than when either agent was used alone. Similarly,
Powell and Webster (2004) studied the effects of S. carpocapsae, S. feltiae, and H.
bacteriophora on the aphid predator A. aphidimyza. The adult emergence was
reduced by H. bacteriophora and S. carpocapsae, but not by S. feltiae. Pupae of A.
aphidimyza within their cocoons were non-susceptible to S. carpocapsae. The pred-
ator inhibited the development of EPNs and ultimately killed them in the host
because of complete destruction of the host body, and its internal desiccation due to
holes opened in the integument (Foltan and Puza 2009).
EPNs have potential to control many pest arthropods. Some reports concerning
effects of EPNs, their symbiotic bacteria or their metabolites on non-target species
under natural conditions do exist. However, most of these studies were laboratory
bioassays, with only a few studies evaluating field effects on non-target species,
indicating a need for more field-based studies.
EPN-bacterium complexes can affect non-target species either through direct
infection or indirectly through effects such as depletion of the target host popula-
tions. The careful selection of host specific EPN strains for the target pests might
help minimize the direct effects. While selecting any EPN species for use in a clas-
sical or inundative biological control program, the risk to non-target organisms like
depletion of host food resources, competition with other pests in regard to scarcity
of food in presence of EPNs, should always be considered. As discussed above,
when EPNs compete with parasitoids inside hosts, death of immature parasitoids
may occur.
The degree of non-target impact will also depend on environmental factors that
affect the EPN efficacy and persistence in the area of application. The use of patho-
gens that are highly virulent to some non-target organisms may not have these
effects, if exposure of non-target species to the nematodes is unlikely to occur or
limited in area. For example, chances of infection of adult parasitoids by EPNs is
minimal because they mostly occur in above ground habitats, eliminating most con-
tact chances with nematodes.
Some factors affecting the degree of potential non-target risks posed by use of
EPNs in biological control include the following:
1. Host Range: The host range plays an important role in the selection of EPN
strains. In the past, most failures in use of EPNs in field are related to a poor
match between the EPN species and target insect pest. It is a challenge to main-
tain balance between host specificity and broad host range for safety of non-
target organisms. Steinernema scapterisci and S. neocurtillae Nguyen and Smart
are host-specific species that have few, if any, non-target effects. However, broad
host ranges are commercially desirable for nematodes marketed for application
as pesticides.
2. Dose: The dose to be used in field should be determined in the laboratory first.
Excessively high doses may be both costly and increase risk of non-target
impacts.
3. Time of application: Strategies such as the avoidance of coincidence of EPN
application with the peak activity of non-target organisms will reduce the chances
of intraguild predation, that will indirectly limit the chances of migration of adult
predators to other areas with more prey options, thus limiting the local extinc-
tions of the natural enemies.
4. Dispersal: Harm to certain non-target organisms may be limited or avoided if
exposure of the non-target species to the pathogen is outside the EPNs dispersal
distance and beyond the treated area.
5. Persistence: The long term persistence of particular EPNs must be determined

as part of the risk assessment. The best example here would be the introduction
of S. glaseri, which was applied on a large scale in New Jersey in 1939–42 to
control Japanese beetle. This nematode could still be found after 50 years, but
only at few locations and in the southern part of the area where nematodes were
applied (Gaugler et al. 1992). However, along with requirement of persistence of
these EPNs, non-target risks associated with this cannot be avoided.
Little literature was found on possible negative effects on non-target species of
EPN symbiotic bacteria when separated from their respective EPNs. These symbi-
otic bacteria require nematodes as carrier for their survival. Therefore, the possibil-
ity of establishment of these bacteria in absence of their particular host nematodes
in natural conditions is highly limited. There is, however, still a need to study the
virulence of these bacteria to non-target organisms in the field, without their EPN
hosts. The formulation of symbiotic bacteria without nematodes, the persistence of
such bacteria, their shelf life, production and application methods, and the effects of
biotic and abiotic factors on these bacteria are all aspects that need to be explored
and studied in detail for interpreting potential non-target effects.
EPNs that show high virulence to non-target arthropods in laboratory studies
may have much lower, or no, effects under natural conditions due to the lack of
contact between the non-target species and the nematodes. Because the chances of
direct contact between nematodes and organisms are maximized in laboratory tests,
it is to be expected that impacts under field conditions will vary, and likely be lower.
Bathon (1996) noted that mortality from EPNs will be temporary, spatially restricted,
and will affect only part of a population. The physiological host range does not
necessarily equal ecological host range of the entomopathogens (Hajek and Goettel
2000). The efficacy of EPNs does not only depend on the infectivity but is also
affected largely because of the environmental factors surrounding the EPNs and the
potential hosts. The host range of EPNs under favorable conditions i.e. adequate
moisture, temperature and no effect of UV radiation will always be different from
their ecological host range. The ecological host range of EPNs in natural environ-
ment can be determined after the physiological host range is known from laboratory
studies. But in laboratory, it is difficult to maintain the ecological conditions as
similar to field conditions and it is not reasonable to apply the laboratory results in
field conditions. According to Bathon (1996), the susceptibility of hosts to EPNs in
the field might be related to habitat requirements and searching behavior of nema-
tode strains. This raises questions about the reliance only on data from laboratory
studies to predict field non-target impacts. However, laboratory observations can be
an important beginning for evaluating the potential effects of these nematodes in
natural populations. The laboratory and semi-field studies should be conducted in a
way to mimic the field conditions as much as possible. In-depth knowledge of field
effects of these parasitic nematodes is required to assess the negative effects.
Overall, the long-term impact of the EPNs on non-target arthropods is much less
severe than that of chemical pesticides, with varying degree of impact among
n on-target arthropods. Current literature as a whole suggests that non-target effects

of EPNs are negligible.
Acknowledgements This material is based upon work supported by the National Institute of
Food and Agriculture, U.S. Department of Agriculture, Multistate-W4185 project “Biological
Control in Pest Management Systems of Plants” (Accn#1014043; Hatch Project#MONB00857).
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Chapter 10
Granuloviruses in Insect Pest Management
Pankaj Sood, Amit Choudhary, and Chandra Shekhar Prabhakar
Abstract Alternative strategies for insect pest management were looked and
microbial pesticides in particular were noticed as attractive candidates. Among bac-
uloviruses, the granulosis viruses are highly specific and safer to non target species.
They are considered potential candidates for use as biological insecticides for con-
trol of economically important insects. These viruses are highly virulent, selective,
stable and environmentally benign, once applied. However, usage and availability of
granuloviruses is limited, worldwide. Slow action, restricted host range and low
persistence are some of major drawbacks of such microbial pesticides, hindering
their large scale usage. Methods must be developed for the unequivocal identifica-
tion of these viruses. Their effects on non target species must be investigated at the
cellular and molecular levels to enhance their role in pest management programmes.
Efforts for identifying potent microbial agents, based on their rich biodiversity,
must be applied extensively to expand the genetic make-up of baculoviruses.
Recombinant DNA technology in baculoviruses could result in accomplishment of
many achievements. In order to increase the uptake of baculoviruses, it is necessary
to develop robust molecular biology techniques, to enhance safe food production for
the nutritional and health security of growing population.
Keywords Biocontrol · GV · Baculovirus · Insect pest management · Organic

agriculture
P. Sood (*)
Krishi Vigyan Kendra, CSK Himachal Pradesh Krishi Vishvavidyalaya, Sundernagar,
Mandi, Himachal Pradesh, India
A. Choudhary
Department of Entomology, Punjab Agricultural University, Ludhiana, Punjab, India
C. S. Prabhakar
Department of Entomology, Veer Kunwar Singh College of Agriculture, Dumraon (Bihar
Agricultural University, Sabour), Buxar, Bihar, India

https://doi.org/10.1007/978-3-030-23045-6_10
276 P. Sood et al.
10.1 Introduction
In the recent years, biological control strategies have gained importance for the
management of agricultural pests. This shift occurred due to the repeated failure of
synthetic pesticides in managing agricultural pests, thereby causing several out-
breaks. The growing knowledge and awareness on the negative impacts of chemical
insecticide usage on the ecosystem and public health led to efforts directed towards
a reduction in chemical control of insect pests, diseases and weeds (Haase et al.
2015). Along with these issues, there are also the problems pertaining to target and
non-target insect species. At the same time, human population is growing at a very
fast rate. To fulfill the nutritional and other requirement of such a huge population
we have to sustain an even more intensive agriculture. In such system, pest prob-
lems will continue to be a challenging factor. Hence, to operate such system in
which synthetic pesticides have to be lowered without any negative effects on yields,
we have to look for viable alternatives. These must confer the idea of sustainable
crop yield by significantly managing the pest populations, helping in preserving the
ecosystem health.
Under the above situations, we look towards biological control of agricultural
pests. These will, individually or in integration with other pest management strate-
gies, help us in attaining the above mentioned objectives. Furthermore, under the
concept of biocontrol we have to consider many agents such as macro and microor-
ganisms. Among the microorganisms category, many entomopathogens can play a
key role. Under natural conditions the populations of many pests are regulated by
epizootics caused by various entomopathogens such as bacteria, fungi and viruses.
Several examples are available showing how entomopathogens have been deployed
successfully under classical biological control strategies, for management of insect-
pests (Kalha et al. 2014).
Among these, insect pathogenic viruses are promising and crucial components,
devising suitable integrated pest management strategy. Viruses of only a few fami-
lies are known to infect insects. Entomopathogenic viruses from a single family
only, i.e. Baculoviridae, have been explored as biopesticides thus far. Insects belong-
ing to orders Lepidoptera, Hymenoptera and Diptera are natural hosts for this virus
family. These entomoptahogens are globally well known for application as biopes-
ticides, to control a number of important insect pest populations of crops and forests
(Sun and Peng 2007). Thus, their use represents an alternative and eco-friendly
approach for pest management. They are safe to human health and the ecosystem,
allowing innovative industrial productions and field applications (Sumathy et al.
1996).
Betabaculovirus, one of the four genera of family Baculoviridae, is a
Lepidopteran-specific granulovirus (GV). The other three genera of Baculoviridae
are Alphabaculovirus (Lepidopteran-specific nucleopolyhedrovirus),
Gammabaculovirus (Hymenopteran-specific nucleopolyhedrovirus), and
Deltabaculovirus (Dipteran-specific nucleopolyhedrovirus) (King et al. 2011).
10 Granuloviruses in Insect Pest Management 277
Baculoviruses have rod-shaped nucleocapsids, each comprising a single circular

genome. They are enveloped, singly or in groups, by a membrane to form virions.
Virions are occluded in a protein matrix, which forms the occlusion body (OB)
(Williams et al. 2017). The OB protects the virions, known as occlusion derived
virions (ODVs), from environmental factors. These viruses can be readily distin-
guished into two groups: nucleopolyhedroviruses and granuloviruses that clearly
differ in the structure of their OBs (Adams and McClintock 1991).
Nucleopolyhedroviruses have polyhedral OBs (0.5–10 μm), mainly comprising of
crystalline polyhedrin protein, which occludes large numbers of virions. In contrast,
granuloviruses have smaller granule-like OBs (∼0.4 μm), that mainly comprise
granulin protein. Each granulovirus OB contains a single virion (Williams et al.
2017).
Baculoviridae (Nucleopolyhedrosis and Granulosis viruses) are exploited for the
development of commercial virus-based biopesticides for insect pest management.
In the present chapter, we discuss the ecology and use of granulosis viruses in the
management of insect pests.
10.2 Classification of Entomopathogenic Virus
Earlier, viruses were classified using a Linnaean (Binomial) taxonomy, initially

based on morphological characteristics and then stretched to genomic phylogeny
with the development of modern molecular biology tools. Actual classification of
viruses, known as the Baltimore classification, is based on nucleic acids and on the
function of capsid (Sparks 2010). This classification separates viruses into seven
classes based on the type of nucleic acids present in the capsid that represent the
viral genome, and on the way the mRNA is coded, for protein production in the cell
(Baltimore 1971). The Baltimore classes are as follows:
I. double strand, dsDNA viruses (herpesviruses, baculoviruses, poxviruses)
II. single strand, ssDNA viruses (+) sense DNA (parvoviruses)
III. dsRNA viruses (reoviruses)
IV. (+) ssRNA viruses (picornaviruses, togaviruses)
V. (−) ssRNA viruses (orthomyxoviruses, filoviruses)
VI. ssRNA-RT viruses (positive sense RNA with a DNA intermediate such as
retroviruses)
VII. dsDNA-RT (DNA viruses that utilize reverse transcription such as
hepadnaviruses)
In view of the general approval accorded by virologists to the uniform taxonomy
of viruses, insect virus classification also follows the direction of the International
Committee on Taxonomy of Viruses (ICTV) (Van Regenmortel et al. 2000). The
general characters considered in the classification of any virus include the type of
genetic material (single- or double-stranded DNA, single- or double-stranded RNA,
positive or negative strand), virion morphology and size (icosahedral, rod-shaped),
278 P. Sood et al.
the presence of an envelope surrounding the virion, and of an occlusion body engulf-
ing the virions, host and host range. The same criteria are followed to classify the
diversity of viral groups that attack insect species.
Insect pathogenic viruses are named with acronyms, according to their host and
the viral group to which they belong to. For example, the cabbage butterfly, Pieris
brassicae granulosis virus, is named PbGV. Therefore, all nucleopolyhedrosis
viruses are named NPV while the granulosis viruses are named GV. Further, lepi-
dopteran baculoviruses were divided in NPVs (group I and II) and GVs on the basis
of genomic nucleic acids sequence data (van Oers and Vlak 2007). The entomopox-
viruses are named EPV, the iridoviruses as IV, and the cytoplasmic polyhedrosis
viruses (cypoviruses) as CPV.
10.3 Granuloviruses of Important Insect Pests
GVs have been reported only from insects belonging to the order Lepidoptera,
except one doubtful case reported in insect belonging to the order Hymenoptera.
Insects may acquire GV inoculum through feeding. The GV infected insect tissues
eventually disintegrate and the body fluid got filled with granulin inclusions. In
India, GVs have been reported to be effectively used in the management of Chilo
infuscatellus (Snell.) (Easwaramoorthy and David 1979; Mala and Solayappan
2001), Chilo sacchariphagus (Bojer) (Easwaramoorthy and Jayaraj 1993;
Easwaramoorthy and Santhalakshmi 2000), Spodoptera litura (Fab.) (Narayanan
1985), Phthorimaea operculella (Zeller) (Pokharkar et al. 1999; Chandla and Verma
2000) and Plutella xylostella L. (Subramanian 2002; Kennedy et al. 2003). GV
infecting Pieris brassicae L. have been reported for the first time in India from
Himachal Pradesh by Sood (2004) and Narayanan and Sood (2006). Several GVs
have been reported from India and abroad (Table 10.1), but only a few have been
studied in details for their potential as biopesticides, in the management of eco-
nomically important insect pests.
10.4 Mode of Action
Insect viruses, as they occur in nature, consist of either double-stranded or single

stranded circular/straight genomes of viral DNA/RNA with/without a double mem-
brane envelop. Mode of action of baculoviruses is well studied. Baculoviruses show
double stranded and circular viral DNA, with a double membrane envelop. They
typically have narrow host ranges, often limited to just one or a few related insect
species, although the most intensely studied member of the family, Autographa
californica multiple nucleopolyhedrovirus (AcMNPV), is able to infect as many as
30 species from several lepidopteran genera. These occlusion-derived viruses
(ODV) contain one or multiple nucleocapsids with each capsid holding a single
Table 10.1 Granuloviruses reported from main insect pests

S. No. Insect Species GV acronyms References
1. Agrotis ipsilon AiGV Chaudhry et al. (1976)
2. Agrotis segetum AsGV Zethner (1980)
3. Achaea janata AjGV Pawar (1980)
4. Adoxophyes orana AdorGV Aizawa and Nakazato (1963)
5. Artogeia rapae ArGV –
6. Clostera anachoreta CaGV –
7. Clostera anastomosis ClanGV –
8. Choristoneura fumiferana ChfuGV –
9. Choristoneura murinana ChmuGV Cunningham (1982)
10. Chilo infuscatellus CiGV Easwaramoorthy and David (1979)
11. Chilo saccharigus indicus CsGV Mehta and David (1980)
12. Cnaphalocrocis medinalis CnmeGV Jacob et al. (1971)
13. Cnephasis pumicana CnpuGV Glas (1991)
14. Cryptophlebia leucotreta CrleGV Fritsch and Hubner (1986)
15. Cydia pomonella CpGV Benz (1981)
16. Dendrolimus sibiricus DesiGV Baranovsky and Litvina (1978)
17. Diatraea saccharalis DsGV Pavan et al. (1983)
18. Erinnyis ello ErelGV –
19. Epinotia aporema EpapGV –
20. Harrisina brillians HabrGV –
21. Helicoverpa armigera HearGV Gitay and Palson (1971)
22. Homona magnanima HomaGV Sato et al. (1980)
23. Hyphantria cunea HycuGV Weiser et al. (1986)
24. Lacanobia oleracea LaolGV Crook and Brown (1982)
25. Mythimna unipuncta MuGV –
26. Pericallia ricini PeriGV Jacob et al. (1972)
27. Pieris brassicae PbGV Billotti et al. (1956); Sood (2004)
28. Phthorimaea operculella PhopGV Chandla and Verma (2000)
29. Plodia interpunctella PiGV Hunter et al. (1977)
30. Plutella xylostella PlxyGV Kao and Rose (1976)
31. Pieris rapae PrGV Jaques (1973)
32. Pseudalatia unipuncta PsunGV –
33. Scripophaga excerptalis ScexGV Singaravelu et al. (1999)
34. Scrobipalpa absoluta SaGV Lipa (1998)
35. Spodoptera litura SlGV Pawar and Ramakrishnan (1977)
36. Spodoptera exigua SeGV Narayanan (2003)
37. Spodoptera frugiperda SfGV –
38. Trichoplusia ni TnGV –
39. Xestia c-nigrum XcGV Lipa and Ziemnicka (1971)
40. Zeiraphera diniana ZdGV Baltensweiler et al. (1977)
280 P. Sood et al.
viral genome. One or more ODVs, in turn, are ‘occluded’ within a protein matrix,
referred to as occlusion bodies, that protect the virus from the environment, as the
the occlusion bodies eventually slowly degrade under UV light (Cory and Myers
2003; Harrison and Hoover 2012).
Baculoviruses are transmitted orally in nature, and the target of primary infection
is the larval midgut epithelium. After being consumed, occlusion bodies get dis-
solved in the midgut lumen, releasing the embedded ODV, which infect midgut
epithelial cells (Clem and Passarelli 2013). The ODV bind and fuse with midgut
cells, releasing the nucleocapsids into the cytoplasm. The nucleocapsids are trans-
ported to the nucleus, uncoat and begin replication, producing budded virus parti-
cles (Miller 1997; Elderd 2013). Progeny BV bud from the basal surface of the
epithelium, cross the basal lamina, and infect most of the remaining tissues of the
larva.
The process of ODV attachment and entry depends on several viral proteins
found in the ODV envelope, referred to as PIFs (per os infectivity factors) (Clem
and Passarelli 2013). Recent results indicate that some of the PIFs form a complex
in the ODV envelope, likely interacting with an unknown midgut receptor, mediat-
ing fusion with the plasma membrane (Peng et al. 2012). Thus, the PIFs constitute
a novel virus attachment/fusion protein complex. Interestingly, PIFs appear to be
ancient genes that are conserved among three related insect virus families, as pif
homologs are also found in the nudiviruses and the polydnaviruses, the latter of
which form mutualistic relationships with parasitic wasps (Bezier et al. 2009; Burke
et al. 2013). In most baculovirus infections, the budded virus then spreads through-
out the larva via the tracheal system and haemocytes. Late in infection, the host’s
tissues are filled with virions that are occluded in millions of occlusion bodies,
which are released upon death when the host liquefies (Harrison and Hoover 2012).
The gross GVs pathologies are similar to those of NPVs, but differences occur
depending upon the type of tissues infected. The first indication of infection in the
larva is the loss of appetite and a progressive color change from the normal color to
a pale whitish or milky yellow appearance, especially on the ventral side (Huger
1966). The whiteness is due to the abundance of OBs in the hypertrophied fat bod-
ies. When the infection is limited to the fat body, the larvae often increases in size,
become white, opaque and mottled at an advanced stage of infection, and later show
a brownish discolouration (Hamm and Paschuke 1963). Such larvae may live longer
and become larger than an uninfected one. In the case of systemic granulosis, the
larva usually dies in a brief period, much shorter than observed for an infection
involving mainly the fat body. At death such a larva is smaller than a healthy one.
10.5 Genome Organization
The super coiled ds DNA genome of GVs varies in size from 100 to 180 kilobase
pair (kbp) with potential to code for over 100 genes. The lowest genome size
(99,657 bp) has been detected in Adoxophyes orana GV (AdorGV), while the
largest (178,733 bp) has been reported in Xestia c-nigrum GV (XcGV) (Hayakawa
et al. 1999; Wormleaton et al. 2003). The estimated G + C content for betabaculovi-
rus members ranged between 32.5% in Cryptophlebia leucotreta GV (CrleGV) and
45.2% in Cydia pomonella GV (CpGV) (Han et al. 2016).
The granulin gene or a restriction site close to it have been used to define the zero
point of GVs restriction maps. Hybridization studies from a large number of viruses
show that most, if not all, granulin gene sequences are highly conserved. The pro-
moter regions of granulin and polyhedron genes are of considerable interest because
of the very high levels of expression of these proteins. The 5′ flanking region of
these genes contains a highly conserved motif of 12 nucleotides (Rohrmann 1986)
which includes the transcription initiation site, followed by an Adenine (A)-
Thyamine (T) rich sequence up to the start of coding region. There are additional
two conserved nucleotides adjacent to this region in CpGV, PbGV and Trichoplusia
ni GV, giving a 14- mer peptide with sequence TTTATAAGGAATTA. The region
following this sequence is shorter and more variable in GVs than in NPVs, though
with a similarly high A + T content of 82–93% and a complete absence of guanine
(G) (Crook and Winstanley 1992).
Studies by Bah et al. (1997) showed that the granulin genes of TnGV, PbGV,
CrleGV showed homologies ranging from 76.7 to 80.5% for nucleotide sequences
and from 84.2 to 88.3% for amino acids, when compared to Choristoneura
fumiferana GV (ChfuGV).
The enhancin genes have been sequenced for TnGV and Pseudaletia unipuncta
GV (PuGV). They have more than 90% amino acid homology. These genes also
contain the consensus late promoter ATAAAG sequence just upstream the coding
sequence (Table 10.2). The TnGV enhancing gene hybridizes under conditions of
high stringency to DNA bands from some other GVs, indicating that these GVs also
contain homologous genes.
10.6 Genetic Manipulation for Improved Efficacy
In general, GVs are not stable under natural conditions, as they readily get inacti-
vated under the influence of UV rays,. Enhancement of NPV infectivity and reduc-
tion in incubation time by the enhancing gene (viral enhancing factor) VEF have
been demonstrated in certain GVs such as AcMNPV. This VEF is ten times more
stable than virions to UV inactivation and also impart heat stability, apart from
reducing the survival time (Granados and Corsaro 1990; Gallo et al. 1991). Studies
with both native and recombinant enhancing showed inactivation by metal ions che-
lators and reactivation with divalent ions, thus identify the enhancing factor as a
metalloprotease.
In CpGV, an inhibitor of apoptosis (Iap) gene has been identified. It blocks apop-
tosis which is a natural defense mechanism in Spodoptera frugiperda, as shown in
cells infected with a mutant AcMNPV lacking a functional p35 gene. The Iap gene
encodes a polypeptide with a predicted relative molecular weight of 31.3 kd. The
282
Table 10.2 Sequences of the promoter regions of granulin genes from PbGV, CpGV and TnGV
Virus Sequencea Nucleotides before ATG
PbGV 15
CpGV 19
TnGV 11
a
Gaps to align the sequences are indicated by dashes. The numbers of nucleotides between the conserved 14–mer and the ATG translation start site are shown
P. Sood et al.
region near the C- terminus of the polypeptide contains a distinctive zinc finger like
motif which is similar to that found in several other genes, with potential to regulate
apoptosis.
Some other genes such as Bam H1’J’ have been subcloned and subjected to
transposon mutagenesis in E. coli cells using a Tn3 derivative (Wonkyung et al.
1997).
There are a number of candidate genes available which, if expressed by the virus
during infection, are likely to result in earlier host death or at least in reduced insect
feeding. Though much work on these lines has been done in NPVs, very little infor-
mation on these aspects pertains to GVs, mainly because of difficulties in develop-
ing a good cell culture system for selection and cloning of recombinant GVs. The
insertion and expression of foreign genes in baculoviruses is mainly performed
through the replacement of the granulin gene coding region with a foreign gene
sequence. Granulin genes from TnGV and PbGV have been cloned and sequenced
by Akiyoshi et al. (1985) and Chakerian et al. (1985), respectively. Crook and
Winstley (1992) constructed a CpGV transfer vector containing the 5′ and 3′ flank-
ing regions of the granulin gene including the entire promoter region, but the entire
granulin coding region that was deleted and replaced by a Bam H1 cloning site. A
cloned strain of CpGV 9-M1 using successive rounds of an in vitro limiting dilution
method was obtained by Crook et al. (1997). The region containing the granulin
gene and an open reading frame (ORF) immediately upstream of it was sequenced.
Lery et al. (1997) established 12 in vitro clones from a Tunisian isolate of
PhopGV. Substitution of granulin for polyhedrin has also been demonstrated by
Eason et al. (1998) for better infectivity. Smith and Goodale (1998) determined the
nucleotide sequence and located the major in vivo transcript termini of the Lacanobia
oleraceae granulosis virus (LoGV) egt gene.
10.7 Mass Production
(i) Culturing of Host Insects

So far, no medium has been developed to culture granuloviruses under laboratory
conditions. The GV can be mass multiplied only in the host insect larvae. The host
insect culture can be maintained on artificial/semi synthetic diet under laboratory
conditions in most of the species. In some cases, i.e. in shoot borer C. infuscatellus,
larvae from 30–90 days old sugarcane crop showing fresh dead hearts were col-
lected in plastic containers with shoot bits, for rearing of larvae for GV mass
production.
(ii) Propagation
Third or fourth instar larvae are suitable for virus multiplication. For inoculation,
a virus suspension containing 107–108 OB/ml of water is prepared and the larvae are
fed with a diet containing the viral inocula for 24 h. Thereafter, the virus fed larvae
284 P. Sood et al.
are reared on normal diet in individual plates or containers. The plastic containers
are provided with filter paper for absorption of excess moisture, and the diet and
filter paper are changed on alternate days. The infected larvae begin to show symp-
toms in 5–8 days and start dying from 8 up to 20 days, depending upon the species.
Dead larvae which die due to infection are collected and stored at -20 °C.
(iii) Purification
The infected larvae are macerated in distilled water and filtered through muslin
cloth to remove the debris. Thereafter the suspension is centrifuged at 500 rpm for
3 min and the sediment is discarded. The supernatant is centrifuged at 10000 rpm
for 30 min to sediment the virus. The supernatant is discarded and the virus pallet is
re-suspended in small volume of water to obtain fairly pure preparation of the virus.
(iv) Storage
The virus is stored, suspended in distilled water in amber coloured bottles in a
cool dark place. If possible it can be stored in the refrigerator at 4–5 °C. The virus
can be stored for three or more years without apparent loss in infectivity.
(v) Safety
Most of the GV isolates have been found to be safe to many adult parasitoids and
predators present in the crop ecosystem, as well as to productive insects such as
honey bee, mulberry etc. So far, no occupational hazard has been reported while
working with GVs.
(vi) Mass Production
Baculoviruses have potential to be used as microbial control agents against insect
pests due to their high pathogenicity, host specificity, environmental benefit and
biosafety (Rahman and Gopinathan 2003; Simon et al. 2004). Till date however,
baculovirus-based products used on a large scale for insect pests control have been
produced on infected larvae either field collected, or to the most reared in the labo-
ratory on an artificial diet. It is very pertinent to determine the optimal parameters
for baculovirus production under in vivo conditions, including diet composition,
rearing conditions (containers used, temperature, humidity), and the age/develop-
mental stage at which to infect larvae (Shapiro 1986; Hunter-Fujita et al. 1998). In
addition, facilities such as equipments, processes for efficient infection of larvae,
collection of cadavers, and isolation of viral occlusions are also a prerequisite
(Wood and Hughes 1996; Black et al. 1997; van Beek and Davis 2007). However,
in vivo mass production on host larvae suffers from certain problems such as the
insurgence of diseases in the primary host production colony (McEwen and Hervey
1960) and contamination of the final product with other microorganisms and poten-
tially allergenic insect parts (Podgwaite et al. 1983; McKinley et al. 1997). Hence,
efforts have also been made for in vitro mass production of baculoviruses using
insect cell cultures. These do not only result in an improved quality product but also
result less expensive, requiring less labour for production at a larger scale. They also
allow for the production of baculoviruses that infect host species for which their
in vivo production is not feasible or desirable (e.g., species with small larvae, such
as P. xylostella, or species with urticacious hairs, such as L. dispar). The develop-
ment of suitable, specific media for cell culture in large scale for in vitro production
of baculoviruses has further been made more feasible (Maiorella et al. 1988;
Murhammer and Goochee 1988).
Baculoviruses used as biocontrol agents, however, suffer from instability after
exposure to solar irradiation, especially due to the quick inactivation of the virus by
sunlight. UV-B (280–310 nm) in particular are responsible for the inactivation of
insect pathogens in general and of insect viruses in particular, under natural condi-
tions (Ignoffo et al. 1977; Asano 2005). The resulting loss of biological activity
affects the rate at which insect-pests are killed and in many instances the pathogens
lose >90% of their original activity within days (Ignoffo et al. 1977; Shapiro et al.
2002). Sometimes, the efficacy of these microbials is greatly reduced within the first
24–48 h post spraying (Ignoffo and Batzer 1971; Young and Yearian 1986). These
factors lower the satisfactory field utilization of GVs for pest management (Timans
1982; Shapiro et al. 2009). Brightners, white carbon and antioxidants have been
found to provide some level of UV protection for insect viruses (Shapiro and
Robertson 1990; Shapiro 1992, Sood et al. 2013b).
(vii) Formulation
Formulation of biopesticides can improve their efficacy to acceptable levels of
pest control, that can be achieved with lower doses, finally representing an impor-
tant reduction in the cost of each application. The efficacy of bioinsecticides that act
by ingestion can be improved by the use of phagostimulant formulations that
increase the consumption by the pathogen. In the case of baculovirus-based insecti-
cides, the use of feeding stimulants that encourage phytophagous larvae to consume
foliage contaminated with OBs can result in an increased prevalence of infection
and improved pest control. However, the added cost of such formulations may not
always be justified by corresponding improvements in crop yields. The most com-
mon baculovirus formulations are water-dispersible liquids or powders that are
applied to crops as aqueous sprays using conventional spray equipment. One limita-
tion of baculoviruses as biopesticides is that they are rapidly deactivated by UV
light exposure (Ignoffo et al. 1989). The incorporation of UV-protectant sunscreens
into baculovirus formulations is therefore a subject of considerable interest (Shapiro
et al. 1983; Williams and Cisneros 2001). Stilbene optical brighteners substantially
enhanced the infectivity of baculoviruses (Hamm and Shapiro 1992; Dougherty
et al. 1996). It appears that they change the pH of the insect midgut and also block
the sloughing of primary infected midgut cells, leading to a higher probability of
establishment of infection in larvae simultaneously fed with virus and optical
brightener, compared with virus alone (Washburn et al. 1998). Several optical
brighteners are known to interfere with chitin fibrillogenesis, increasing the perme-
ability of this structure which normally acts as a defensive barrier to microbial inva-
sion (Wang and Granados 2000). The inclusion of optical brighteners in formulations
may substantially increase the viability of baculoviruses used for biocontrol as
higher pest mortality can be achieved with lower applications of virus (Hamm et al.
286 P. Sood et al.
1994; Thorpe et al. 1999). Although optical brighteners are widely used for domestic
and industrial applications, they were not previously tested in the environment. If
they were used in bio insecticide-based control systems, they could be applied to
crops over large areas but little is known on the potential consequences of such
actions, including possible effects on crops growth.
The ingredients of a biopesticide formulation must: i) ensure stability during
biopesticide production, processing and storage, ii) assist application, iii) protect
the biopesticide from unfavorable environmental conditions iv) promote its activity
at the target site. Formulations are composed of i) an active ingredient ii) carriers,
often an inert material used to support and deliver the densely populated active
ingredient to the target and iii) adjuvants, compounds that: promote and sustain the
function of the active ingredient by protection from UV radiation, ensure rain fast-
ness on the target, retain moisture or protect against desiccation, or promote the
spread and dispersal of the biopesticide. Most of the viral biopesticides are formu-
lated as dusts because of a number of advantages over liquid formulations. List of
GV based products commercialized worldwide (Modified after Haase et al. 2015) is
provided in Table 10.3.
Table 10.3 List of GV based products commercialized worldwide (modified after Haase et al.
2015)
Producer
Virus name Target insects Crops Product names companies Country
Cydia Cydia Apple, Carpovirus Plus, NPP-Arysta Argentina,
pomonella GV pomonella, pear, Madex, Life Science, Uruguay
Grapholita walnut Carpovirusine, Andermatt Chile
molesta Apple, Madex Twin Biocontrol
peach NPP-Arysta
Life Science
Erinnyis ello Erinnyis ello Cassava, Baculovirus Empresa de Brazil,
GV rubber erinnyis Pesquisa Colombia
trees Agropecuária e
Extensão, Rural
de Santa
Catarina,
BioCaribe,
CORPOICA
Phthorimaea Phthorimaea Potato Baculovirus, CORPOICA Colombia,
operculella operculella PTM SENASA Peru, Peru, Costa
GV Tecia solanivora baculovirus INTA Costa Rica
Corpoica Rica
Phthorimaea Phtorimaea Potato Matapol Plus, PROINPA Bolivia,
operculella operculella Bacu-Turin Foundation, Ecuador
GV + BacillusTecia solanivora INIAP
thuringiensisSymmetrischema
tangolias
Codling moth Codling moth Vegetable Madmex, Andermatt Switzerland,
GV crops Granupom Biocontrol, Germany
10.8 Field Applications
Since many years, GVs have been observed to be responsible for severe epizootics
in various insect populations (Huger 1963). The GV of P. rapae excised an unusual
high regulation degree of the pest during 1947–1948 and 1958 in New Zealand. The
same GV was also observed in Hawaii to cause severe epizootics. Similar observa-
tions were made in Japan (Ito et al. 1960). Other GVs that cause high mortality in
the field have been observed in populations of Choristoneura murinana on spruces,
Hyphantria cunea on walnuts and Cydia pomonella on apples, pears and walnuts. In
Chilo infuscatellus, the GV prevalence ranged from 16 to 36.8% in 1981 and from
17 to 42% in 1982 (Easwaramoorthy 1984). Similarly in Chilo sacchariphagus
indicus, the GV infection ranged from 17.4 to 35% in 1982 and from 18.8 to 45.0%
in 1982.
A number of GVs underwent field testing for management of pests and the
important ones are reviewed below.
The most famous GV which has been developed as a commercial product is
‘Capex’ which is derived from AdorGV and got registered in Switzerland in 1989
(Andermatt 1991).
A successful use of the GV (AsGV) has been reported against Agrotis segetum
on root crops in Denmark (Zethner 1980), on maize in Spain (Caballero et al. 1990,
1991) and lettuces and dwarf asters in Germany (Fritzsche et al. 1991). Working on
improvement of Virin-OS (AsGV), Kitik (1989) during 1987–1989 produced about
20 kg of formulated material which was used in Uzbekistan and Moldovia to control
the pest on cotton, sugar beet, vegetables and winter cereals. A mixture of GV and
NPV has also been tested with reasonable success in the Former Soviet Union. In a
joint project between Denmark and Pakistan, a Danish strain of AsGV was tested in
Pakistan on tobacco. Spraying tobacco seedlings with concentrations from 5 × 107
to 1 × 109 OBs/ml, at 60–250 ml/m reduced second instar larval damage by 72–100%
(Chaudhry et al. 1976; Shah et al. 1979; Zethner et al. 1983).
Potato is an important food crop which is infested with Phthorimaea operculella.
A granulovirus of this insect (PhopGV) has been isolated from Sri Lanka and was
later found also in Tunisia, Yemen, Egypt and elsewhere in Africa. Several isolates
have been characterized (Vickers et al. 1991; CIP, 1993). Its production methodolo-
gies were developed in South America. These technologies appeared highly suitable
and thus were successfully adopted in Tunisia and Egypt, where facilities capable of
production enough virus to treat 10,000 tonnes of tubers were built (Abol-Ela et al.
1996). In Tunisia, trials were conducted to reduce infestation in tubers at harvest
and in stores thereafter, by field applications of PhopGV to the soil surface before
harvest. The spray formulation were reported to reduce field infestation of tubers by
73%. In storage, pest infestation failed to develop from tubers subjected to field
treatment. Trails in Egypt demonstrated a much better rate of post-harvest protec-
tion (95%) using 2 LE (larval equivalent) in 10 ml water with 0.001% Tween 20 per
kg of tubers. The virus was also found effective in Australia (Reed 1969; Reed and
Springnett 1971), Peru (de Oliveira 1998) and Bolivia (Calderon and Andrews
288 P. Sood et al.
1994). In Peru, a PhopGV isolate was developed as a microbial pesticide through an

initiative of the International Potato Center (CIP). This virus provided effective con-
trol of the pest when 20 virus infected larvae were mixed with one kg of talc, and
used as a suspension in 1 L of water. A dry product applied at a dose of 5 kg per
tonne of stored tubers also provided high levels of control (ca. 95% mortality)
(Raman et al. 1987).
Chilo infuscatellus is an important pest of sugarcane. The application of CiGV at
1 × 107 to 109 OBs/ml at 35, 50, 65 and 80 days after planting reduced pest inci-
dence below the economic injury level (Easwaramoorthy 1984, 2002;
Easwaramoorthy and Santhalakshmi 1988). Parameswaran et al. (1991) reported
that application of CiGV (107 OBs), twice at 35 and 50 days after planting, effec-
tively reduced the shoot borer population. Application at a high volume was more
effective than at a low volume (Easwaramoorthy and Jayaraj 1991). The CiGV was
commercially produced by the Tamil Nadu Agriculture University (TNAU),
Coimbatore, India for more than a decade.
The GV of the codling moth, Cydia pomonella has been extensively tested in
various countries. CpGV has been shown to be a potent agent for codling moth
control. The application of CpGV has been especially more useful in regions where
codling moth is univoltine, as in most of Europe. Where there is more than one
annual generation e.g. in California, and especially where such generations have not
exactly synchronized phenology, the pest appeared more difficult to manage. The
use of pheromone trapping to reveal adult emergence patterns permits improved
accuracy of time of spraying. Developmental programmes have been conducted in
many countries such as USA and inside Europe in Austria, Germany, the Netherlands,
United Kingdom and Switzerland. Under an IOBC/WPRS working group, the latter
group of nations, together with Hungary tested the virus in more than a dozen
orchards in 1976–1977, using material produced in Darmstadt, Germany (Huber
1981).
A C. pomonella granulovirus (CpGV) identified by Tanada (1964) was capable
of causing epizootics in populations infesting pome fruits and walnuts. Field tests
data proved this virus to be a potential candidate in providing an effective alterna-
tive for control of codling moth larvae in integrated and organic pome fruit and
walnut production in several countries (Huber and Dickler 1977). In Argentina,
Carpovirus Plus® (Quintana and Alvarado 2004) and Madex® (Haase et al. 2015)
showed highly satisfactory results when the virus was applied at doses of 1013 OBs/
ha at intervals of 8–10 days.
An interesting separation of two strains of the AdorGV replicating in either the
nucleus or the cytoplasm has been made, but it is not known which one was
employed in field tests (Huber 1998).
In field collected Cnephasia spp., Glas (1991) found a GV that infected both C.
longana and C. pumicana. This author showed that it is possible to spread the virus
disease by spraying GV suspensions on the bark of trees. A dose of 2 × 1010 OBs per
trunk was sufficient to induce more than 90% larval mortality, leading to a complete
breakdown of the pest population. In a field where the population was already
p resent, spraying of GV on the eggs present on the trees was still useful, as the
population collapsed 3 weeks earlier than in the untreated plots.
Several commercial products of CpGV have been marketed in Europe. They
include Carpovirisine (Burgerjon and Sureau 1985), Madex, Granupom, Granusal
etc. In the Netherlands, Granusal was as effective as a chemical regime (Helsen
et al. 1992). In Italy, field comparisons of Carpovirisine and Granupom suggested
that both were as effective as a standard insecticide (Pasqualini et al. 1994). Studies
on the non-chemical control of C. pomonella in France over a 10 year period were
reviewed by Andemard (1986), who concluded that mating disruption and CpGV
were the most promising management approaches.
The fall armyworm Spodoptera frugiperda is another pest of economic impor-
tance. It is a polyphagous insect that causes economic losses in several important
crops, such as maize, sorghum, rice, cotton, and pastures. Since this pest showed
resistance to other control tactics, baculoviruses were evaluated for effectiveness in
Argentina (Yasem de Romero et al. 2009), Brazil (Valicente and da Costa 1995),
Mexico (Ríos-Velasco et al. 2012) and Peru (Vásquez et al. 2002). The efficacy in
controlling the pest was similar to that of chemical insecticides, 22 days post emer-
gence of the pest (Gómez et al. 2013).
The epizootiology of a naturally occurring GV of Dendrolimus sibiricus was
studied in the former Soviet Union by Baranovsky and Litvina (1978), and subse-
quently this GV was commercialized. Applications are best made in the autumn
against early instars (Orlovskaya 1989). Erinnys ello, although known to attack over
35 plant species in South America, is known especially as a pest of cassava and rub-
ber. In Brazil, the ErelGV has been used on over 2000 ha of cassava and rubber (de
Oliveria 1998). A standardized formulation consists of 20 ml (18 g) of a crude and
filtered macerate which is diluted for spraying in 200 L water/ha or for ULV in 3 L
water/ha. In Colombia, 50–70 ml of larval macerated in 200 L water with 0.2 ml
Triton-ACT is sprayed per ha. The speed of action in Colombia is phenomenal, as
>80% mortality was recorded in 48 h. In Southern Brazil, a mortality of 90% was
attained in 4 days after spraying of 20 ml of larval macerate. An increase in numbers
of natural enemies following GV application is thought to explain the observed
spread of infection from sprayed areas.
Hypanthia cunea is a serious defoliator of popular and other deciduous trees.
Experimental Hypanthia cunea granulosis virus (HycuGV) insecticides were devel-
oped and tested in Bulgaria with the commercial name of Hifantrin and in Russia
and Ukraina with the name of Virin-ABB. Satisfactory results were reported from
Ukraina (Sikura and Smetnik 1980; Kransnitskaya 1980). At present Virin-ABB-3
is in commercial production in Moldavia (Chkhirii 1993).
Limited greenhouse trails indicated that Lacanobia oleracea GV has a strong
potential as a pest control agent on tomato (Crook and Brown 1982).
The effectiveness of spraying the homologous GV of Pieris rapae alone or in
combination with Bacillus thuringiensis (Bt) var. kurstakii was reported in Taiwan
(Su 1986, 1991). Control was effective for 14 days in the field with persistence of
GV exceeding that of Bt in China, using 1 g of the powered GV product in 30 ml
water, presumably sprayed to get a reasonable droplet coverage with 90% control of
290 P. Sood et al.
P. rapae on cabbage. Rituma (1990) reported that virin-KB gave 70–96% control
and increased the yield value by 150 roubles/ha in Lativa. In North America, the GV
is recommended at a dose of 7.5 × 1012 OBs/ha in about 100 L/ha. Two to six appli-
cations per year are made on cole crops, depending on date of planting and harvest.
The control of P. rapae by GV measured by crop protection equals or exceeds con-
trol by either chemical insecticides or Bt (Cunningham 1998).
A freeze-dried preparation was found to be effective for protection of almonds
and raisins against attacks by the Indian meal moth, Plodia interpunctella Hubner.
The virus was extensively tested on dried fruits and nuts and, to a lesser extent, on
grains. It is used as a prophylactic material and persistence is adequate to provide
protection from packaging of foods for consumption. The population of P. inter-
punctella was reduced by 95–100% and damage to products was reduced to a non-
detectable level. A formulation of this virus has been patented by the USDA
Agricultural Research Service.
Cabbage butterfly, Pieris brassicae (Linnaeus) (Lepidoptera: Pieridae), is a seri-
ous pest of cabbage, cauliflowers and many crucifer crops of the world (Feltwell
1978; Bhandari et al. 2009). A P. brassicae granulovirus (PbGV) strain was isolated
and characterized from Himachal Pradesh, India by Sood (2004), which was effec-
tive in managing P. brassicae larvae (Sood et al. 2011; Sood and Prabhakar 2012).
The effective formulation technology is another challenge for the development
of a stable insect virus product under field conditions. Usually, active ingredients
i.e. virus OBs/POBs are mixed with various adjuvants which improve the efficacy,
stability and handling of the pesticide. The most common process of formulating a
virus is drying the infected larvae, either through dehydration, lyophilization or by
an air flow, in order to generate a powder. Lactose was also added to improve the
viruses stability and infectivity. A suitable carrier depending upon its compatibility
with the virus particles is then added to achieve the uniform concentration. Silica
and clays are commonly used for this purpose. Surfactants, adherents, thickeners,
binders, phagostimulants, UV protectants or optical brighteners, boric acid as stress
causative, etc. may also be added to make a good formulation (Sood et al. 2013a, b).
Formulation in the form of microencapsulation has also been developed.
10.9 Conclusion
Under natural conditions insect populations are regulated by epizootics caused by

various entomopathogens such as bacteria, fungi and viruses etc. These may offer
viable pest management solutions in most agroecosystems. The growing knowledge
and awareness on the negative impacts of chemical insecticide usage on the ecosys-
tem and public health led to efforts directed towards usage of biopesticides over
synthetic pesticides. Among various entomopathogens, baculoviruses in particular
are promising alternatives. Many virus-based formulations have been used in the
past and are available worldwide, but their usage has been still restricted compared
to other pesticides. Their increased utilization therefore requires enhanced virulence
coupled with speed of kill and stability in the ecosystem. Accordingly, viruses ini-
tially found in nature need to be improved in terms of virulence and stability to
make them more potent, before field usage. A number of granuloviruses infecting
major insect pests have been isolated and tested for management, but only a few
have been commercialized owing to their variable potency and stability. To counter
this problem, some researchers manipulated and formulated stable products.
However, still a lot has to be done to popularize their usage in large scale pest man-
agement programmes, which can be achieved by developing more virulent and sta-
ble formulations, apart from ease of the registration procedure.
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Chapter 11
Interactions of Entomopathogens
with Other Pest Management Options
Surendra K. Dara
Abstract As the demand for sustainably produced food is increasing, the use of
biopesticides has also been increasing in the recent years. While biopesticides based
on entomopathogens have been successfully employed for pest management around
the world for decades, there are still some concerns about their efficacy relative to
chemical pesticides, limiting their widespread adaptation. Microbial pesticides
could be an important part of all integrated pest management (IPM) strategies and
understanding their interactions with other control options can help promote their
use. Several studies demonstrated improved pest control efficacy when different
kinds of entomopathogens were used. Entomopathogens were combined or rotated
with chemical or botanical pesticides, or virulence of certain entomopathogens was
synergized by chemical pesticides. Certain fungicides are also compatible with
entomopathogenic fungi enabling pest and disease management treatments at the
same time. Improved microbial control can help reduce chemical pesticide use and
the associated environmental and human health risks.
Keywords Entomopathogens · Microbial control · Integrated Pest management ·

Synergy
11.1 Introduction
Entomopathogens such as bacteria (e.g., Bacillus, Serratia, and Yersinia), fungi

(e.g., Beauveria, Conidibolus, Entomophaga, Entomophthora, Erynia, Hirsutella,
Isaria, Lecanicillium, Metarhizium, Neozygites, and Pandora), microsporidia (e.g.,
Brachiola, Endoreticulatus, Nosema, and Vairimorpha), nematodes (e.g.,
Heterorhabditis and Steinernema), and viruses (e.g., granuloviruses and nucleo-
polyhedroviruses) have been used for pest management in agriculture, forestry, ani-
mal husbandry, aquatic or urban environments, to manage a variety of arthropod
S. K. Dara (*)
University of California Cooperative Extension, San Luis Obispo, CA, USA

https://doi.org/10.1007/978-3-030-23045-6_11
300 S. K. Dara
pests. While some of these pathogens manage target pest populations through natu-
ral infections, others are mass-produced and applied through inundative releases.
Several entomopathogens have been developed into a number of commercial
biopesticide formulations and used for pest management, alone or in combination
or rotation with other management options.
Traditionally, entomopathogen-based microbial pesticides had limited use com-
pared to chemical pesticides and were used in high value crops, greenhouse pro-
ductions systems, or when control with chemicals or other conventional options
was difficult. Some of the factors that may limit their widespread use are related to:
a higher cost of microbial pesticides compared to chemical pesticides and the need
of special storage and handling, shorter shelf life, poor quality control, slow rate of
pest control, a lack of understanding of microbial control mechanisms, and the
general perception of variability or unreliable control. While a few manufacturers
of microbial pesticides maintained their share in some niche markets, complexities
of mass-production of entomopathogens and the high cost of registering pesticide
formulations restricted the development of new microbial pesticides, although
research around the world identified several virulent isolates. However, indiscrimi-
nate use of chemical pesticides that frequently leads to the development of resistant
pest populations as well as raises concern for environmental and human health,
brought attention, in recent years, to sustainable food production using safer pest
control options. Because of the increased demand for organically produced food,
organic acreage has been steadily increasing around the world, ultimately creating
a demand for non-chemical control options including microbial pesticides. For
example, between 2007 and 2012, there was an 83% increase in the sales of organic
products by the farms in the United States (USDA-NASS 2016). To maintain their
share in the pesticide market, several large chemical pesticide companies are now
adding microbial pesticides, among other non-chemical tools, to their product
portfolios. An emphasis on sustainable food production is also promoting the use
of microbial pesticides on conventional farms, as a part of integrated pest manage-
ment (IPM), in addition to their common use in organic farms.
In light of the demand for sustainably produced foods and increasing popularity
and use of microbial pesticides, research studies are evaluating the efficacy of vari-
ous microbial pesticides and their incorporation into IPM strategies. This chapter
focuses on the interaction of entomopathogens among themselves and with other
pest management options, to understand their efficacy and role in IPM.
11.2 Entomopathogen Combinations
Similar to the combinations of chemical pesticides aiming at improving pest control

efficacy, certain combinations of entomopathogens can also be employed for pest
management with additive or synergistic effects. A number of studies evaluated the
combination of entomopathogenic nematodes (EPN) with entomopathogenic fungi
(EPF) or bacteria against various pests. Additive effect of Metarhizium anisopliae
11 Interactions of Entomopathogens with Other Pest Management Options 301
with Heterorhabditis indica at two application rates, and Beauveria bassiana with
the bacterium Serratia marcescens at a higher rate, were observed against the pecan
weevil, Curculio caryae resulting in increased pest mortality (Shapiro-Ilan et al.
2004). Similarly, the combination of M. anisopliae and Steinernema kraussei as a
soil drench resulted in 100% mortality of the overwintering larvae of the black vine
weevil, Otiorhynchus sulcatus in strawberry compared to 50–88% mortality by M.
anisopliae and 60–69% mortality by S. kraussei, when applied alone (Ansari et al.
2010). Acevedo et al. (2007) reported that the combination of a Brazilian isolate of
H. bacteriophora (JPM4) and an isolate of M. anisopliae (LPP39) improved the
mortality of the sugarcane borer, Diatraea saccharalis, with reduced LT50 and LT95
values. However, production of the infective juveniles (IJ) was reduced in the pres-
ence of M. anisopliae. In such cases, applying EPF prior to the application of EPN
appeared to be ideal for IJ production (Shaurub et al. 2016). Application of B. bassi-
ana before applying S. riobrave or H. bacteriophora improved IJ production in the
Egyptian cotton leafworm, Spodoptera littoralis. Combination of different EPN or
EPN with B. bassiana also resulted in increased S. littoralis mortality. Similar
improved control from the co-application of EPF and EPN was seen in the large
pine weevil, Hylobius abietis in Ireland (Williams et al. 2013) and the chestnut
weevil, Curculio elephas in Turkey (Asan et al. 2017). Fungal infections probably
weaken the pest and increase infection by EPNs.
The combination of EPF and bacteria was also found effective in different studies
where bacterial infection reduced insect feeding, weakening the pest, delaying molt-
ing and increasing the chances of fungal infection. The combination of B. bassiana
and B. thuringiensis subsp. tenebrionis increased the control efficacy by 6–35% in
Colorado potato beetle, Leptinotarsa decemlineata in field studies in the United
States (Wraight and Ramos 2005). Similarly, improved mortality in a shorter time
was achieved by the combination of B. thuringiensis subsp. morrisoni (Btm) with
lower conidial concentrations of M. anisopliae or B. bassiana vs L. decemlineata in
Kazakhstan (Kryukov et al. 2009). Yaroslavtseva et al. (2017) demonstrated that sub-
lethal doses of Btm var. tenebrionis significantly suppressed the cellular immunity
and inhibited the detoxification enzymes in L. decemlineata resulting in a synergy
with M. robertsii. Preliminary laboratory studies in China showed that B. bassiana
infection in the second instar larvae of the spotted asparagus beetle, Crioceris quatu-
ordecimpunctata, increased from 42% to 78% in the presence of a sublethal dose of
Cry3Aa Bt toxin (10 μg/ml) (Gao et al. 2012). In an earlier study, synergism was
seen between M. anisopliae and Serratia entomophila against the second instar lar-
vae of the New Zealand grass grub, Costelytra zealandica (Glare 1994).
In a Greek study, Mantzoukas et al. (2013) evaluated different combinations of
Bt subsp. kurstaki (Btk), B. bassiana, and M. robertsii against the Mediterranean
cornborer, Sesamia nanogrioides and found synergistic, additive, and negative
interactions depending on the combination and time of observation. Sayed and
Behle (2017) evaluated different proportions of Btk spores and crystals and B.
bassiana blastospores against the cabbage looper, Trichoplusia ni, in the United
States and found that 50:50 ratio was synergistic. A recent study in Pakistan
demonstrated that EPF-Bt combination can also be used against sucking pests
302 S. K. Dara
(Jugno et al. 2018). Commercial formulations of M. anisopliae and Bt were tested

alone and in combination against the Indian cotton jassid, Amrasca biguttula and
the cotton aphid, Aphis gossypii on eggplant. The combination killed 83% of the
jassids and 72% of the aphids compared to 53% and 52% mortality by Bt and 67%
and 59% by M. anisopliae in jassid and aphid populations, respectively.
Combining entomopathogens with different modes of infection can enhance the
pest control efficacy by taking advantage of multiple routes of infection. EPF infec-
tions are primarily by contact, bacterial infections are by ingestion, and EPN infec-
tions occurs through natural openings or cuticle by IJs actively seeking their host.
Compared to younger pest larvae, older ones are more resistant to bacterial infec-
tions while EPF can be more effective against adults than immature stages, that
might escape infection through molting. Combination of pathogens can also help
address these situations by targeting multiple life stages.
11.3 Entomopathogens with Natural Enemies
Natural enemies of arthropod pests coevolved with entomopathogens and devel-

oped their own ways of resisting or avoiding infections. However, the susceptibility
of natural enemies to entomopathogens depends on the arthropod species or the
strain of the pathogen. For example, the B. bassiana isolates from the native cocci-
nellid predator, Olla v-nigrum in Southeastern United States were highly patho-
genic to the native predator, but not to the invasive Harmonia axyridis (Cottrell and
Shapiro-Ilan 2003). However, the commercial GHA isolate of B. bassiana was rela-
tively less pathogenic to both lady beetle species. A laboratory study in Austria
showed that a local isolate of B. bassiana was pathogenic to the bark beetle, Ips
sexdentatus, but appeared safe to its predator, the European red-bellied clerid
(Thanasimus formicarius) at the same concentration (1 × 107 conidiophores/ml).
Only when a very high dose of dry conidia of unspecified concentration was applied
to the entire ventral side of the adult predators, 47% of them died from 24 to 48 days
after inoculation (Steinwender et al. 2010). Similarly, Bourassa et al. (2001) reported
that some African isolates of B. bassiana and M. anisopliae caused 77–85% mortal-
ity in adult larger grain borer, Prostephanus truncatus, compared to a 13–21% mor-
tality in its predator, Teretriosoma nigrescens, 6 days after treatment. While the pest
mortality went up to 91–96% in 14 days, that of the predator increased up to
42–56%. A Canadian study evaluated 10 isolates of B. bassiana from North
America, Europe, and West Africa against L. decemlineata, the green peach aphid,
Myzus persicae, and a predatory coccinellid, Coleomegilla maculata lengi (Todorova
et al. 2000). While six of the isolates were highly pathogenic to both the pest and
predatory species, two Canadian isolates, a Bulgarian and a Beninese isolate were
highly pathogenic to the pest species, but not the coccinellid predator.
Jacobson et al. (2010) reported that application of B. bassiana effectively con-
trolled populations of the western flower thrips, Frankliniella occidentalis, in
glasshouse cucumbers without having any negative impact on the predatory mite,
Neoseiulus cucumeris, which was prophylactically released to prevent the buildup

of whitefly populations. There was no difference in predatory mite numbers on
treated plants, compared to untreated plants, even 3 weeks after B. bassiana applica-
tion. Similarly, B. bassiana did not affect the predation of Phytoseiulus persimilis
on the twospotted spider mite, Tetranychus urticae (Wu et al. 2018). Studies con-
ducted in California strawberries also did not show any negative impact of B. bassi-
ana on P. persimilis and Neoseiulus spp. when evaluated against T. urticae (Dara
2015a). No impact was also observed for B. bassiana, I. fumosorosea, and M. brun-
neum on a variety of arthropod natural enemies, when tested against the western
tarnished plant bug, Lygus hesperus (Dara 2016). However, an isolate of M. brun-
neum was highly pathogenic to the predatory mites P. persimilis and Neoseiulus
californicus (Dogan et al. 2017) while B. bassiana and I. fumosorosea caused 43
and 31% mortality in P. persimilis, respectively, and about 16% mortality in N. cali-
fornicus in laboratory assays (Vergel et al. 2011). In studies conducted in Benin, the
predatory mite Typhlodromalus aripo consumed equal numbers of healthy cassava
green mites (Mononychellus tanajoa) and those infected with the entomophthora-
lean fungus Neozygites tanajoae (Ariori and Dara 2007; Agboton et al. 2013).
Combined use of T. aripo and N. tanajoae in screenhouse experiments showed
reduced efficacy of T. aripo (Agboton et al. 2013) in the presence of the fungus. The
preference of T. aripo to feed on fungus-infected pest mites and subsequent loss of
survival and oviposition were thought to be responsible for the reduced efficacy of
the predatory mite, in the presence of the fungus.
In other studies, combined use of biological and microbial control agents
improved the control efficacy. For example, the combination of the mosquito preda-
tor, Toxorhynchites brevipalpis and M. brunneum against Aedes aegypti larvae
showed additive and synergistic effect at higher conidial concentrations (Alkhaibari
et al. 2018). Since M. brunneum is pathogenic to T. brevipalpis, their combination
was antagonistic at the concentrations of 105 and 106 conidia or blastospores/ml.
However, the addition of the mosquito predator had an additive effect on the median
LT50 of Ae. aegypti caused by the fungus at 107 conidia/ml and a synergistic effect at
108 conidia/ml. However, such an effect was not seen with blastospores because
they were more virulent to the predator. In a 1981 study in the United States, para-
sitization of the gypsy moth (Lymantria dispar) by Apanteles melanoscelus
increased 6–12 times in plots treated with Bt showing a synergistic effect (Weseloh
et al. 1983). Bt reduces the size of surviving caterpillars and A. melanoscelus para-
sitism is higher in smaller caterpillars.
Natural enemies can also help to spread an entomopathogen infection. Kryukov
et al. (2017) reported that the ectoparasitoid Habrobracon hebetor, did not distin-
guish between healthy greater wax moth (Galleria mellonella) larvae and those
infected with B. bassiana and significantly increased fungal infections through pas-
sive vectoring. It also appeared that envenomation by the parasitoid increased the
conidial germination on the insect cuticle and reduced the fungal encapsulation
(insect immune response) in the hemolymph. Roy and Pell (2000) discussed syner-
gistic and antagonistic interactions of EPF and various natural enemies. For
example, foraging natural enemies increased the infections of the entomophthora-
304 S. K. Dara
lean fungi Pandora neoaphidis in pea aphid, Acyrthosiphon pisum, and Zoophthora
radicans in the diamondback moth, Plutella xylostella, in laboratory studies, in
some cases spreading the infection to healthy pest populations on different plants.
Although some species of natural enemies are physiologically susceptible, espe-
cially under laboratory conditions, to hyphomycetous fungi that have a wide host
range, they are ecologically less susceptible under field conditions.
These studies show entomopathogen and natural enemy combinations that are
either compatible or more effective. Understanding these interactions will help
management decisions such as those considering the timing of natural enemies or
entomopathogens release or certain combinations to avoid.
11.4 Entomopathogens with Semiochemicals, Traps,

and Netting
Unlike chemical and botanical pesticides, which kill the target arthropods either
through direct contact or systemic activity in the plant, entomopathogens can per-
sist, proliferate, and be disseminated in pest populations and habitats. Depending on
the insect species, bait stations or pheromone traps equipped with entomopatho-
gens, or insect netting treated with EPF, can be used to kill the visiting insects or
inoculate them to disseminate inocula or infection in the rest of their populations.
An earlier study in the United States by Nordin et al. (1990) explored autodis-
semination of two nucelopolyhedroviruses (NPV) from treated males to mated
females and eventual transovarial transmission. This was a preliminary step to
develop a technique using sex pheromones. Virus-treated males of tobacco bud-
worm, Heliothis virescens, mated with females resulting in 69% virus-induced mor-
tality in the progeny in the case of Autographa californica multiple
nucleopolyhedrovirus (AcNPV), and 53% in the case of Helicoverpa zea single
nucleopolyhedrovirus. However, in a two-year follow up field study using phero-
mone traps, male H. virescens transmitted AcNPV to females and caused subse-
quent larval mortality in the next generation, but to a limited extent (Jackson et al.
1992). Dispersal of H. virescens moths beyond the experimental plots was thought
to be a reason for observing limited efficacy.
Frankliniella occidentalis acquired twice as many M. anisopliae conidia in the
presence of a semiochemical (5.0 × 104) in an autoinoculation device, compared to
not having the semiochemical (2.2 × 104) in French bean fields, resulting in a 42%
increase in thrips mortality in a Kenyan study (Niassy et al. 2011). However, semio-
chemical volatiles drastically reduced the conidial viability within 7 days, with a
gradual decline in daily mortality. In a field study in Canada, control of the artificial
infestations of the common click beetle, Agriotes obscurus, by band applications of
M. brunneum conidia on rice granules significantly improved when pheromone-
impregnated cellulose-based granules were also applied (Kabaluk et al. 2015).
Some studies evaluated the mating competitiveness of males treated with EPF and
found that fungus-treated males were equally competitive as untreated males for the
first few days after inoculation and that their competitiveness declined as the infec-
tion progressed (Dimbi et al. 2009; Thaochan and Ngampongsai 2018). In their
laboratory study conducted in Spain with an attractant and M. anisopliae against the
Mediterranean fruit fly, Ceratitis capitata, San Andrés et al. (2014) also showed that
the fitness and attractiveness of sterile or wild type males were not affected by fun-
gal infection.
Entomopathogens such as EPF can be used for managing non-agricultural pests
such as ticks, flies, and mosquitoes. In Kenya, a 64% reduction in field populations
of the tropical bont tick, Amblyomma variegatum, was achieved by using M. aniso-
pliae in a semiochemical-baited trap (Nchu et al. 2010). A simple contamination
device made with a clear plastic water bottle helped infect >90% of the visiting
tsetse flies (Glossina spp.) with a Kenyan isolate of M. anisopliae (Maniania 2002).
Conidial viability lasted for 31 days under field conditions in this study. Mosquito
nets treated with M. anisopliae and B. bassiana were successfully used for control-
ling different species of mosquitoes (Mnyone et al. 2010; Howard et al. 2010).
11.5 Entomovectoring
Entomovectoring refers to the use of pollinators such as bees to deliver entomo-

pathogen inocula to pest habitats. Although pollinators are not directly involved in
pest management, this section is included to provide a brief overview of how the
foraging behavior of bees can be used for microbial control. Beehives or nesting
units are attached with compartments containing entomopathogen inocula at the
exit points. Bees walk through and pick up inoculum as they leave the hives and
passively disperse it as they visit different plants. When bees return to the hives,
they carry very low levels of inoculum and the high temperatures in the hives are
generally detrimental to the entomopathogens, preventing infections in bee colo-
nies. While this technology is now commercially available (e.g. Flying Doctors® by
Biobest) to deliver beneficial microbes for pest and disease control, several earlier
studies explored different entomovectoring concepts initially for controlling plant
pathogens, and later for arthropod pests.
In 1989, Gross et al. (1994) developed a ‘honey bee hive compatible pathogen
applicator device’ to deliver Heliothis NPV (HNPV) in crimson clover (Trifolium
incarnatum), in Southeastern United States. HNPV-induced mortality in corn ear-
worms (Helicoverpa zea) was significantly higher (74–87%) in fields foraged by
Apis mellifera with HNPV compared to control fields (11–14%). HNPV-induced
mortality was also higher in Heliothis spp. collected from the treated fields and held
in the lab (27–42%) compared to those from the control fields (2%). Similarly, mor-
tality of H. zea that fed on cranesbill (Geranium carolinianum) fruits and flowers
from the treated fields or on the honey from HNPV exposed beehives was also very
high compared to controls. In 1996 and 1997 Jyoti and Brewer (1999) used A.
mellifera to deliver Bt in North Dakota sunflower fields to control the banded sun-
306 S. K. Dara
flower moth, Cochylis hospes. They did not evaluate the pest mortality in the field,
but fed the larvae with diet containing wash water from sunflower capitula collected
in fields foraged by A. mellifera carrying Bt, fields sprayed with Bt, and untreated
fields resulting in 87–88%, 58–68% and 8–10% mortality, respectively. Seed yield,
seed weight, oil content, and damage control from bee-vectored Bt fields were equal
or superior to those measured in Bt sprays. Butt et al. (1998) reported using A. mel-
lifera to deliver M. anisopliae to control the pollen beetle, Meligethes aeneus, in
oilseed rape during the winter and spring of 1997, in field cages (2.7 × 2.7 × 1.8 m)
in the United Kingdom. When beetles collected from the cages were incubated in
the lab, the mortality on different sampling dates during the two seasons varied from
23–99% in the presence of bees with M. anisopliae, compared to 0–23% in the pres-
ence of bees without the fungus. In a Canadian study, when B. bassiana was deliv-
ered by the bumble bee Bombus impatiens in greenhouse sweet peppers, it resulted
in 34–45% mortality in the tarnished plant bug, Lygus lineolaris, and 34–40% in F.
occidentalis, compared to 9–15% and 3% in controls, respectively (Al-mazra’awi
et al. 2006a). In another Canadian study in greenhouse tomato and sweet pepper, B.
bassiana delivered by B. impatiens provided a significant control of the greenhouse
whitefly, Trialeurodes vaporariorum (49%) in tomato, and of L. lineolaris (73%) in
sweet pepper (Kapongo et al. 2008). Bees also delivered another fungus,
Clonostachys rosea (Gliocladium roseum), resulting in a significant suppression of
the grey mold caused by Botrytis cinerea in tomato and sweet pepper. In a follow up
study in greenhouse tomato and sweet pepper, Shipp et al. (2012) found no negative
impact of B. bassiana delivered by B. impatiens on parasitoids Encarsia formosa,
Eretmocerus eremicus, and Aphidius colemani and the predatory mite Amblyseius
swirskii. Although mortality of the predator Orius insidiosus was higher in B. bassi-
ana treatment, there was no difference in overall parasitism or predation by different
species.
Bumble bee B. terrestris picked up 9.3 × 106 M. anisopliae conidia on their body
as they walked through the inoculum containing 107 conidia/g and retained 28% of
those spores after flying for a minute, with no negative impact on survival (Smagghe
et al. 2013). Similarly, Lin et al. (2017) explored the idea of delivering B. bassiana
with predatory mites Stratiolaelaps scimitus, N. cucumeris and Amblyseius swirskii.
Predatory mites retained conidia mixed in the substrate (inert material used for
transporting and delivering predatory mites) with minimal or no negative effect on
their survival by the fungus. However, evaluation of the efficacy of B. bassiana on
the pest mite was not within the scope of this study. In a Canadian study, B. bassiana
transported by honey bee, A. mellifera, in canola (Brassica napus) resulted in a
higher mortality (48–56% in 2002 and 22–45% in 2003) in L. lineolaris, compared
to those in control cages visited by fungus-free bees (9–10% in 2002 and 15–22%
in 2003) (Al-Mazra’awi et al. 2006b).
Entomovectoring is now more common in greenhouses than in open fields. Since
bee pollination is utilized in a number of field crops and orchard systems, growers
can take advantage of the bees to deliver inocula of entomopathogens for arthropod
pests and other beneficial microbes for disease control. This practice can reduce the
cost of applying pesticides and increase the control efficacy.
11.6 ntomopathogens with Botanical and Chemical

E
Pesticides
Using entomopathogens with botanical and chemical pesticides is a good strategy

to: (i) maintain pest control efficacy, (ii) reduce the risk of resistance development,
(iii) extend the life of effective chemical pesticides, (iv) minimize the impact on
nontarget organisms, (v) optimize the cost of pest management, and (vi) maintain
environmental and human health. Employing a variety of control options is an
important component of IPM, and entomopathogens can be important for many
IPM programs. Several studies demonstrated improved pest management with com-
bining or rotating entomopathogens with botanical and chemical pesticides.
Shi and Feng (2006) reported that combining ~5% of the labeled rate of pyrida-
ben 15% with an emulsifiable formulation of B. bassiana provided a very effective
and prolonged control of the citrus red mite, Panonychus citri, in citrus orchards in
southeastern China, compared to pyridaben treatment alone at the labeled rate.
Exposing the house fly, Musca domestica, to sequential applications of imidacloprid
and B. bassiana at LC30 rates resulted in a significant increase in the mortality than
the individual treatments in both insecticide-resistant and susceptible adults, espe-
cially when B. bassiana followed imidacloprid application (Farooq et al. 2018).
Applying imidacloprid after B. bassiana increased the mortality by 7–9% in the
susceptible strain and by 8–10% in the resistant strain, whereas fungal treatment
after imidacloprid increased 16% mortality in the susceptible strain and 18–22% in
the resistant strain. Although the resistant strain had higher expression of detoxifica-
tion genes and elevated levels of detoxification enzymes, adding the fungus signifi-
cantly improved fly mortality. In a West African study, imidacloprid also improved
the conidial germination of another hypocrealean fungus, Hirsutella thompsonii,
but did not significantly improve the mortality of M. tanajoa, in combination (Dara
and Hountondji 2001). Assays conducted in Georgia showed that simultaneous or
sequential treatment of B. bassiana and the insect growth regulators (IGRs) difluben-
zuron and novaluron had additive effects on the mortality of the second instar
nymphs of the migratory locust, Locusta migratoria migratorioides (Bitsadze et al.
2013). Nymphs were exposed to B. bassiana by dipping in conidial suspensions and
to IGR by feeding pieces of IGR-treated corn leaves. Except when diflubenzuron-
treated leaves were fed first followed by the fungal treatment after 48 hours, simul-
taneous exposure or exposing the nymphs to B. bassiana first resulted in additive
effect. Hiromori and Nishigaki (1998) found improved control of the first instar
larvae of the oriental beetle, Anomala cuprea, when a Japanese isolate of M. aniso-
pliae was used with some chemical insecticides and IGRs in laboratory assays,
although such an improvement could not be noticed in the field. Studies conducted
in California on strawberries and vegetables, and the feedback received from several
vegetable growers showed that combining azadirachtin – a botanical insect growth
regulator, insecticide, and antifeedant – with B. bassiana is more effective than
individual treatments of the fungus or azadirachtin (Dara 2015b, 2016). Infestations
of the honeysuckle aphid, Hyadaphis foeniculi, and the rice root aphid,
308 S. K. Dara
Rhopalosiphum rufiabdominale, in organic celery showed 18 and 129% increase

after two applications of azadirachtin or B. bassiana alone, respectively, while their
combination resulted in a 62% reduction (Dara 2015b). Similarly, a better control of
aphids (unknown species) and L. hesperus was achieved in strawberry by the com-
bination of B. bassiana and azadirachtin or a higher label rate of azadirachtin alone
than individual treatments of B. bassiana or a lower rate of azadirachtin (Dara
2016). When the invasive Bagrada bug, Bagrada hilaris, infested several vegetable
crops in California, several organic growers who were advised to use the combina-
tion of B. bassiana and azadirachtin reported satisfactory control (Dara, pers. com-
munication with farmers). Mohan et al. (2007) tested the compatibility of neem oil
(with 1.5% azadirachtin) with 30 B. bassiana isolates from around the world. They
found that some isolates of B. bassiana were sensitive to neem oil (with 0.15%
azadirachtin) and among compatible isolates, one had a synergistic impact on the
mortality of second instar larvae of the tobacco cutworm, Spodoptera litura.
Combination of B. bassiana and azadirachtin also improved the control of the
sweetpotato whitefly, Bemisia tabaci, on eggplant with improved egg and nymphal
mortality and lower LT50 values, than their individual treatments, in China (Islam
et al. 2010). Since immature stages of insects can escape fungal infection due to
molting before cuticle penetration of the germ tubes, a combination of chemical or
botanical IGRs with EPF can work in two ways: i) IGRs target immature stages
while EPF target adults and susceptible immatures, and ii) IGRs interfere with molt-
ing and make immatures more vulnerable to fungal infections. Some pesticide man-
ufacturers are now marketing B. bassiana formulations with pyrethrums and/or
azadirachtin (e.g. BotaniGard MAXX, XPECTRO by LAM International) or pyre-
thrins with azadirachtin (e.g. Azera by Valent USA) among other active ingredient
combinations.
Laboratory studies in Russia found synergy between lower rates of M. robertsii
(at 5 × 105 conidia/ml) and a commercial formulation of avermectins (metabolites
of Streptomyces avermitilis at 0.005%) against L. decemlineata larvae (Tomilova
et al. 2016). Avermectins significantly suppressed the insect immune responses and
helped with increased fungal infections resulting in 1.2 to 1.6-fold increases in con-
trol efficacy.
Niu et al. (2016) reported additive and synergistic interaction of S. marcescens
with spirotetramat and thiamethoxam, depending on the concentration, in labora-
tory and greenhouse studies in China, against the third instar nymph of the brown
plant hopper, Nilaparvata lugens. In a study conducted in the United States, Wang
et al. (2013) found out that feeding a low dose of the chitin synthesis inhibitor,
lufenuron to the Formosan subterranean termite, Coptotermes formosanus, fol-
lowed by the exposure to Pseudomonas aeruginosa had a synergestic effect.
However, lufenuron did not have such an interaction with S. marcescens and Bt
subsp. israelensis. Similarly, a chitinase producing bacterium, Paenibacillus sp. D1
and chitinase extracted from the bacterium, had a synergistic interaction with
acephate against the cotton bollworm, Helicoverpa armigera, in laboratory studies
in India (Singh et al. 2016).
Laboratory, greenhouse, and field studies in California evaluated entomopathogen-

based, botanical, and chemical pesticides as standalone applications or combina-
tions and rotations to develop IPM strategies against various pests, with emphasis
on the western tarnished plant bug, Lygus hesperus (Dara et al. 2013, 2018; Dara
2016). When a low rate of B. bassiana (0.2 lb./acre) was used with 1/5 the label rates
of azadirachtin, fenpropathrin, naled, and thiamethoxam in the 2010 laboratory
assays, the combination with fenpropathrin and thiamethoxam was higher than their
individual treatments, during the first 3 days after application (Dara et al. 2013). In
the follow up field studies in 2012, 2013 and 2014, various combinations and rota-
tions of chemical pesticides, azadirachtin, pyrethrins, B. bassiana, M. brunneum
and the bacterium Chromobacterium subtsugae were applied thrice at weekly inter-
vals (Dara 2016). In the 2012 study, while L. hesperus numbers increased by 12%
in untreated control by the end of the study, B. bassiana with fenpropathrin, azadi-
rachtin, and 0.5 rate of acetamiprid caused 79, 69 and 67% reductions, respectively,
compared to a 7.4% reduction by B. bassiana alone, 63% by novaluron + bifenthrin,
and 86% by the full rate of acetamiprid. In the 2013 study, L. hesperus increased by
47% in untreated control, 17% in the chemical standard, acetamiprid, and 56% in B.
bassiana + azadirachtin, followed by C. subtsugae and flonicamid. However, B.
bassiana applied with low rates of acetamiprid, flonicamid, and avermectin in con-
secutive weeks, resulted in a 61% reduction in L. hesperus followed by 50% reduc-
tion from one application of novaluron + bifenthrin and two applications of B.
bassiana + azadirachtin, and 47% from two applications of sulfoxaflor high rates
followed by B. bassiana + C. subtsugae. Other chemical pesticide rotations pro-
vided 46–53% control in this study. In a 2014 study, none of the treatments was able
to reduce L. hesperus populations and only their ability to limit the population
buildup compared to pre-treatment counts could be observed. Three applications of
the high rate of sulfoxaflor restricted the increase in L. hesperus to 14% and the high
rate of diatomaceous earth (DE) followed by low rate of B. bassiana + acetamiprid
and M. brunneum + azadirachtin to 17%, compared to 383% increase in untreated
control and 1083% in the chemical standard of acetamiprid. Two applications of B.
bassiana + azadirachtin after novaluron + bifenthrin limited the L. hesperus buildup
to 54%. In a latter study, Dara et al. (2018) expanded the control options to multiple
EPF (B. bassiana, I. fumosorosea, and M. brunneum), botanical pesticides (azadi-
rachtin and pyrethrum), chemical insecticides from different mode of action groups,
and mechanical removal by vacuuming. While a rotation of three chemicals (sulf-
oxaflor, flupyradifurone, and flonicamid) was the only treatment that reduced the L.
hesperus numbers by 22%, the remaining treatments varied in their ability to limit
or not limit the population buildup after three weekly applications of treatments.
Compared to untreated control where L. hesperus increased by 190.8% post-
treatment, the grower standard of acetamiprid limited the increase to 54.9%. While
two sulfoxaflor applications followed by vacuuming resulted in the highest increase
(403.6%), two flupyradifurone applications followed by vacuuming limited the
increase to 25.5%. There was only 9.5% increase in L. hesperus numbers when a
310 S. K. Dara
formulation of B. bassiana with pyrethrum was followed by vacuuming and

application of novaluron + bifenthrin. Two other treatments that included two appli-
cations of M. brunneum or I. fumosorosea with azadirachtin products limited the
pest build up to 59–79%. These data show that chemical pesticides are not always
effective and EPF can be effectively used with other control options. Dara et al.
(2018) also observed, in a different study, that the combination of B. bassiana and
bifenazate at the lowest label rates resulted in a 46.7% reduction in T. urticae after
two applications compared to 39.9% by the full rate of bifenazate, 33.3% by abam-
ectin, 47.2 and 50.9% by two formulations of fenpyroximate, and 62.4% by cyflu-
metofen. These extensive studies show that there are unlimited possibilities for
chemical and non-chemical pesticide combinations and that all control options vary
in their efficacy from time to time. They also demonstrated that EPF, when used
with certain chemical or botanical pesticides at regular or lower rates, provided
control similar or even superior to chemical pesticides.
Synergistic combinations of EPF with DE and chemical insecticides have been
effectively used for controlling a variety of stored-product pests as discussed in a
recent review of Rumbos and Athanassiou (2017). While diatomaceous earth is
abrasive to the insect cuticle and increase the chances of conidial adhesion and pen-
etration, chemical insecticides weaken the insect and increase the chances of fungal
infections. Some of the examples that Rumbos and Athanassiou discussed include
DE with B. bassiana against the lesser grain borer (Rhyzopertha dominica), the red
flour beetle (Tribolium castaneum), and weevils (Sitophilus spp.), DE with M.
anisopliae against R. dominica and the rice weevil, S. oryzae, and fenitrothion with
B. bassiana and M. anisopliae against S. oryzae.
It is also important to understand and avoid antagonistic combinations such as B.
bassiana with the acaricide, triflumuron (benzoylphenyl urea) which reduced the
fungal efficacy against T. urticae (Irigaray et al. 2003). Similarly, imidacloprid was
antagonistic to N. tanajoae (= N. floridana) and reduced the germination of primary
conidia and formation of infective capilliconidia (Dara and Hountondji 2001).
Studies conducted by Neves et al. (2001) evaluated the impact of neonecotinoids
acetamiprid, imidacloprid, and thiamethoxam against B. bassiana, M. anisopliae,
and Paecilomyces sp. at the field application rate, a rate above and below. Depending
on concentration, these chemicals either improved or negatively affected the conid-
ial production, germination and vegetative growth.
One of the concerns for using EPF for pest control is their compatibility with
fungicides in cropping systems where the latter are frequently applied for disease
control. To save the cost of application, growers frequently use tank-mix pesticides
and fungicides. While some combinations are compatible, others affected
EPF. Multiple studies explored the compatibility of B. bassiana and M. anisopliae
with fungicides (Samson et al. 2005; Bruck 2009; Akbar et al. 2012; Dara et al.
2014; Roberti et al. 2017). Samson et al. (2005) reported adverse effect of methoxy
ethyl mercuric chloride, flusilazole, prochlorax, and propiconazole on the readial
growth of two isolates of M. anisopliae in vitro, but the conidial viability and
recovery in the soil were not affected. They also found that when the greyback cane-
grub, Dermolepida albohirtum, and the negatoria canegrub, Lepidiota negatoria,
were exposed to the soil treated with M. anisopliae and fungicides, the mortality of
D. albohirtum was affected, but not that of L. negatoria. Bruck (2009) reported that
several fungicides, except etridiazole, propamocard, and mafanoxam, significantly
inhibited the conidial germination and mycelial growth of M. brunneum in vitro.
When Norway spruce (Picea abies) cuttings were planted in soil containing M.
brunneum and two applications of the fungicides were applied, there was no nega-
tive impact on M. anisopliae in bulk soil, but only captan and triflumizolet (which
had shorter intervals between the applications) had a negative impact on M. aniso-
pliae in the rhizosphere. Akbar et al. (2012) reported that a metalaxyl + mancozeb
combination was highly toxic to the conidial germination and mycelial growth of
M. anisopliae. In studies with B. bassiana and some common fungicides used in
strawberry, Dara et al. (2014) found that captan and thiram affected the fungus.
When mealworm (Tenebrio molitor) larvae were exposed to B. bassiana with fun-
gicides applied at different time intervals, captan caused a 43–70% reduction and
thiram caused a 26–67% reduction in larval mortality regardless of the interval.
Other fungicides (fluxapyroxad + pyraclostrobin, sulfur, pyraclostrobin + boscalid,
myclobutanil, iprodione, and cyprodinil + fludioxonil) from different mode of
action groups were compatible with B. bassiana (BotaniGard ES, strain GHA)
resulting in 93–100% larval mortality. It also appeared that larval mortality was
higher in treatments with fluxapyroxad + pyraclostrobin and cyprodinil + fludioxo-
nil, during the first 2 days after treatments, compared to B. bassiana alone. In a
different study, Roberti et al. (2017) observed that mepanipyrim and spiroxamine
reduced B. bassiana (Naturalis, strain ATCC 74040) colony growth by 9.7 and
6.9%, respectively. When the fungicides and B. bassiana were sprayed on zucchini
plants for controlling T. vaporariorum, boscalid + pyraclostrobin and cyprodinil +
fludioxonil affected the pathogenicity by reducing the efficacy against nymphs by
91 and 87%, respectively. These studies show that several fungicides are compatible
with EPF depending on species and strains.
11.7 Conclusion
As the world is gradually moving towards sustainable pest management approaches,

entomopathogens are becoming increasingly popular as control options. To develop
an effective and long-lasting IPM strategy, one should consider a variety of tools
including chemical and non-chemical alternatives. Understanding their interactions
to identify additive and synergistic combinations and avoiding antagonistic ones
helps with effective pest management and reinforces trust in the potential of non-
chemical alternatives.
312 S. K. Dara
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Chapter 12
Toxicological Prospects on Joint Action
of Microbial Insecticides and Chemical
Pesticides
A. R. N. S. Subbanna, J. Stanley, V. Venkateswarlu, V. Chinna Babu Naik,

and M. S. Khan
Abstract Microbial insecticides or entomopathogens are effective and eco-friendly

insect pest management options. But slow mode of action and lack of a visual pest
control, as expected by a farmer, mostly limits their wide commercial usage. The
present day regular and high incidences of insect pests, due to intensive monocul-
tures, warrant inevitable use of high doses of chemical pesticides. However, their
judicious application depends on the diverse environmental threats associated. So,
deployment of both entomopathogenic microbes and chemical pesticides together is
considered to reduce the risk to the environment. Various studies also reported more
efficient synergistic interactions in combined use than for independent applications.
Synergism has the ability to reduce the pesticide doses. Most importantly, the com-
bined application due to synergism can effectively tackles the pest problem and also
helps in establishment of an entomopathogen in a given ecosystem. Once estab-
A. R. N. S. Subbanna (*)
Crop Protection Section, ICAR-Vivekananda Institute of Hill Agriculture (VPKAS),
Almora, Uttarakhand, India
Department of Entomology, College of Agriculture, Govind Ballabh Pant University of
Agriculture and Technology (GBPUA&T), Pantnagar, Uttarakhand, India
J. Stanley
Crop Protection Section, ICAR-Vivekananda Institute of Hill Agriculture (VPKAS),
Almora, Uttarakhand, India
V. Venkateswarlu
Division of Crop Protection, ICAR-Central Tobacco Research Institute,
Rajahmundry, Andhra Pradesh, India
V. Chinna Babu Naik
Division of Crop Protection, ICAR-Central Institute of Cotton Research,
Nagpur, Maharashtra, India
M. S. Khan
Department of Entomology, College of Agriculture, Govind Ballabh Pant University of
Agriculture and Technology (GBPUA&T), Pantnagar, Uttarakhand, India

https://doi.org/10.1007/978-3-030-23045-6_12
318 A. R. N. S. Subbanna et al.
lished, the entomopathogens can effectively manage the pest population build up in
an eco-friendly manner, and over the years they can evade the use of pesticides or,
if not so, reduce their dosage. The present chapter critically discusses possible syn-
ergism between entomopathogens and chemical pesticides and the present status of
pest management achieved through this approach, in the context of latest research
findings.
Keywords Entomopathogens · Chemical pesticides · Joint action · Compatibility ·

Synergism
12.1 Introduction
Insect pests continue to be limiting factors of agricultural production destroying an

estimate around one-fifth of total global crop production annually, making crop
protection an inevitable issue for agricultural production systems. An annual invest-
ment of US$ 40 billion on 3 million metric tonnes of pesticides (Atwal and Dhaliwal
2015) was unable to manage this level of damage. Application of this huge amount
of pesticides raised complicate environmental and human health issues, besides
becoming economically demanding against resistant populations of pest species.
Despite of all these concerns, from the farmers’ point of view chemical pesticides
are the competent pest management options available, due to their easy accessibil-
ity, storability, reliability of results, ready to use formulations and, especially, the
‘fast solution’ they provide against any pest problem.
Keeping in view the different environmental threats associated with the use of
chemical pesticides, the concept of pest control was tuned towards pest manage-
ment by combination of different tactics for pest suppression. This underpins the
idea of Integrated Pest Management (IPM) based on economic thresholds. The
essence of IPM is the use of all the tactics of pest suppression including cultural,
mechanical, physical, biological and chemical methods. In the present context of
increased pest problems, in most cases IPM relies on mixtures or sequential applica-
tions of pesticides, with different modes of action. Although non-intensive, the
application of alternative non-chemical tactics require through knowledge about
biology and ecology of target pests, which might also be location-specific. So, use
of pesticides is continued to be a key option for pest management in current chemi-
cal intensive cropping systems. Besides, pesticides are a global million dollar indus-
try and in situations like emergencies and epidemics they are the only quick option
available. Still, their use should be minimized to safeguard both biotic and abiotic
environments. Although some chemical synergists are reported to improve bioeffi-
cacy and lower field doses, their impact on environment is also deleterious.
Insect pathogens such entomopathogenic bacteria (EPB), fungi (EPF) and nema-
todes (EPN) are thought to be coevolved with insect pests and have the capability to
cause epizootics. They are considered as one of the major natural regulation factors,
maintaining the pest population density in a given ecosystem. Biological control
using entomopathogens is considered as a biologically safe and environment-
12 Toxicological Prospects on Joint Action of Microbial Insecticides and Chemical… 319
friendly alternative to pesticides. In organic and sustainable agriculture systems,

these approaches represent the major strategy of pest management and are gradually
gaining significance. Although potentially bioactive, major drawbacks is their slow
and imperceptible mode of action. Besides, indiscriminate use of pesticides defi-
nitely over mask their existence and sometimes may be also lethal. It is also impor-
tant to note that some strains of entomopathogens are capable to resist lethal action
of pesticides, sometimes found to be compatible or even synergizing each other in
killing target pest (Feng and Pu 2005; Jia et al. 2016; Ali et al. 2017; Meyling et al.
2018). Entomopathogens, besides being direct lethal agents, can also be used as
biosynergists to chemical pesticides. A clear understanding of biology, joint ecol-
ogy and resulting physiological pathogenicity effects may give an idea about their
combined utilization in successful pest management programs. The insecticide
resistance developed by the majority of key pests also demands an alternative strat-
egy with improved efficacy. It is also known that resistance against entomopatho-
gens is ostensible in insect pests and can provide stable pest control under favorable
environments.
Keeping in view the reliability of both entomopathogens and chemical pesti-
cides, their joint application offers an excellent strategy against many pest problems
with reduction in doses (Wang et al. 2013; Singh et al. 2016). In this chapter, we
discuss the possible combinations, synergism and modes of action of synergism,
apart from further implications and knowledge gaps.
12.2 Methodologies Used for Compatibility Analysis
In vitro assays are the first dependable methodology to envisage the levels of com-
patibility between two pesticidal components. However, dealing with a broad range
of unicellular (i.e. EPBs), multi-cellular (EPF and EPN), non-cellular [entomo-
pathogenic viruses (EPVs)] and symbionts (EPN), different entomopathogenic
groups warrant specific methodologies for compatibility assays. The laboratory
compatibility of common microbes with that of pesticides is measured by median
inhibitory concentration (IC50) (Stanley and Preetha 2015). This section portrays
different methodologies adopted by various authors in compatibility analysis
between diverse entomopathogens with chemical insecticides.
(i) For EPF
In general, compatibility of EPF with insecticides was estimated by evaluating
conidia germination after their incubation (about 24 h) in required concentrations of
the pesticide to test. Alizadeh et al. (2007) reported use of field recommended doses
(FRD) of insecticide at 0.5× FRD, 1× FRD and 2× FRD, in combination with
conidia (at 106 conidia/ml) in potato dextrose broth at 25 ± 2 °C. After 24 h, number
of conidia germinated out of total counted was estimated to get percent germination
in comparison with the control treatment, without insecticide used. Yii et al. (2015)
proposed a plate assay where a 107 conidia/ml solution was evenly spread onto dif-
ferent concentrations in intoxicated potato dextrose agar (PDA), and allowed to

germinate. Subsequently, 5–6 replications of 1 cm2 discs were cut and counted for
germination percent.
Poisoned food technique (Moorhouse et al. 1992) is one of the most widely used
methodologies to estimate effects of pesticide poising on vegetative growth of fungi.
In this technique, a required concentration of pesticide is aseptically mixed with
warm PDA (± 45 °C) and plated in a petri dish. After solidification, a disc (~8 mm)
of actively growing fungal mycelium is placed at the center (Stanley et al. 2010) and
allowed to grow at 25 ± 2 °C. The data on radial growth of fungal mycelium is mea-
sured either at regular intervals or when the mycelium in control plates reaches
borders, to estimate percent inhibition.
Based on conidia germination, radial vegetative growth inhibition and inhibition
in conidia production on intoxicated media, Alves et al. (2007) proposed the follow-
ing biological index (BI) formula, to categorize toxicological effects of pesticides
on EPF.
10 ( GR ) + 47 ( VG ) + 43 ( SPR )
BI =
100
in which GR = reduction in conidia germination, VG = reduction in vegetative

growth, and SPR = reduction in conidia production or sporulation. All these param-
eters were initially corrected for the controls and then fed to the formula for calcu-
lating BI. A BI value less than 42 represents toxicity, between 42 and 60 is considered
moderately toxic, and above 60 represents compatible EPF and pesticide.
The BI formula represents the actual toxicity status of a given pesticide by con-
sidering all the three growth stages of EPF, i.e. germination, vegetative growth,
sporulation to an extent of 10, 47 and 43%, respectively, instead of considering their
maximum levels. Due to this the formula is widely accepted and used in estimating
compatibility between EPF and pesticides (Silva et al. 2013; Yii et al. 2015).
(ii) For EPN
The toxicological effects of pesticides on EPNs are manifested in the form of
reduced viability and infectivity (Negrisoli et al. 2010a). Viability of EPN was esti-
mated by incubating them in an equal volume of test concentration of the pesticide.
After 48 h, the viability is estimated under a stereomicroscope by observing move-
ments of infective juveniles (IJs). The infectivity of IJs at different incubation peri-
ods is estimated after removing the pesticide component by repeated washings and
decanting. Thus obtained IJs are used for bioassay against Galleria mellonella
(about 250 IJs per 10 final instar larvae) and compared with control mortality, once
confirming the infection by EPNs after cadaver dissection. The toxicological effects
(E %) of pesticides on EPN are calculated as follows.
E% = 100 - (100 - %corrected mortality ) × (100 - RI )
where RI = reduction in infectivity of IJs, which was calculated as

RI = (1 - M t / M c ) × 100
where Mt = mortality in treatments and Mc = mortality in control. The E% value

represents the toxicological classification of test pesticide viz., < 30% is non toxic,
between 30–79% is slightly toxic, between 80–99% is moderately toxic and > 99%
is highly toxic (Negrisoli et al. 2010a).
(iii) For EPB
No standard and specific protocols were reported to estimate compatibility of
EPBs with chemical pesticides. However, some qualitative techniques as that of
EPF by intoxicated media are reported (Mishra and Tandon 2003). A required insec-
ticide concentration was mixed in a specific growth medium, after solidification,
100 μL of approximately 104 cfu/ml solution was evenly spread on to plates. After
incubation, the colonies formed were counted and compared with control plates.
For Pseudomonas, its fluoresce nature was measured every 24 h of incubation for
3 days and compared with that of the control (Stanley et al. 2010).
In a similar qualitative assay, specific media is spread with 100 μL of bacterial
culture or mixed with 2% of actively growing bacterial cells and, after solidification,
a paper disc impregnated with required concentration of pesticide is placed above.
After incubation, any inhibition zone of bacterial growth in and around the disc
represents incompatibility.
12.3 Methodologies Used for Estimation of Joint Action
Majority of studies reported joint action of entomopathogens with commercial for-

mulations of insecticides. The entomopathogens are laboratory prepared for their
infective stages viz., colony forming units (CFUs) for EPB, conidia for EPF, IJs for
EPN and POBs for EPV. Different combinations are tested against lepidopteran,
homopteran and coleopteran pests using specific Insecticide Resistance Action
Committee (IRAC) approved methods. In all the bioassays, especially with EPB
and EPF, a detergent (i.e. Tween 80 or Triton X100 at a concentration of 0.01%) is
included for measuring equal dispersal of conidia or CFUs and wetting of feed sur-
face area.
Against termites, Yii et al. (2015) proposed a bait formulation for estimating
joint action of EPF and insecticides. In this bioassay, sawdust was initially immersed
in required concentrations of an insecticide, following air drying the saw dust was
mixed with dry harvest conidia at required concentration. Thus prepared bait formu-
lation was fed with worker termites and data on mortality was recorded up to 8 days.
The dead insects were also kept for development of mycosis. Wang et al. (2013) also
used similar methodology by using EPB (Pseudomonas aeruginosa, Serratia marc-
escens and Bacillus thuringiensis) with a stack of 6 filter paper discs (9 cm diame-
ter) as feed instead of sawdust baits.
M E = M I + M F (1 - M I / 100 )
Where MI = mortality by insecticide alone and MF = mortality caused by EPF alone.

Mansour et al. (1966) proposed a cooperative virulence index (cf) to classify the
joint action of insecticides as
Mm - Mi - Me
cf =
Mi - Me
Where Mm = mortality caused by mixture, Mi = mortality caused by insecticide

alone, Me = mortality caused by the entomopathogen alone.
This can also be written as co-toxicity factor, as given by Mansour et al. (1966)
and Abbassy et al. (1979) as
Observed mortality - Expected mortality

Co toxicity factor =
Expected mortality
A co-toxicity factor value < −20 indicates antagonism, > 20 indicates synergism
and between −20 to +20 indicates additive interaction.
Although different co-toxicity and synergistic coefficients are proposed,
Mansour’s cooperative virulence index was widely used for estimating joint action
between insecticides and entomopathogens (Zou et al. 2014). This is due to the use
of lethal concentrations by former methods where an estimate of active ingredient
(ai) is based on weight upon volume (mg/lt or mg/kg), which is suitable for chemi-
cal pesticides. But in case of entomopthogens active ingredients are CFU/ml (in
case of EPB) or conidia/ml (in case of EPF) or POB/ml (in case of EPV). The
Mansour’s method relies on mortalities of individual components and mixtures, to
estimate joint action which fits with both insecticides and entomopathogens.
Majority of the studies reported linear relationship between concentration-
mortality (CM). However, time factor is also important to realize the effects of toxin
components due to the use of lowered concentrations than those recommended
(Nowierski et al. 1996). Feng and Pu (2005) proposed a time–concentration–mor-
tality (TCM) modeling, which is both statistically and biologically robust, to esti-
mate synergistic interactions between insecticide and entomopathogens. In some
instances, the TCM model showed an acceptable goodness of fit with reliable esti-
mates of LC50 and LC90, where the commonly used CM model based on probit
analysis failed, due to uncontrolled bias. Besides, it is also important that scaled-up
range of toxins should be investigated for realizing the exact interaction effects
between pesticides and entomopathogens. Using overdoses of insecticide easily
overwhelm the activity of entomopathogen counterpart and at the same time the
maximum mortality realized cannot exceed 100% in any combinations, which may
lead to misinterpretation of antagonism. Feng and Pu (2005) also indicated that
sublethal doses of pesticides below LC50 should be considered first, and reported a
reduction in imidacloprid field dose from 300 to 30 g ai/ha in combination of EPF,
Beauveria bassiana, to realize a similar effect.
Different authors used various combinations and mixtures of insecticides and

entomopathogens for field evaluations. For example, Negrisoli et al. (2010b) used
half the recommended concentrations of insecticides and entomopathogens in com-
bination. Morales-Rodriguez and Peck (2009) used ½ and ¼ doses of pesticides
against white grub species, Amphimallon majale and Popillia japonica. Feng and
Pu (2005) used sublethal doses of the insecticide imidacloprid against Nilaparvata
lugens. Niu et al. (2018) evaluated approximate LC20 concentrations of S. marces-
cens (108 to 109 CFU/ml) with two insecticides (spirotetramat and thiamethoxam),
at LC25 and LC50 concentrations, against N. lugens, with mortality data up to 9 days.
The primary objective of using mixtures of insecticides and entomopathogens is the
minimization of chemical pesticides and promotion of entomopathogens activity, in
a given ecosystem. In view of this we recommend field applications/evaluations of
mixtures containing insecticides at their sublethal doses at which they can simply
act as stressor against target pest, to enhance the infectivity of the entomopathogen
component rather than acting as one of the lethal factor.
12.4 Compatibility Between Entomopathogens

with Chemical Pesticides
Studies on the compatibility between targeted combinations of entomopathogens

and pesticides under in vitro conditions are one of the preliminary requirements to
assess the fate of both pest management options. However, mixing entomopatho-
gens (biological organisms) with toxic chemical compounds, in a huge variety of
modes of action, appears as a complicated issue and requires through evaluation
(Zou et al. 2014). Field application of bacteria, fungi and nematodes usually relied
upon environment-resistant stages (i.e. spores, conidia and infective juveniles),
which usually show a minimal tolerance level to different stresses including toxico-
logical compounds like pesticides. However, their combined use requires through
investigations on retaining their native activity, survival and bioactivity. Many
researchers proposed different methodologies/techniques (discussed in previous
headings) which targeted these basic issues.
Actually, most compatibility studies were reported with fungal bioagents.
Conidia viability, growth of mycelium in intoxicated media, further sporulation and
infectivity are the critical factors considered for fungal bioagent compatibility
(Alves et al. 2007). Asi et al. (2010) reported that vegetatively growing fungi are
more resistant than their conidial stages. However, variations in methodologies may
contribute to differential results. For example, Yii et al. (2015) reported that solvent
used for insecticides, i.e. acetone, significantly reduced spore germination and
increased vegetative growth and spore yield of Metarhizium anisopliae. Similar
alterations in results can be expected when using commercial formulations which
contain a variety of additives (Anderson and Roberts 1983), apart from the technical
a.i. tested. Studies also reported that entomopathogenic nematodes are sensitive to
surfactants which are commonly used in most commercial formulations of insecti-

cides (Kaya et al. 1995; Krishnayya and Grewal 2002).
The in vitro compatibility tests generally use direct exposure of bioagents to high
concentrations of toxin, as well as allowing long term contact. The latter is mostly
not a situation often encountered under field conditions (Neves et al. 2001).
Additionally, fate of an insecticide under field conditions (half life, degradation fac-
tors, plant absorption, relative contact with bioagent etc.) ultimately decides its del-
eterious effects on bioagent. As a consequence, the realized laboratory toxicity does
not always mean that the pesticide is detrimental. Sometimes, the mode of action of
a given pesticide can also signify possible impairment to the target bioagent. For
example, abamectin damages sensory organs of nematodes leading to poor infectiv-
ity of IJs (Head et al. 2000). The primary constituents of nematode cuticle include
collagens, cuticulins and other proteins (Negrisoli et al. 2010a, b) which are not
affected by pesticides with chitin synthesis inhibition activity (i.e. triflumurom,
diflubenzuron etc.) (Hara and Kaya 1982; Rovesti and Deseo 1990; De Nardo and
Grewal 2003). Similarly, the presence of butyrylcholinesterase, instead of acetyl-
cholinesterase, in synapses of EPNs makes them compatible with organophosphous
insecticides such as chlorpyrifos (Zimmerman and Crashaw 1990; Selkirk et al.
2001; Alumai and Grewal 2004; Gutierrez et al. 2008). Such interpretation in respect
of basic knowledge about the pesticide and bioagents may avoid tedious experimen-
tal procedures on compatibility and joint action.
Various studies on compatibility reported contentious results with respect to
same species of bioagents and insecticide. The reason is mainly related to the use of
native strains or isolates, which may have an innate capacity to counteract the toxic
effects resulting from environment and local isolation conditions. Such physiologi-
cal mechanisms can be expressed in the form of increased survival (Shumacher and
Poehling 2012), reproduction and even utilization of pesticides or metabolites as
nutrients (Moino and Alves 1998). Adaptability of a bioagent to toxic chemicals is
also an important factor which warrants time lapse studies and further understand-
ing of biological adaptations.
The toxicological effects of pesticides on bioagents is manifested in form of
reduced spore viability, inhibition in germination, lower vegetative growth, limited
sexual reproduction or loss of infectivity etc. All these noxious effects appear dose-
dependent and require intensive investigations to understand dose-damage relation-
ships. These studies may also provide required informations about temporal and
spatial separation of both pest management options, for synergism to be observed.
12.5 J oint Action of Entomopathogens with Chemical

Pesticides
In general, most studies reported the use of compatible chemical pesticides for fur-
ther evaluation of their joint action against insect pests. However, the negative
results in laboratory compatibility tests may not always lead to poor biotoxicities,
especially under field conditions, which is governed by a variety of ecological and
biological parameters (some examples of positive and negative interactions between
entomopathogens and pesticides are detailed in Table 12.1 and 12.2, respectively).
Moreover, use of minimal concentrations of chemical pesticides ― approximately
between LC10 - LC30 ― reported high synergistic interactions under both laboratory
and field conditions. Field evaluation of these negative interactions or further testing
at lesser dosage of pesticide may hence yield a real picture of bioactivities. Joint
action studies should hence be planned with a range of pesticide and bioagent doses
starting from lower sublethal doses to a maximum of individual mortality up to
50%. It is also important to note that no generalized behavior can be expected from
single chemical group of insecticides to a single bioagent. Differential synergistic
interactions between imidacloprid and clothianidin (neonicotinoid group) against P.
japonica and A. majale (Morales-Rodriguez and Peck 2009) or of imidacloprid and
thiamethoxam with Heterorhabditis bacteriophora against Cyclocephala hirta, C.
pasadenae and Exomala orientalis (Koppenhöfer et al. 2002) evidently showed this
feature. A synergistic joint action can also be achieved by a temporal separation of
the insecticide and bioagent (Negrisoli et al. 2010b). Meyling et al. (2018) reported
synergism of entomopathogens and insecticide as a function of sequence and time
of application.
Conventional mortality analysis carried out using factorial experiments may pro-
vide data on target pest mortality over a fixed and minimal time period (generally
72 h). However, the lethal action of both mortality factors is a function of the inter-
actions between time and concentration (Nowierski et al. 1996). Most studies
ignored time factors, which may yield a bias when compared with individual mor-
talities (Nowierski et al. 1996; Feng and Pu 2005). This bias is greatly overcome by
a robust TCM relationship, that takes into account the time and concentration into a
single model, enabling us to separate the interaction effects from the individual
effect of time and concentration on pest mortality. This estimate of TCM is not only
robust from the mathematical point of view but also have a high biological relevance
on the relative potency of both the entomopathogen and the chemical. The median
lethal time (LT50) is estimated by linear interpolation in such TCM models. Relative
potential are also tested using LC50 values of entomopathogens and chemicals (Feng
and Pu 2005). In case of virus-insecticide mixtures, the standard probit or logit
analysis do not holds good as the concentration mortality data do not follow a logis-
tic or Gaussian distribution, due to the differences in the mode of action and/or
interactions between the virus and the insecticide.
Table 12.1 Example of positive interactions between entomopathogens and insecticides
326
Entomopathogens Insecticide Target Pest References Remarks

Entomopathogenic Nematodes
H. bacteriophora Imidacloprid and Clothianidin Amphimallon Morales- Synergism was discernible from laboratory,
majale, Popillia Rodriguez and greenhouse and field trials
japonica Peck (2009)
Imidacloprid A. orientalis Polavarapu et al. Synergism was with low dose but not with high
(2007) doses
Imidacloprid Thiamethoxam P. japonica, Koppenhöfer et al. Imidacloprid provided stronger and more
Cyclocephala hirta, (2002) consistent synergism with nematodes than
C. pasadenae, E. thiamethoxam.
orientalis
H. bacteriophora, Imidacloprid E. orientalis Koppenhöfer et al. Synergism
H.megidis, H. (2002)
marelatus, S. glaseri,
S. feltiae.
Heterorhabditis sps., Triflumuron, Deltamethrin, Tuta absoluta Sabino et al. Compatible with both nematode species
S. carpocapsae dimethylamino-propyl, lambda- (2014)
Cyhalothrin + chlorantranilprole,
Clorantranilprole, Thiamethoxam +
lambda-cyhalothrin.
H. indica, S. Chlorpyrifos, Cypermethrin, S. frugiperda Negrisoli et al. Additive (H. indica with cypermethrin,
carpocapsae, S. Spinosad, Methoxyfenozide, (2010a) spinosad, methoxyfenozide and
glaseri Deltamethrin+Triazofos, Lufenuron deltamethrin+triazofos; S. carpocapsae with
lufenuron, chlorpyrifos and cypermethrin) and
synergistic (H. indica with chlorpyrifos; S.
glaseri with chlorpyrifos and lufenuron)
S. carpocapsae Azadiractin Western flower Otieno et al. Synergism
thrips (2016)
A. R. N. S. Subbanna et al.
12
Entomopathogenic fungi
M. anisopliae Permethrin, Imidacloprid, NeemAzal, NA Schumacher and Compatible with conidial germination,
Amitraz Poehling (2012) vegetative growth and sporulation
Thiamethoxam, lambda-cyhalothrin Rice pests Silva et al. (2013) No effects on conidial germination, vegetative
growth and sporulation
Chlorpyrifos, Propetamphos, German cockroach Pachamuthu and Additive and syngergistic
Cyfluthrin Kamble (2000)
Fipronil Coptotermes Yii et al. (2015) Greatest synergism
curvignathus
Imidacloprid, Clothianidin Popillia japonica Morales- Discernible in the laboratory and greenhouse,
Rodriguez and but not in field.
Peck (2009)
Imidacloprid Diaprepes Quintela and Synergism
abbreviates McCoy (1997)
Imidacloprid Diaprepes Quintela and Low conidial attachment to larval cuticle at
abbreviates McCoy (1998b) higher dose of chemical
Imidacloprid C. bergi Jaramillo et al.
(2005)
Blatella germanica Pachamuthu and
Kamble (2000)
Thiamethoxam Rice stalk stink bug Quintela et al. Synergism
(2013)
Endosulfan, Imidacloprid, Lufenuron, Spilarctia obliqua Purwar and Synergism
diflubenzuron, Dimethoate, Sachan (2006)
Oxydemeton methyl
M. brunneum Imidacloprid Anoplophora Russel et al. Compatible
glabripennis (2010)
Toxicological Prospects on Joint Action of Microbial Insecticides and Chemical…
(continued)
327
328

M. anisopliae, B. Azadiractin Western flower Otieno et al. Synergism
bassiana thrips (2016)
M. anisopliae, B. Acetamiprid, Imidacloprid, NA Neves et al. (2001) No negative effect on conidia germination,
bassiana, Thiamethoxam growth and vegetative growth
Paecilomyces
M. Anisopliae, B. Imidacloprid, Clothianidin Amphimallon majale Morales- Synergism
bassiana and Popillia Rodriguez and
japonica Peck (2009)
M. Anisopliae, B. Imidacloprid, Fipronil NA Moino and Alves No negative effect
bassiana (1998)
B. Bassiana Imidacloprid NA Neves et al. No effects on conidial germination, vegetative
(2001), Alizadeh growth and sporulation
et al. (2007) and
Abidin et al.
(2017)
Alpha-cypermethrin Tenebrio molitor Meyling et al. Synergistic effect when only the interval
(2018) between applications was >48 h. with 72 h
between exposures, mortality had increased to
100% after 8 days
Neem Spodoptera litura Mohan et al. Compatible and synergistic
(2007)
Endosulfan Spilarctia oblique Purwar and 4.9 times increased toxicity
Sachan (2006)
Imidacloprid and clothianidin Popillia japonica Morales- Discernible in the laboratory and greenhouse,
Rodriguez and but not in the field.
Peck (2009)
Imidacloprid Diaprepes Quintela and Low conidial attachment to larval cuticle at
abbreviatus McCoy (1998b) higher dose of chemical
A. R. N. S. Subbanna et al.
12
Lipaphis erysimi Purwar and Synergism

Sachan (2004)
Botanical compounds Ephestia kuehniella Shakarami et al. Synergism
(2015)
Lecanicillium Matrine Bemisia tabaci Ali et al. (2017)
muscarium
Isaria fumosorosea Spirotetramat, Acetamiprid, B. tabaci Zou et al. (2014) Lethal time and pathogenicity increased
Imidacloprid, Thiamethoxam and Tian et al.
(2015)
B. brongniartii, M. Azadiractin NA Vyas et al. (1992) Compatible
anisopliae
Entomopathogenic bacteria
B. thuringiensis Thiodicarb H. armigera Khalique and Synergism observed at high thiodicarb and low
Amhed (2005) Bt-toxin concentration
S. marcescens Spirotetramat, Thiamethoxam Nilaparvata lugens Niu et al. (2018) Both synergistic and additive effects
B. Thuringiensis Botanicals Cnaphalocrocis Nathan et al. Synergism
medinalis (2004)
Entomopathogenic virus
Nuclear polyhedrosis Spinosad S. littoralis Khattab (2007) Reduction in lethal dose
virus
Nuclear polyhedrosis Spinosad S. frugiperda Mendez et al. Synergism
virus (2002)
Nuclear polyhedrosis Carbamates, Methomyl and Heliothis virescens Mccutchen et al. Synergism
virus Pyrethroids (1997)
Toxicological Prospects on Joint Action of Microbial Insecticides and Chemical…
329
Table 12.2 Examples of negative interactions between entomopathogens and insecticides

Entomopathogens Insecticide References Remarks
H. bacteriophora, Thiophanatemetil, Negrisoli et al. Reduced the viability
S. carpocapsae Thiametoxam, (2008) and infectivity of
Imidacloprid, Aldicarb, EPNs
Carbofuram
M. anisopliae Fipronil Schumacher and Moderate toxicity at
Poehling (2012) higher doses
Imidacloprid Abidin, et al. Moderately inhibited
(2017) and the conidia
Shumacher and germination and
Poehling (2012) vegetative growth
B. Bassiana Imidacloprid James and Elzen Antagonism
(2001) and Abidin
et al. (2017)
Deltamethrin, Abidin et al. (2017) Conidia germination
Chlorpyrifos, Thiodicarb, inhibited
Imidacloprid,
Cypermethrin
Fenitrothione Zibaee et al. (2009) Fungal infection
decreased the
susceptibility.
In view of available data, joint action studies of different bioagents with pesti-
cides resulted in maximum additive and few antagonistic or synergistic interactions
(Morales-Rodriguez and Peck 2009). Bacterial bioagents yield minimal number of
synergistic interactions and are very particular with pesticide. Overall maximum
compatibility and synergism responses of bioagents was in the order:
EPN > EPF > EPB. At the same time, maximum number of studies was in the order
of EPF > EPN > EPB. The strength of synergism diminishes from laboratory to
greenhouse, to field (Morales-Rodriguez and Peck 2009).
Amongst EPB, B. thuringinsis is well studied, having more than a century of
history in pest management. However, only minimal studies reported its synergism
with chemical pesticides (Chen et al. 1974; Salama et al. 1984; Morales-Rodriguez
and Peck 2009; Subbanna et al. 2019). With respect to chemical groups, B. thuringi-
ensis is compatible and showed more synergism with pyrethroids than with carba-
mates and organophsphous insecticides (Salama et al. 1984). The carbamate group
had no significant effect on bacterial growth at lower concentrations, and no signifi-
cant synergism was observed (Sutter et al. 1971). Likewise, the sister species, B.
popilliae and B. sphaericus were found to be susceptible to most commonly used
pesticides at high concentrations (Dingman 1994; Mishra and Tandon 2003). Other
insect pathogens such as Serriatia marcescens should be studied for synergistic
properties in view of their reported field performance, along with spirotetramat or
thiamethoxam (Niu et al. 2018).
EPFs are highly variable in their synergistic potential with pesticide groups,
which is a function of the strain or isolate under testing. Abidin et al. (2017) opined
that the EPF adaptability to toxins should also be taken into consideration in design-
ing joint applications. Neonicotinoids (especially imidacloprid) are reported to be
synergistic with EPFs against the white grubs such as P. japonica (Morales-
Rodriguez and Peck 2009), the weevil Diaprepes abbreviatus (Quintela and McCoy
1997, 1998a), the caterpillar Spilarctia obliqua (Purwar and Sachan 2006), the bug
Cyrtomenus bergi (Jaramillo et al. 2005), the termites Heterotermes tenuis (and
Reticulitermes flavipes (Ramakrishnan et al. 1999; Moino and Alves 1998), the
German cockroach (Kaakeh et al. 1997), the burrower bug Cyrtomenus bergi
(Jaramillo et al. 2005) and the rice stalk stink bug Tibraca limbativentris (Quintela
et al. 2013, with thiomethoxam). These synergistic interactions can reduce the
insecticide dose from 25% to 14% or even less of the recommended rate (Feng and
Pu 2005; Quintela et al. 2013). Sometimes, compatible insecticides may show posi-
tive correlation between insecticide dose and induced mortality. Other insecticide
groups are also safe and can be used with EPF at appropriate concentrations (Li
et al. 1996; Pachamuthu and Kamble 2000; Chen and Feng 2003). Synergistic inter-
actions are also prominent between sucking pest specific EPFs and pesticides such
as Isaria fumosorosea with spirotetramat (Zou et al. 2014), or Lecanicillium mus-
carium with matrine against Bemesia tabaci (Ali et al. 2017).
The commercial use of EPNs is mainly concentrated towards white grubs man-
agement. Most joint action studies using EPNs and insecticides was restricted
against white grubs (Negrisoli et al. 2010b). In case of EPNs also, imidacloprid
showed high synergistic interactions at appropriately lower doses. Higher doses of
imidacloprid and nematodes sometimes showed some antagonistic interactions
(Koppenhöfer et al. 2000; Polavarapu et al. 2007; Sabino et al. 2014). Nematode
strain and species-dependent responses were highly prevalent. Major studied with
white grub species involved P. japonica, Cyclocephala hirta, C. pasadenae,
Anomala orientalis, Exomala orientalis, A. majale, Maladera castanea etc. All
these species showed differential responses to varying combinations of neonicoti-
oids with EPNs (Koppenhöfer et al. 2002; Koppenhöfer and Fuzy 2003).
Minimal studies reported joint action of EPVs with insecticides. So the potential
interaction of EPVs with pesticides is little understood. McCutchen et al. (1997)
opined that insecticide acting on nervous system (such as pyrethroids, carbamates
etc.) perform well along with EPVs even though their mode of action is not clear.
Spinosad is also reported to act synergistically under field conditions against
Spodopetra frugiperda (Mendez et al. 2002) and S. littoralis (Khattab 2007)
although minimal positive interactions were observed in laboratory.
Majority of the antagonistic interactions in entomopathogen-insecticide complex
are the result of high doses of chemical pesticides which may engulf the activity of
the bioagent counterpart. At the same time it may not be safe to test only low doses,
as often reported. It is also important to note the subsequent effects of an insecticide
on a bioagent. For example, Russel et al. (2010) showed reduction in fungal myco-
sis and conidia yield by M. brunneum when combined with low doses of imidaclo-
prid, although the mixture was synergistic against Anoplophora glabripennis. In
EPNs this antagonism is manifested in the form of reduced parasitism (Baweja and
Sehgal 1997). Besides, the laboratory antagonism may not always yield a negative
response under field conditions. Neonicotinoid insecticides (imidacloprid and

thiomethoxam) were also found to be antagonistic in many cases (James and Elzen
2001; Koppenhöfer et al. 2002; Morales-Rodriguez and Peck 2009).
The joint action of entomopathogens was mostly studied with neem products
such as commercially available formulations, extracts, seed kernel extracts, oils etc.
The target pests used for estimating a joint action belonged to diverse groups,
including sucking pests such as B. argentifolii (James 2003), Thrips tabaci (Mohan
et al. 2007), B. tabaci (Shakarami et al. 2015) and Frankliniella occidentalis (Otieno
et al. 2016), lepidopteran larvae such as Spodoptera litura (Shakarami et al. 2015)
and storage pests such as Tribolium castaneum (Akbar et al. 2005). Most studies
targeted the joint action of EPFs with neem products and supported improvements
in lethal concentrations and time of kill (Mohan et al. 2007). Despite of these syn-
ergistic interactions, the active botanical insecticidal compounds, i.e. phytoalexines,
triterpenoids and sulfurade compounds etc., have a mycotoxic capacity and other
associated compounds may also influence germination and growth of bioagents.
Entomopathgenic bacteria such as B. thuringiensis (Nathan et al. 2004), with nema-
todes (Otieno et al. 2016) and viruses (Muralibaskaran et al. 1999; Bhandari et al.
2009) also showed substantial synergistic activity with neem products, although
dose- dependent. Extracts from other pesticidal botanicals from Annona squamosa,
Prosopis juliflora, Eupatorium and Artemisia also showed relative synergism with
different bioagents.
12.6 Mechanism/Mode of Action of Synergisms
The combined mode of action of two different pest management options based on a
pure biological and a chemical toxin is difficult to ascertain. However, some models
and predictions can be done after knowing the individual biological targets of the
associated components. In general, majority of the studies reported apparent
decrease in lethal concentrations and time required to kill target pests. Both inde-
pendent and combined actions of the components can explain the reduction in time
required to kill. In independent action, the insecticides generally kills insect pest
before 72 h of exposure, a period during which the bioagents colonize the site of
action but are not lethal. Subsequent lethal actions by bioagents further improve
mortality, which is not the situation in independent actions. On the other hand, bio-
logical explanations on synergistic joint action may involve similar target sites (ner-
vous system, ion exchange channels etc), concurrent actions on different tissues or
binding sites, diverse actions on the same tissue or different sites, and other physi-
ological irregularities etc.
Physiological interactions play an important role where one component wanes a
biological system or pest mechanism, thereby enhancing or providing a way for an
elevated lethal action of the associated component. For instance, any pathogenic
infection to nerve cells improves their sensitivity to insecticides. Similarly, patho-
genic S. marcescens strains (Ishii et al. 2012) kill host immune cells. A low dose of
imidacloprid was found to affect the grooming behavior of soil inhabiting pests
(termite, Heterotermes tenuis and white grub, Diaprepes abbreviatus) thus facilitat-
ing the attachment of EPF conidia and further infection (Quintela and McCoy,
1998a, 1998b). Type, concentration and formulation of an insecticide are the archi-
tectural factors governing the joint action and its extent. Other effects include
extended developmental periods, reduced larval feeding, instar intermediates,
abnormal pupae, malformed adults, reduced adult longevity and fecundity, poor
survival of new born etc. which weaken insect biological systems promoting the
toxic actions. However, reduced food intake is a physiological mechanism to resist
or avoid the action of systemic insecticides, and some enterobacteria (Morales-
Rodriguez and Peck 2009).
Some physiological interactions induced by insecticides may also favor the ento-
mopathogenic counterpart, thereby exerting synergism. Sublethal doses of some
insecticides weaken the immune system, facilitating fungal invasion (Hiromori and
Nishigaki 2001). Quintela et al. (2013) reported that sublethal concentrations of
thiamethoxam reduced fungistatic aldehyde production by rice stalk stink bug, thus
improving infectivity by M. anisopliae. Some indirect interactions may also facili-
tate survival, development and infectivity of entomopathogens. For example,
Anderson et al. (1989) reported growth enhancement of B. bassiana by some pesti-
cides due to the adjuvants in the formulation, which helped in scattering of conidial
piles thereby promoting propagule numbers. Similarly surfactants and solvents may
also be involved in synergistic interactions.
The basic biological functions of any given living organisms rely on enzymes,
which is also true for insects. Any external toxicological intervention definitely
alters different enzymes associated to target systems, viz. digestive tract, nervous
system etc. In insects, tolerance towards toxic compounds is a function of the effi-
ciency in their detoxification by carboxylesterases and glutathione-S-transferase
(Claudianos et al. 2006). Other enzymes involved in repair of damage to biomem-
branes are superoxide dismutase, catalase and peroxidase. Enzymes such as chitin-
ases are directly involved in defense against invading pathogens, especially against
EPF. Studies reported high levels of these enzymes in response to pesticides and
entomopathogen applications in different insects i.e. B. tabaci (Tian et al. 2015; Jia
et al. 2016), Plutella xylostella (Luo and Zhang 2003) etc. The increase in enzyme
activity is prominent during initial periods of application (mostly up to 72 h), later
restrained due to overhaul by toxicity of the external agent (pesticides or entomo-
pathogen). In joint applications the sublethal or even non-lethal doses of insecti-
cides act as stressor facilitating the entomopathogen invasion (Vallet-Gely et al.
2008).
Metabolism of acetylcholine is also a primary target for most of the insecticides,
particularly neurotoxic. Synergistic interaction between nerve toxins and entomo-
pathogens can be explained by chemical disturbance in acetylcholine balance and
its degrading enzyme, acetylcholine esterase (Liu et al. 2008; Zibaee et al. 2009; Ali
et al. 2017).
Insect immune system is governed by hemocytes, which attack any foreign bod-
ies’ directly, and phenoloxidase cascade, a major defense of humoral reaction (for
more details see Chaps. 8 and 13). Amongst hemocytes, the plasmatocytes and
granular cells are responsible for phagocytosis and encapsulation of intruders.
Hiromori and Nishigaki (2001) reported decrease in granulocytes and inhibition in
humoral activity by reduced phenoloxidase activity in the whitegrub A. cuprea, in
response to mixed application of M. anisopliae and an insecticide. Insect growth
regulators used as insecticides also have synergistic interactions, causing disrup-
tions in chitin lamellae thereby facilitating infection by EPF (Hassan and Charnley
1987, 1989).
Both compatibility and joint interactions of any given pesticide and entomopatho-
gens, under both laboratory and field conditions, represent the outcome of complex
systems and cannot be generalized. Their interaction is function of the dynamic
uniqueness of both components. The determining factors of entomopathogen are
species, strain/isolate, insecticide resistance level, existing biotic and abiotic condi-
tions in the area of isolation, innate biochemical and physiological potency etc. The
pesticide factors to be considered are chemical group, mode of action, concentra-
tion, type of formulation, ingredients of formulation etc. Target species, evaluation
methods, timing of assay can also be influential parameters which are under the
researcher’s hand. Scientific understanding of all these factors could predict the
interaction but not the actual performance. Amongst entomopathogens, to the best
of our knowledge no study reported compatibility and synergism of EPVs with
insecticides which might be due to their obligate parasitism. However, they repre-
sent an environmentally competent group of biopesticides due to their ability to
cause epizootics and ease of formulation preparations. Intensification of research
efforts in this area is hence required.
The synergistic interactions always showed waning trends towards field, which
might be due to increased size of experimental units as well as other biotic and abi-
otic factors which are excluded under laboratory conditions. Correspondingly, some
antagonistic and additive interactions also proved to be effective under field condi-
tions. Although biochemical and physiological interactions of synergism are known,
performance of minimum possible interactions, including antagonistic and additive
effects under field conditions, could give a clear picture of a joint action between
pesticides and entomopathogens. Similar studies on temporal and spatial separation
of incompatible mixtures may also explain possible combinations for enhanced and
ecofriendly pest management. The possible effects on non-target species and natu-
ral enemies should not be ignored, as the final applications are targeted to agroeco-
systems where a complex of species coexists.
Understanding the entomopathogens and insecticides interplay may lead to pos-
sible new strategies for management. This strategy uses manifold and complex
attack approaches in killing target insect pests. Besides, due to direct toxicity effects
on pest populations after receded insecticidal activity of pesticides, the entomo-
pathogen may establish on the residual host population offering subsequent pest
management in targeted agro-ecosystem. This process may also eliminate subse-
quent pesticidal applications and safeguard the biodiversity. There is a possible
development of resistant or tolerant strains of entomopathogens that can survive at
high pesticide concentrations. These strains offer possible development of a com-
posite commercial formulation containing combined preparations, that can also be
used in insecticide resistance management. Despite of relative, partial successes, no
study reported large scale field successes of joint applications, which needs to be
done to exploit their full potential.
Acknowledgments This study was supported by the Indian Council of Agricultural Research
(ICAR), New Delhi. Authors are thankful to Director, ICAR-VPKAS, Almora.
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Chapter 13
Entomopathogen and Synthetic Chemical
Insecticide: Synergist and Antagonist
Arash Zibaee
Abstract The use of synthetic insecticides and biological agents are the main tools
to control agricultural pests, each with its own advantages and disadvantages.
Although the use of chemical compounds is inevitable in some cases, attitudes of
consumers and agricultural experts towards healthy products and lower environ-
mental contamination have increased the prevalence and preference for biological
agents. Among them, insect pathogens have been considered as the unique and
widely distributed components in many ecosystems, due to their diverse virulent
mechanisms. The entomopathogenic fungi (EF) and nematodes have been commer-
cialized as biologically active insecticides against a wide range of pests. Although
many environmental benefits for these compounds have been identified, the disad-
vantages such as low virulence due to behavior or habitat of target pest, delayed
killing performance and sensitivity to environmental factors, lead to simultaneous
use of entomopathogens with one or more chemical insecticides in reduced doses.
In this review, the possibility of simultaneous use of chemical insecticides from dif-
ferent classes with EF and nematodes were discussed by indicating severally up-to-
date studies. The effects of insecticides on cessation or induction of germination and
conidiation of fungi have been reported, depending on the concentrations of used
chemicals. Field or laboratory experiments have shown synergism or antagonism of
EF with some insecticides. In case of entomopathogenic nematodes, the effects of
insecticides from different classes have been investigated on mobility and survival
of nematodes, as well as on synergistic or additive effects. Generally, the possibility
of simultaneous use of chemical insecticides with these two groups of entomo-
pathogens depends on target pest, spraying method, insecticide class or formulation
and origin of entomopathogens.
Keywords Entomopathogen · Insecticide · Interaction · IPM
A. Zibaee (*)
Department of Plant protection, Faculty of Agricultural Sciences, University of Guilan,
Rasht, Iran

https://doi.org/10.1007/978-3-030-23045-6_13
342 A. Zibaee
13.1 Introduction
To produce healthy food for growing population of world has been one of the most
important requirements in recent decades. In addition to limiting factors such as
proper planting, climate and processing costs, insect pests, plant diseases and weeds
have caused the significant negative effects on agricultural production (Oerke et al.
1994). Insects as main, occasional and secondary pests have had enormous impacts
on the yields of agricultural products and induce much of production costs (Chandler
et al. 2011). Although different methods have been applied to reduce insects eco-
nomic impact over years, biological and chemical controls have been assigned the
largest share (Pretty 2008; Lacey et al. 2015). Although the use of biological agents
has led to production of healthy or so-called organic products, chemicals from dif-
ferent groups of organochlorine, organophosphorus, carbamates, neonicotinoids,
insect growth regulators, botanical insecticides and other nature-based insecticides
have been inevitable in many cases (Talebi-Jahroumi 2012). Data gained based on
the essential chemical industry-online indicate that chemical insecticides comprise
24% of used agrochemicals, in which different classes have their own proportions
from 3% to 22% (Fig. 13.1 a, b, c). Despite fast and efficient impacts of chemicals
on suppressing pest population outbreaks, some issues led to limitations on their use
as summarized by Chandler et al. (2011): (i) damages on environment and human
during manufacturing, handling and application because of not judicious use of
broad-spectrum insecticides, (ii) issues such as pest resurgence, emergence of sec-
ondary pests and development of heritable resistance following prophylactic use of
insecticides, (iii) withdrawal of several groups of insecticides because of new health
and safety legislations, (iv) concerns on chronic toxicity of insecticides due to resid-
uals in food, on all consumers (human and livestock).
Such concerns and disadvantages of synthetic chemical insecticides forced
trends to use other control procedures against insect pests. One of the promising and
long-lasting ways could be to use natural enemies in different agroecosystems.
Although natural enemies may result in 50–90% efficiency of pest control in crop
fields, potential application and reliability require the extended scientific knowledge
and concerned economic and social attitudes (Pimentel 2005). Nevertheless, there
are several advantages that make biological control as one of the major control pro-
cedures in sustainable agriculture including: (i) the high efficiency at low cost, (ii)
self-perpetuation at little or no cost, (iii) free of harmful effects on non-target organ-
isms, (iv) ability of biocontrol agents to rapid reproduction, (v) self-searching the
target hosts, (vi) survival at relatively low host densities in addition to (vii) adapt-
ability to several climatic conditions (Mason and Huber 2002; Neuenschwander
et al. 2003; Omkar 2016; Khan and Ahmad 2015; Khan et al. 2016). Smith (1919)
was the first to define the term of biological control (biocontrol) as the “top-down”
action of natural enemies to equilibrate the density of pest population at a level
lower than when they are absent. Later, DeBach (1974) represented it as “biocontrol
is the use of living organisms/natural enemies to suppress the population density or
impact of a specific pest organism, making it less abundant or less damaging than
13 Entomopathogen and Synthetic Chemical Insecticide: Synergist and Antagonist 343
Fig. 13.1 The percentage of chemical insecticides used vs other chemicals and their different
classes compared to groups of entomopathogenns. (a) Proportion of pesticide used in agroecosys-
tems, (b) Proportion of different classes of insecticides used in agroecosystems, (c)
Commercialization proportion of entomopathogens against insect pessts. (Based on: www.essen-
tialchemicalindustry.org/materials-and-applications/crop-protection-chemicals. Html; The 2010
Worldwide Biopesticides Market Summary, vol. 1. CAB International Centre, Wallingford.)
it would otherwise be”. Based on these definitions, biological control relies on

proper action of organisms which are known as biocontrol agent acting vs pests.
Predators, parasites, parasitoids and entomopathogens are the biocontrol agents
engaged in biological control tactics over decades. Each agent adopts its unique way
to concur and subsequently decrease population density of hosts, but several factors
such as their high adaptability, host specificity (monophagous), voracious feeding
capacity, synchronized development with pest species, more spatial and temporal
dispersal, multiplication capacity, short life cycle with multiple generations, unend-
ing host search capacity, being non-palatable for predators, and resistance to other
pathogens and parasites do influence their appropriate effectiveness in (IPM)
(Omkar 2016). Among the biocontrol agents, entomopathogens are the infectious
microorganisms that invade insects through different portal of entries, e.g. integu-
ment and mouth, reproduce within hemocoel and spread to infect other individuals
or insects. Spanning over the prokaryotic and eukaryotic Kingdoms they include
viruses, bacteria, microsporidia, fungi and nematodes (Kaya and Vega 2012). These
agents are used to control several insect pests and have shown to be successful
344 A. Zibaee
depending on virulence, ease of application and production, low cost, good storage
properties, safety to farmers, and colonization in environment (Lacey et al. 2015;
Omkar 2016). Among the entomopathogens, the bio-insecticides based on the bac-
terium Bacillus thuringiensis Berliner and its derivate comprise the highest ratio of
the market, with fungi in the second rank and nematodes in the least ratio (Fig. 13.1c).
In this chapter, initially a background on use of entomopathogenic fungi (EF)
and nematodes as well as their infectious mechanisms in insects will be presented,
then, their limitations in biological control programs and reasons for integration
with insecticides will be discussed by reporting the results of laboratory and field
studies. Eventually, the synergistic and antagonistic mechanisms between EF/nema-
todes with chemical insecticides will be compared in several case studies.
13.2 Potential of Entomopathogenic Fungi Against Insects
EF are the dominant pathogens among populations of insect pests which are almost
ubiquitous in both terrestrial and aquatic ecosystems. These biocontrol agents have
shown a high potential to regulate population densities of insects not only agricul-
tural pests but also insect-vector like mosquitoes. EF cause infections throughout
insect populations as obligatory to facultative pathogens but their applications in
ecosystems might be possible through mass production, formulation and proper
dosage in the framework of microbial insecticides (Lacey et al. 2015). EF are cate-
gorized into several groups that are located in the Hypocreales order of Ascomycota,
including imperfect taxa such as Beauveria, Metarhizium, Nomuraea, Isaria. and
Hirsutella spp. Other important species such as Entomophthora, Zoophthora,
Pandora and Entomophaga spp. belong to the order of Entomophthorales from
Zygomycota (Araujo and Hughes 2016; see Chaps. 2 and 3 for more details).
Species belonging to Hypoceales such as Beauveria spp. and Metarhizium spp. are
suitable for mass production and to be used against insect pests of crops, orchards,
greenhouses and forests because of their possibility to grow on low-cost cultivated
environments, utilizing simple technologies to be formulated and marketed (Jenkins
et al. 1998; Maniana et al. 2002).
Once a conidium attaches to the insect cuticle, it germinates through it and mul-
tiplies after reaching the body general cavity. Ultimately, blastospores are prolifer-
ated and lead to host death by production of secondary metabolites and consumption
of food sources within tissues, mainly fat bodies (Brownbridge et al. 2001; Ortiz-
Urquiza and Keyhani 2013). The pathogenic fungi which are able to penetrate faster
the hemocoel can quickly spread within an insect population and cause earlier
death. They utilize an enzymatic complex with main roles of chitinases and prote-
ases to penetrate through integument (St. Leger et al. 1986; Zibaee and Bandani
2010; Ortiz-Urquiza and Keyhani 2013; Firouzbakht et al. 2015). Typically, at the
beginning of infection, trypsin- and subtilisin-like proteases synthesize and infil-

trate the fungus hypha to the body while after several hours, the synthesis of chitin-
ases increases the penetration and contamination efficiency (St. Leger et al. 1986).
Therefore, in addition to bioassay of fungal pathogens on insects, it is possible to
determine their efficacy by comparing expression of the involved genes, especially
proteases and chitinases (Pedrini et al. 2007; Ortiz-Urquiza and Keyhani 2013).
There are several advantages which make EF suitable candidates in biological
control programs: (i) they are easily cultured on standard artificial media such as
potato-dextrose agar or malat extract agar (however Entomophthora species require
a medium containing animal materials), (ii) they are safe for humans and livestock
because the best growth temperatures are between 20 and 25 °C, and their growth is
inhibited at high temperatures, till 37 °C, (iii) ease of release in a culture medium or
on hosts in wet conditions that facilitates the production of non-sexual spores, that
are easily dispersed by wind, rain, and water droplets, (iv) resistance to peripheral
conditions by producing highly resistant or inactive spores.
Nevertheless, there are some disadvantages which make it difficult to recom-
mend EF as the most powerful biocontrol agents in agroecosystems. Several envi-
ronmental factors are involved in formation of a fungal epizootic in a pest infested
area, thus providing favorable environmental conditions is essential for inoculum
success in agroecosystems (Lacey et al. 2015). The spores of EF are sensitive to
ultraviolet radiation and drought. Another problem is a provision of appropriate
types of inoculum to be used in fields, although those can persist in soil and organic
matter residues (Goettel et al. 2010). Moreover, several EF may be susceptible to
high doses of conventional fungicides. Finally, specificity to target insect, dose of
conidia and slow killing are the other factors which limit use of EF as myco-
insecticides, in some cropping systems. Hence, the use of these agents should be
reasonably made along with a complete understanding of where they are aimed to
be used (Goettel et al. 2010; Lacey et al. 2015). It is imperative to highlight that
advances in molecular biology of EF provide a fundamental platform on improve-
ments of fungal survival and virulence by transforming techniques (Ortiz-Urquiza
et al. 2015; Zhao et al. 2016). In fact, these techniques produce recombinant fungi
with new characteristics which requires appropriate transformation systems to iso-
late specific pathogenic genes, investigate virulence determinants leading strains
with enhanced virulence or tolerance to environmental stress (Ortiz-Urquiza et al.
2015; Zhao et al. 2016). But these techniques are costly, require a lot of time to
study biometrics and hosting allocation, finally including the legal rules governing
genetically modified organisms. Therefore, the use of other methods is necessary to
increase efficacy or durability of EF in agroecosystems. In this step, combining the
application of EF with reduced concentrations of authorized insecticides can effec-
tively control insect pests and reduce the environmental and biological risks due to
chemical compounds.
346 A. Zibaee
13.3 I n vitro Interactions of Entomopathogenic Fungi

and Chemical Insecticides
Like natural environmental factors, chemical insecticides do directly affect life

characteristics of EF. This would be crucial if a compound turned into an obstacle
on fungal inoculum establishment in agroecosystems. Such incompatible insecti-
cides suppress vegetative and reproductive performance of fungi and decrease fun-
gal efficiency in biological control or IPM (Zibaee et al. 2009). It is hence mandatory
to determine potential compatibility of EF and chemical insecticides prior to field
application by assessing germination, vegetative growth and conidia production.
These parameters may vary depending on species and isolates, as well as on the
nature of the insecticide (Goettel and Inglis 1997; Pachamuthu et al. 1999; Antonio
et al. 2001). Germination is an important factor for fungal penetration and subse-
quent host colonization as any decline in its percentage may negatively influence
fungal induced mortality in the host population (Alves 1998). Increasing the yield
of conidia and improving their quality are the main objectives to consider EFs as
suitable biocontrol agents. This is of highly importance as conidia are the main
infective units against insect pests and contribute significantly in fungal survival and
dispersal in the environment (Muñiz-Paredes et al. 2017). In vitro studies on interac-
tions between chemical insecticides and EF have been concentrated on possible
inhibition of mycelial growth, sporulation and conidial germination although these
traits are not necessarily correlated together (da Silva et al. 2013). For example,
Tamai et al. (2002) reported inhibition of mycelial growth in B. bassiana whereas
conidia production increased due to stress imposed by the chemical pesticides or
initial reduction of mycelial growth. Even, a chemical may have no negative effects
on mycelial growth but may inhibit conidiation. Among the mentioned fungal traits
in response of chemical treatments, conidial germination may be an indicator of
successful epizootics among insect populations (da Silva et al. 2013). Hence, any
suppression in conidial germination may lead to lower efficiency and persistence of
EF in the target insect habitat. In the following, some examples are given to eluci-
date in vitro interactions between chemical insecticides and EF in laboratory
assessments.
Li and Holdom (1994) showed that organochlorine or organophosphorus insecti-
cides were more toxic to entomopathogens than other insecticides. They reported
that carbamate insecticides such as carbofuran, methoxyl and oxymyl were moder-
ately toxic while chloropyriphos, malathion and tempephos were extremely toxic.
Asi et al. (2010) evaluated the effects of 13 insecticides including chlorpyrifos,
methomyl, thiodicarb, chlorfenapyr, indoxacarb, emamectin benzoate, lufenuron,
profenofos, abamectin, triflumuron, flufenoxuron, methoxyfenozide and spinosad
on mycelial growth and conidial germination of Metarhizium anisopliae
(Metchnikoff) and Paecilomyces fumosoroseus (Wize). Although all used insecti-
cides significantly decreased mycelial growth and conidial germination, with high-
est toxicity showed by chlorpyrifos. In contrast, spinosad had the least effects on
mycelial growth and conidial germination of both fungi (Asi et al. 2010). Marzieh
et al. (2010) reported drastic reduction in conidial germination of M. anisopliae

when fipronil, pyriproxyfen and hexaflumuron were amended to Sabourad Dextrose
Agar (SDA) at the highest concentration (0–15%). They also found higher negative
effects on hexaflumuron than other two insecticides (fipronil and pyriproxyfen)
indicating its incompatibility with M. anisopliae. Archana and Ramaswamy (2012)
added different concentrations of three organophosphorus insecticides (phorate,
malathion and chlorpyrifos) and the two synthetic pyrethroids (deltamethrin and
permethrin) into the three culture media (Potato Dextrose Agar, Sabourad Dextrose
Agar and Czapek Dox Agar) of P. fumosoroseus to check their effects on vegetative
growth and colony development. At first, the authors reported that PDA and SDA
were the more appropriate media for vegetative growth and spore production of P.
fumosoroseus than CDA. Then, they found phorate as the most incompatible insec-
ticide compared to other ones as it caused the least colony growth and spore produc-
tion. A moderate growth of the fungus was observed in PDA plates treated by
melathion, permethrin, deltamethrin and chlorpyriphos, while the growth of P.
fumosoroseus in SDA plates treated by the mentioned insecticides showed no sig-
nificant difference, compared to control. These four insecticides had no effects on
spore production in both PDA and SDA media (Archana and Ramaswamy 2012).
In vitro toxicity of conventional agrochemicals used in rice crops including eight
insecticides (fipronil in two formulations, methyl parathion, cypermethrin, thia-
methoxam, thiamethoxam+Lambsa-cyhalothrin, lambda-cyhalothrin, methamido-
phos), four fungicides (difenconazole, propiconazole, trifloxystrobin, azoxystrobin)
and five herbicides (glyphosate, 2,4-dichlorophenoxyacetic acid, bentazon, imaza-
pic + imazapyr, pendimethalin) were investigated on germination, vegetative growth
and conidia production in a Brazilian strain of M. anisopliae (CG 168) (da Silva
et al. 2013). The field recommended doses of each chemical was added into PDA
culture media. Results demonstrated that the fungicides difenoconazole, propicon-
azole, trifloxystrobin and azoxystrobin had the most toxic effects on the given
parameters of M. anisopliae. In contrast, all used insecticides showed the least tox-
icity and the herbicides only decreased mycelial growth. The authors concluded that
methyl parathion, thiamethoxam, lambda-cyhalothrin, glyphosate, bentazon and
imazapic + imazapyr were the compatible chemicals with the Brazilian strain of M.
anisopliae and could be used in IPM of rice pests.
Shaabani et al. (2015) investigated the potential compatibility of an Iranian iso-
late of M. anisopliae (DEMI 001 strain) with imidacloprid through determining
conidial germination, mycelial growth and sporulation. Different concentrations of
imidacloprid were added into SDA and yeast extract prior to add 0.5 mL of conidial
suspension (105 conidia/mL). The authors reported that imidacloprid had no nega-
tive effects on conidial germination, mycelial growth and sporulation, compared
with control.
In a recently published study, the in vitro interaction between five insecticides of
Archer PlusTM (gamma-cyhalothrin 15% [w/v]), LambdaTM (lambda-cyhalothrin
25% [w/v]), CoragenTM (rynaxypyr 20% [w/v]), MatchTM (luphenuron 5% [w/v])
and IntrepidTM (methoxyfenozide 24% [w/v]), and different strains of Beauveria
bassiana (Balsamo-Crivelli), M. anisopliae and M. robertsii Bisch et al. were
348 A. Zibaee
s tudied under laboratory conditions (Pelizza et al. 2018). The significant differences
were found via conidial-viability tests among the assayed insecticides and the fun-
gal strains. Germination of conidia in all fungal isolates significantly decreased
along with increasing insecticide concentrations although γ- and λ-cyhalothrin
showed the least effects on germination. Also, the vegetative growth of different
fungal strains was influenced by chemical insecticides with the most inhibitory
effects on the colony area of B. bassiana strains LPSc 1082, LPSc 1098 and M.
anisopliae strain LPSc 907, when exposed to luphenuron. In addition, the highest
concentrations of γ-cyhalothrin, λ-cyhalothrin, and rynaxapyr had no effects on the
vegetative growth of strain LPSc 1067. Finally, the authors found the highest inhibi-
tory effects on conidia production for methoxyfenozide, whereas γ-cyhalothrin and
λ-cyhalothrin showed the lowest effects.
13.4 I n vivo Interactions of Entomopathogenic Fungi

13.4.1 Selected Case Studies
Because of some potential deficiencies of EF to control insect pests, combination

with insecticides (mainly low risk ones) might be recommended in agroecosystems.
Although such a combination has been shown through synergistic or additive mech-
anisms, there are some studies indicating antagonistic interactions between EF and
synthetic insecticides. In this section, these findings will be exemplified and their
mechanisms will be discussed.
Earlier studies demonstrated that combinations of dichlorodiphenyltrichloroeth-
ane (DDT), azinphos-ethyl, carbaryl, fenvaralate, abamectin, triflumuron, and
thuringiensin with B. bassaina caused no synergistic or additive effects on mortality
of Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera:
Chrysomelidae) (Fargues 1973, 1975; Anderson et al. 1989). In contrast, Quintela
and McCoy (1997) prepared different concentrations of B. bassiana and M. aniso-
pliae, and treated them via dipping assay against first instar larvae of weevil,
Diaprepes abbreviates L. (Coleoptera: Curculionidae). Meanwhile, the larvae
treated with these EF were orally and topically exposed to 0 and 100 ppm concen-
trations of active ingredient as well as 500 ppm of formulated imidacloprid. The
authors reported no mortality caused by the highest concentrations of the fungi
while the larval mortality and mycosis gradually increased once the concentrations
were applied with imidacloprid, in both contact and oral exposures. Such a syner-
gism was more significant when the highest concentrations of fungus and insecti-
cides were administrated against the larvae. Also, the authors suggested additive
combination of the fungus and imidacloprid when ecdysis of the contact-treated
larvae decreased by increasing both insecticide and EF doses. No larval ecdysis was
found in oral exposure of imidacloprid. The LT50 value of the fungus significantly
decreased in both contact and oral administrations with imidacloprid although oral
administration led to the lower LT50 value compared to contact exposure. The find-
ings on combination of formulated imidacloprid with M. anisopliae revealed that
carriers in formulation had negligible effect on larval mortality, suggesting that the
active ingredient was the key component causing synergism between imidacloprid
and the EF.
The sole and combined effects of M. anisopliae and sublethal concentrations of
chlorpyrifos, propetamphos and cyfluthrin, all in commercial formulations, were
determined against Blattella germanica L. (Blattodea: Ectobiidae) by Pachamuthu
and Kamble (2000). The authors topically treated the adult cockroaches with 108
conidia/ml of M. anisopliae along with low and high concentrations of each insec-
ticide. The combination of M. anisopliae with the three mentioned insecticides led
to the significant higher mortalities in all eight concentrations except for 100 ppm.
The mortality increased in a dose-dependent manner so that the highest values were
found in the combination of M. anisopliae with 300 ppm concentration of chlorpyri-
fos, propetamphos and cyfluthrin. In details, the combination of M. anisopliae with
200 and 300 ppm of chlorpyrifos, 300 ppm of propetamphos, and 20, 30, and
40 ppm of cyfluthrin led to the faster mortalities than M. anisopliae alone, while
other concentrations of the insecticides caused no statistical difference or even
antagonistic effects on LT50 value. Finally, the authors concluded that the increased
mortality was influenced by the concentration and not the type of each insecticide,
showing an additive interaction with the EF.
James and Elzen (2001) reported antagonism in the combined application of B.
bassiana and imidacloprid against Bemisia argentifolii Bellows (Hemiptera:
Aleyrodidae) as the fungus inhibited the insecticide effectiveness. Collectively, they
found the mixture of imidacloprid and B. bassiana increased whitefly mortality but
less than in a direct additive way, whereas the insecticide showed no negative
impacts on spore germination and colony formation. The authors concluded that B.
agnetifolii treated by B. bassiana recruited a behavioral response to decline feeding
and uptake of imidacloprid so that it might be useful to apply imidacloprid in soil
rather than foliage. Their data were in contrast to findings of Steinkraus (1996) and
Brown et al. (1997) on the combined effects of these two components against tar-
nished plant bug.
Shapiro-Ilan et al. (2011) set a series of laboratory experiments to find potential
combination of B. bassiana with the two common commercial insecticides, cyper-
methrin and carbaryl, to increase control efficiency of the pecan weevil, Curculio
caryae (Horn) (Coleoptera: Curculionidae). After initial experiments to find effec-
tive concentrations of both microbial and chemical agents against larvae and adults
of weevil, different combinations were considered using low and high concentra-
tions. Mortality was monitored for 9 and 5 days for the larvae and adults, respec-
tively. Results of combined effects on both stages showed synergistic and
antagonistic interactions among B. bassiana, cypermethrin and carbaryl, respec-
tively. B. bassiana and carbaryl led to synergistic mortality on the pecan weevil
while an antagonistic interaction was found once a combination of B. bassiana and
cypermethrin was used against the larvae. The authors found their results differed
350 A. Zibaee
from some other reports and concluded that the type of interaction between micro-
bial agents and chemical insecticides might be dependent on microbial species,
chemical structure of insecticides, host species and habitat.
In a factorial study, different combinations were prepared by Sharififard et al.
(2011) using two concentrations of M. anisopliae and the three concentrations of
spinosad to find their effects on the larvae and the adults of Musca domestica L.
(Diptera: Muscidae). All treatments including fungal inoculation, insecticide and
their combinations led to significant mortalities of adults with the highest values
caused by the fungus-insecticide mixture. In details, a fungal concentration of 105
conidia/g with 0.5, 1 and 1.5 mg/g of spinosad led to the highest mortality compared
to other fungus concentrations. These findings indicate synergism between spinosad
and M. anisopliae at 107 conidia/g with additive effects on adults mortality. Apart
from mortality, LT50 values of the fungus/spinosad combinations were significantly
lower than each component alone. Likewise, the highest larval mortality was evalu-
ated the combination of M. anisopliae at 108 conidia/g and 0.002, 0.004 and
0.006 μg/g of spinosad, showing synergism.
Apart from chemical insecticides, there are a few studies indicating synergism of
using EF and horticultural oils against some hemipterans. Initially, Cuthbertson and
Collins (2015) demonstrated that mixed application of a petroleum horticultural oil,
Tri-Tek, and B. bassiana increased mortality of Bemisia tabaci (Gennadius) com-
pared to sole use of these components under glasshouse conditions. Kumar et al.
(2017) tested possible compatibility and efficiency of Isaria fumosorosea Wize with
six horticultural oils, vs the Asian Citrus Psyllid, Diaphorina citri Kuwayama
(Hemiptera: Liviidae). A blastospore formulate of the fungus was combined with
Orchex 796, Sun Pure Spray Oil 435, Conoco Blend Spraybase 435 Oil (90 and
100%), JMS Stylet Oil and PFR-97 and applied against D. citri via leaf disk assay,
under laboratory condition. The results clearly demonstrated that horticultural oils
significantly increased the fungus efficiency by 100% mortality caused by Orchex
796 and JMS, at 8 and 12 days. Meanwhile, other oils significantly decreased sur-
vival time of the psyllids mainly Conoco Blend 435 (100%) although no cadaver
colonization was observed in D. citri treated by the oil. The authors concluded that
these oils were compatible with I. fumosorosea, increased its efficiency against
adult psyllids and fungal development index (FDI), the latter being crucial for fun-
gal persistence in the site of spraying, for horizontal transmission of spores to other
individuals (Kumar et al. 2017).
Pelizza et al. (2018) evaluated the combined effects of five insecticides and three
EF (five strains) against soybean defoliating pest, Rachiplusia nu Guenee
(Lepidoptera: Noctuidae). Three concentrations of γ-cyhalothrin, λ-cyhalothrin,
rynaxypyr, luphenuron, and methoxyfenozide were prepared based on 100, 50 and
25% of the average field recommended doses and combined 1:1 with the three con-
centrations (104, 106 and 108 conidia/ml) of B. bassiana (isolates LPSc 1067, LPSc
1082, LPSc 1098), M. anisopliae (LPSc 907), and M. robertsii (LPSc 963) to test
their potential synergistic, antagonistic or additive effects on larvae. Results revealed
significantly different mortalities caused by insecticides/fungi combinations on the
third larval instars after 10-day exposure using half concentration of the field
recommended dose. Larval exposure to LPSc 1067, LPSc1082, LPSc 1098, LPSc
907 and LPSc 963, combined with all insecticides at the highest field concentration,
led to 100% mortality showing additive interactions. Similar results were observed
except for the combination of λ-cyhalothrin/LPSc-1098 and M. robertsii strain
LPSc 963. At the same concentration, the synergistic interaction was found in case
of γ-cyhalothrin/LPSc-1067 or LPSc-963, λ-cyhalothrin/LPSc-1067, rynaxypyr/
LPSc-1067 or LPSc-1098, and luphenuron/LPSc-1067. Moreover, antagonistic
interaction was obtained when the combinations of λ-cyhalothrin/LPSc-1098 or
LPSc-963, rynaxypyr/LPSc-963, luphenuron/LPSc-963, and methoxyfenozide/
LPSc-907 or PSc-963 were used against the larvae of soybean defoliator. The com-
bination prepared based on 25% of the highest field concentration showed an addi-
tive interaction except for γ-cyhalothrin/LPSc-1067, λ-cyhalothrin/LPSc-1067,
rynaxypyr/LPSc-1067, with methoxyfenozide/LPSc-1082 indicating synergism and
rynaxypyr/LPSc-1098 showingantagonism.
13.4.2 echanisms Underlying EF and Insecticides

M
Interaction
Although fungal species, sensitivity to environmental factor, conidia formulation,

target insect species, chemical structure of insecticide and mode of action are among
the factors influencing outcomes of fungi/insecticides combinations, there are some
physiological traits within insects which determine whether these combinations
may be synergistic or antagonistic. Serebrov et al. (2001) infected the fifth larval
instars of greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae) with B.
bassiana, M. anisopliae and P. fumosoroseus.Hemolymph of the larvae was then
collected, determining esterase activity with specific substrates.. In next step, the
authors treated the larvae with a concentration of deltamethrin via topical applica-
tion to determine their sensitivity. The authors found that all fungi induced protein-
aceous patterns related to esterase activity in gel electrophoresis, related to the
virulence of each entomopathogenic fungus and treatements with deltamethrin.
Such changes were observed during mycosis, mechanical damage of cuticle, chill-
ing at –4 °C for 30 min and larval treatment by deltamethrin. Moreover, the LC50
concentration for deltamethrin on the intact larvae was 1.8 mg/g and 2.1 mg/g in the
larvae infected by highly virulent strain.
Serebrov et al. (2006) tried to determine whether fungal infection could elicit
insect resistance against malathion, an organophosphorus insecticide. Initially, the
fifth larval instars of G. mellonella were infected by M. anisopliae, then hemo-
lymphs of control and infected larvae were collected to assay activities of esterase,
glutathione S-transferase and phosphatases. In a further assay, the control and
already infected larvae were exposed to malathion. Finally, enzyme inhibitors were
used to confirm the role of detoxification enzymes in the insect resistance to the
entomopathogenic fungus. The activity of esterases significantly increased almost
352 A. Zibaee
two folds in the hemolymph of the infected larvae by M. anisopliae, while no sig-
nificant changes were observed in fat body and intestine preparations. Similar
results were found in the activity of glutathione S-transferase while acid- and alka-
line phosphatases showed no significant changes (Serebrov et al. 2006). Results on
malathion treatment against control and M. anisopliae infected larvae revealed that
LC50 required to kill 50% of the infected larvae was 1.5 times higher than in control,
showing a certain degree of resistance to malathion in these larvae. Results on using
inhibitors of detoxifying enzymes including S,S,S-tributyltrithiophosphate (tribu-
fos, a non-specific esterases inhibitor), piperonyl butoxide (cytochrome P450-
dependent monooxygenases inhibitor), and diethyl maleate (glutathione
S-transferases inhibitor) showed that the M. anisopliae-infected larvae exposed to
tribufos and piperonyl butoxide had higher mortality compared to control. No sig-
nificant difference was observed when diethyl maleate was used against the larvae.
Consequently, the authors attributed the higher activities of detoxifying enzymes
during mycosis to intoxication with metabolites of M. anisopliae or with products
present in the degraded host tissues (Serebrov et al. 2006). In a similar study, Zibaee
et al. (2009) investigated the effects of B. bassiana and its secondary metabolite on
toxicity of fenitrothione against sunn pest, Eurygaster integriceps Puton (Hemiptera:
Scutelleridae). Both fungal spores and secondary metabolites significantly increased
glutathione S-transferase activity using CDNB and DCNB as substrates, 3–5 days
after inoculation. Similar results were found in the activity of esterase in the infected
hemipteran compared to control, using α- and β-naphtyl acetate as substrates. In the
last experiment, results on fenitrothione treatment on control and treated hemip-
teran with B. bassiana spores and secondary metabolites showed LC50 values of
9.4 ml/mg and 22.5 ml/mg compared to 5.9 mL/mg of control, almost two to four-
fold higher. These results clearly showed resistance of E. intergriceps to fenitrothi-
one after fungal treatment.
Apart from above-mentioned findings indicated the possible mechanisms under-
lying antagonistic interactions between EF and insecticides, there are studies on
explaining why synergism occurs between these agents. In the sets of experiments,
Jia et al. (2016) tried to find whether chlorantraniliprole and a M. anisopliae strain
had synergism in control of the migratory locust, Locusta migratoria L. (Orthoptera:
Acrididae). Then they determined what might be the mechanism underlying such an
interaction. In the first two experiments, the efficiency of M. anisopliae and chloran-
traniliprole was evaluated separately. In the third assay, different proportions of M.
anisopliae and chlorantraniliprole were prepared to test their combination efficiency
against L. migratoria. In the last experiment, the insects were initially treated with
M. anisopliae conidia then after 4 days, and fed on wheat sprouts treated with dif-
ferent concentrations of chlorantraniliprole, under laboratory conditions. The
insects exposed to combined proportion of the entomopathogenic fungus and insec-
ticides were considered for biochemical analysis of esterase, glutathione
S-transferase, monoxygenase, acetylcholine esterase, phenoloxidase, superoxide
dismutase, peroxidase, catalase, chitinase and aryl acylamidase. A higher mortality

was found when the mixed application of M. anisopliae and chlorantraniliprole was
used against L. migratoria with LC50 values of 0.01 and 0.02 mg/L in comparison
with 0.15 mg/L of M. anisopliae alone. Also, the locusts initially treated with the
fungus showed co-toxicity after treatment with chlorantraniliprole. Separately
treated L. migratoria with M. anisopliae and chlorantraniliprole showed the higher
activity of esterase compared to control, but the enzyme activity significantly
decreased following treatment with the mixed solution. A similar trend was reported
for glutathione S-transferase only in the earlier periods after treatments. L. migrato-
ria treated with the mixture of M. anisopliae and chlorantraniliprole showed the
lower activity of phenoloxidase at earlier periods that increased later. Separate treat-
ment with these two agents increased the enzyme activity compared to control. The
sole and mixed exposure on L. migratoria led to the higher activity of monoxygen-
ase somehow similar to the activities of acetylcholinesterase, chitinase and aryl
acylamidase. The activities of superoxide dismutase, catalase and peroxidase
increased in the locusts treated with M. anisopliae and chlorantraniliprole alone, but
the mixed combination increase the activities of superoxide dismutase and peroxi-
dase, with a lower activity of catalase. The authors concluded that L. migratoria
recruits enzymatic defenses against M. anisopliae or chlorantraniliprole, while their
mixed application may disable these enzymes regarding time after exposure. The
authors believed that these agents were able to disrupts calcium ion within insect
cells which was critical to normal activity of enzymes, mainly phenoloxidase.
Moreover, mixed action of M. anisopliae and chlorantraniliprole disabled a com-
plex of biochemical processes leading to synergistic interactions in L. migratoria
(Jia et al. 2016).
In addition, Ali et al. (2017) reported data on toxicological and biochemical char-
acteristics of synergism between Lecanicillium muscarium Zare & Gams and
matrine, following treatment on B. tabaci. Experimental design and assayed bio-
chemical enzymes were similar to Jia et al. (2016). Results showed higher mortality
with the lower LC50 values when different proportions of L. muscarium and matrine
were prepared and applied on B. tabaci, showing synergistic interactions between
the two agents. The esterase and glutathione S-transferase activities initially signifi-
cantly increased after L. muscarium and matrine treatment. However the enzyme
activity sharply decreased in B. tabaci treated with mixture of both agents. In con-
trast, the activity of acetylcholinestrase decreased in B. tabaci treated with the sole
or mixture of both agents, regardless of post-treatment intervals. Finally, the activi-
ties of chitinase and the three enzymes involved in antioxidant responses (superox-
ide dismutase, catalase and peroxidase) increased in the insects treated with sole or
mixture of L. muscarium and matrine, 3 and 4 days after treatments, respectively.
The authors attributed the synergistic interactions of L. muscarium and matrine to
alterations of acetylcholineesterase activity, leading to disturbance of the acetylcho-
line balance in the treated B. tabaci.
354 A. Zibaee
13.5 otential of Entomopathogenic Nematodes

P
Against Insects
Entomopathogenic nematodes (EPN) have a unique symbiotic relationship with

bacteria (Shapiro-Ilan et al. 2017). They enter host body via natural openings like
mouth and anus then continue pathogenesis inside the body as endoparasites (Lacey
et al. 2015, see also Chap. 9). These agents are found in different ecological habitats
infecting several agricultural insect pests although their relationship with insects
varies from phoresis to parasitic/virulent interactions (Shapiro-Ilan et al. 2017). The
entomopathogenic nematodes have been described in 23 families of which seven
families contain species with potential for biological control of insects (see also
Chap. 9). It is notable that species from the two most important families
Steinernematidae and Heterohabditidae have been commercialized for use in agro-
ecosystems (Shapiro-Ilan et al. 2017). It has been reported that EPN distribution is
limited by the availability of sensitive hosts although virulent have the ability to
quickly kill their hosts (1–4 days depending on nematode and host species), coexist-
ing with bacteria (Xenorhabdus bacteria for steinernematidae and Photorhabdus
bacteria for heterohabditidae) (Dowds and Peters 2002; Shapiro-Ilan et al. 2017).
The common feature of all EPNs is to have a third stage infective juvenile (Dauer
Juvenile) which exists outside of host body, carries the cells of symbiont bacteria,
finds the host and enters into hemocoel. In host body, the abandoned bacteria prolif-
erate and kill the host through several mechanisms. Nematodes within infected
body will produce 1–3 generations, and then in the next generation, the contami-
nated juveniles will exit to infect new hosts (Kaya and Gaugler 1993). Infectious
nematodes can be identified with different symptoms. In case of steinernematids,
the killed host larvae are brown, red, and black, while the larvae killed by hetero-
habditids are red, brick, purple, orange, yellow, and green with differences depend-
ing on the bacterial species (Shapiro-Ilan et al. 2017).
The range of insect hosts for the entomopathogenic nematodes is extensive in
laboratory, but it is much more limited in natural environments, depending on host
and environmental conditions. Nevertheless their impact on non-target organisms is
negligible (Lacey et al. 2015). One of the factors limiting their host range is the type
of behavior required to get into the host body. Some species have a sit-and-wait
strategy (ambush) close to the soil surface, where they infect hosts with a special
nictation and jumping behavior. Other species are explorers, active in a wide range
compatible with relatively low-mobility hosts. Some species may also choose
between these two strategies (Shapiro-Ilan et al. 2017).
13.6 I nteractions of Entomopathogenic Nematodes

The EPN that are used against agricultural pests will definitely be exposed to other
materials applied in agro-ecosystems affecting their survival, distribution and effi-
ciency (Shapiro-Ilan et al. 2017). Irrigation methods, type and amount of fertiliza-
tion, type of pesticides used and their formulation are among the factors that affect
interactions of insects and their pathogenic nematodes (Lacey et al. 2015).
Depending on time and type of chemical used, and physical and chemical character-
istics of soil, EPN can show antagonistic to synergistic interactions with insecti-
cides which influence effectiveness of the control method and its cost.
Nishimatsu and Jackson (1998) evaluated the combined effects of terbufos, fono-
fos and tefluthrin with the entomopathogenic nematode Steinernema carpocapsae
Weiser against the larvae of western corn rootworm, Diabrotica virgifera virgifera
LeConte (Coleoptera: Chrysomelidae). The combination of nematode and terbufos
or fonofos resulted in additive effects on mortality of D. virgifera while tefluthrin
and S. carpocapsae showed synergism by increasing 24% of total mortality. Similar
findings were reported in combination of tefluthrin and Heterorhabditis bacte-
riophora Poinar. The authors proposed that the paralytic and convulsive responses
of D. virgifera due to the insecticide treatment might enhance the insect susceptibil-
ity to nematode infection or establishment within the host. In fact, the increased
metabolic activity and reduced directional movement led to higher production of
CO2 which facilitates host finding by the nematode (Nishimatsu and Jackson 1998).
Koppenhöfer et al. (2000) found the synergistic interaction between imidacloprid
and the two entomopathogenic nematodes, Steinernema glaseri (Steiner) and H.
bacteriophora as well as additive interaction with S. kushidai Mamiya against the
larvae of white grubs.Imidacloprid negatively affected mortality, speed of kill and
establishment of S. kushidai while these parameters were positively affected in case
of S. glaseri and H. bacteriophora. The authors reported the general disruption of
normal nerve function leading to lower biological activity of larvae, including their
grooming and evasive behavior, as the main phenomenon responsible for the syner-
gistic interactions observed.
Radova (2010) determined the interaction of Steinernema feltiae Filipjev with
eight insecticides (kinoprene, lufenuron, methomyl, metoxyfenozide, oxamyl,
piperonyl-butoxide, pyriproxyfen, tebufenozide), seven acaricides (azocyclotin,
clofentezin, diafenthiuron, etoxazole, fenbutatinoxide, fenpyroximate, tebufen-
pyrad) and four fungicides (captan, fenhexamid, kresoxim-methyl, nuarimol) evalu-
ating survival and infectivity of the infective juveniles. Treated insecticides caused
a low mortality (2–18%) on infective juveniles of S. feltiae after 3 days of applica-
tion, in which tebufenozide and piperonylbutoxide showed the lowest and highest
mortalities, respectively. Similar findings were reported in case of acaricides except
356 A. Zibaee
for fenpyroximate that caused a >20% mortality after 3 days. Mortalities around
7–8% were observed in S. feltiae after treatment with fungicides, with a lower value
than control for nuarimol. No negative effects were observed in virulence of S.
feltiae after insecticidal treatments as the mortality of 80–100% were imposed on
red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) after
5 days, similar to fungicides, showing no significant effects on EPN virulence. In
contrast, the two acaricides, fenpyroximate and tebufenpyrad, significantly
decreased S. feltiae virulence to 95% and 85%, respectively (Radova 2010).
Negrisoli et al. (2010a) determined the compatibility of the three entomopatho-
genic species including Heterorhabditis indica Poinar, Steinernema carpocapsae
Nguyen & Smart and S. glaseri Nguyen & Smart with 18 conventional insecticides
used against fall armyworm, Spodoptera frugiperda (Smith) (Lepidoptera:
Noctuidae) [namely betacyfluthrin, cypermethrin, chlorpyrifos (two commercial
products), deltamethrin, deltamethrin + Triazofos, diflubenzuron, gamacyhalothrin,
lambdacyhalothrin, lufenuron, methoxyfenozide, methylparathrion, permethrin
(two commercial products), spinosad, triazofos, triflumuron and alphacyperme-
thrin]. Initially, the infective juveniles of each nematode species were exposed to
insecticides, then their viability and infectivity against S. frugiperda were evaluated
in laboratory conditions. One of the commercial formulations of chlorpyrifos
(LorsbanTM) and lufenuron showed the lowest mortality on the infective juveniles of
all EPN. A <15% mortality was found after exposure of H. indica to chlorpyrifos
(LorsbanTM), deltamethrin, lufenuron, deltamethrin + triazofos, diflubenzuron and
gamacyhalothrin, demonstrating no significant differences with control, The two
insecticides, betacyfluthrin and lambdacyhalothrin, showed the highest mortality
against the nematode species (57.8% and 52.6%). Spinosad, lufenuron, chlorpyrifos
(LorsbanTM), chlorpyrifos (VexterTM), gamacyhalothrin and deltamethrin + triazofos
showed the lowest mortality against S. carpocapsae while betacyfluthrin showed
the highest level. The infective juveniles of S. glaseri showed the lowest mortality
after exposure to spinosad, lufenuron, chlorpyrifos (LorsbanTM), chlorpyrifos
(VexterTM), gamacyhalothrin, deltamethrin + triazofos, cypermrthrin, triflumuron,
triazofos, methoxyfenozide and permethrin (Negrisoli et al. 2010a). In this study,
the authors defined infectivity as the EPN ability to kill larvae of G. mellonella.
They reported the infectivity >88% after exposure to chlorpyrifos (LorsbanTM) and
lufenuron, similar to control, while the lowest one was scored for H. indica after
exposure to methylparathion. The infectivity of S. carpocapsae was high after expo-
sure to gamacyhalothrin, chlorpyrifos (LorsbanTM) and lufenuron while it was sig-
nificantly lower than control in response to triazofos. Finally, the insecticides
cypermethrin, lambdacyhalothrin, triflumuron, methoxyfenozide, triazofos and
alphacypermethrin caused the highest S. glaseri infectivity (Negrisoli et al. 2010a).
These authors attributed the insensitivity of nematodes to some insecticides to the
presence of butyrylcholinesterase in their synapses, that protects acetylcholinester-
ase toward inhibitors such as organophosphorus insecticides. Also, they found that
nematodes had no chitin in their cuticle so they are insensitive to diflubenzuron.
These findings and conclusions indicated compatability of H. indica, S. carpocap-
sae and S. glaseri with some registered chemicals to be used against S. frugiperda
at least in laboratory conditions (Negrisoli et al. 2010a).
In a subsequent study, Negrisoli et al. (2010b) tested the efficacy of the above-
mentioned insecticides and the EPN to control S. frugiperda in laboratory and field
conditions. All used insecticides showed equal mortalities against S. frugiperda lar-
vae with values significantly higher than control. After 2 days insecticides including
cypermethrin, spinosad, methoxyfenozide and deltamathrin + triazofos led to addi-
tive interaction with H. indica while chlorpyrifos (LorsbanTM) showed a synergic
interaction. Similar results were observed in combination of S. carpocapsae with
lufenuron, chlorpyrifos (LorsbanTM), cypermrthrin, methoxyfenozide, lambdacyha-
lothrin and chlorpyrifos (VexterTM). S. glaseri demonstrated synergism with chlor-
pyrifos (VexterTM), chlorpyrifos (LorsbanTM) and methoxyfenozide while the
nematode had antagonism with deltamethrin + triazofos, diflubenzuron, matylpara-
thion and alphacypermethrin. Other insecticides caused additive interactions with S.
glaseri (Negrisoli et al. 2010b). After 4 days, chlorpyrifos (VexterTM) showed the
additive interaction with S. glaseri whereas the other insecticides showed nematode
antagonism. Chlorpyrifos (LorsbanTM) and gamacyhalothrin showed synergism
with H. indica while the nematode had antagonism with deltamethrin and methyl-
parathion. A synergism was found in the interaction of S. carpocapsae with alphacy-
permethrin and gamacyhalothrin. The authors suggested that time of exposure
would definitely affect the EPN interaction type with insecticides.
Such synergistic or additive effects have been already reported in combination of
nematodes with malathion (Baweja and Sehgal 1997), organophosphorus, carba-
mates, pyrethroids, cartap and imidacloprid (Zhang et al. 1994; Koppenhöfer and
Kaya 1998; Koppenhöfer et al. 2002). In field experiments, the combined applica-
tion of the insecticides and the EPN caused higher mortalities against S. frugiperda
compared to the nematodes alone, with additive interactions. It is essential to indi-
cate that chlorpyrifos (LorsbanTM) and methoxyfenozide showed no statistical dif-
ference from EPN alone (Negrisoli et al. 2010b).
Bortoluzzi et al. (2013) evaluated the efficiency of sixteen steinernematids and
heterohabditids EPN against banana weevil borer, Cosmopolites sordidus Germar
(Coleoptera: Curculionidae) and their interactions with a conventional insecticide,
carbofuran. In combination experiments, 400 ml/L of the insecticide were mixed
with a solution containing 2000 infective juvenile of each species to check for
potential interactions. All nematode species caused significant mortality on C. sor-
didus. No negative effects of carbofuran was observed on viability of infective juve-
niles but a significant decrease by 72% was recorded in the nematodes exposed to
the insecticide. The authors concluded that carbuforan as a carbamate insecticide
inhibited acetylcholinesterase and disrupting the foraging behavior and entering
capability of the treated nematodes toward host body.
In a regional study, the interactions among three conventional insecticides,
dinotefuran, indoxacarb and imidacloprid, were investigated to the two Arizona-
native entmopathogenic nematodes, Heterorhabditis sonorensis Stock et al. and
Steinernema riobrave Cabanilas et al., by measuring survival, virulence and repro-
duction rates (Navarro et al. 2014). The authors set up three bioassay including
358 A. Zibaee
initial EPN application prior to insecticidal exposure after 24 h, initial exposure to

insecticides prior to nematode application after 24 h and simultaneous application
of all agents. The survival of both nematodes was not affected by the insecticides.
Even one of them, indoxacarb, showed a synergistic effect on the S. riobrave
virulence, although the reproduction of both nematodes decreased by two-fold,
compared to control. Antagonistic interactions on virulence were observed in com-
binations of imidacloprid with S. riobrave compared to an additive effect of the
insecticide on H. sonorensis virulence. Finally, dinotefuran promoted additive
effects on virulence of both nematodes against corn earworm, Helicoverpa zea
Boddie (Lepidoptera: Noctuidae). The authors concluded that synergism between
indoxacarb and the EPN might be due to non-systemic effects of the insecticide on
the insects nervous system, by blocking Na+ channels, leading to larval paralysis
that makes them more susceptible to nematodes. Additionally, antagonism between
imidacloprid and one of the used nematodes was due to its unsuitability against lepi-
dopteran insects (Navarro et al. 2014).
Laznik and Trdan (2014) determined the possible compatibility of eight insecti-
cides (abamectin, lufenuron, toxin of Bacillus thuringiensis var. kurstaki, pymetro-
zine, azadirachtin, imidacloprid, λ-cyhalotrin and pirimicarb) with commercial
products of S. feltiae, S. carpocapsae and S. kraussei Steiner. The two species of S.
carpocapsae and S. kraussei appeared incompatible with all assayed insecticides
while only abamectin and lufenuron negatively affected viability of H. bacte-
riophora. In contrast, S. feltiae was compatible with azadirachtin, toxin of Bacillus
thuringiensis var. kurstaki and imidacloprid. The authors believed that compatibility
might be even strain-specific rather than species-specific. The combination of S.
feltiae and H. bacteriophora were compatible with azadirachtin and pirimicarb
leading to a cost-effective alternative to control vegetable pests.
Sabino et al. (2014) studied the interaction of S. carpocapsae and H. amazonen-
sis with insecticides (abamectin, triflumuron, deltamethrin, dimethylamino-propyl,
chlorpyriphos, lambda-cyhalotrin +chlorantranilprole, chlorantranilprole and thia-
methoxan + λ-cyhalothrin) in field condition. No significant effect was found on
EPN viability except for dimethylamino-propyl which caused a mortality higher
than control (24.6%). Abamectin and chlorpyrifos caused 26–30% and 20–50%
mortalities against the larvae of G. mellonella in combination with H. amazonensis
and S. carpocapsae, respectively. No significant differences for EPN viability and
infectivity were observed after combination with thiamethoxan + λ-cyhalothrin and
anthranilamide, while all pyrethroids imposed significant effects compared to con-
trol. This was likely due to the presence of surfactants in the formulations of these
insecticides. Similarly, Guo et al. (2017) designed the laboratory and field experi-
ments to find interactions of chlorantraniliprole, diflubenzuron and imidacloprid
with S. longicaudum Shen & Wang and H. bacteriophora, against the white grub,
Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae). Different insecticide
concentrations showed no effect on survival or virulence of S. longicaudum and H.
bacteriophora. In laboratory experiment evaluating the effect of nematode–insecti-
cide combinations against larvae of H. oblita, mortality rates of the larvae in all
nematodes treatments were significantly higher than that found in the insecticide
treatments, at 4, 7, and 14 days. Combinations of nematodes and insecticides

showed higher mortalities of target insect compared to the nematodes alone, show-
ing synergism after 4 days. The combinations of S. longicaudum–imidacloprid, H.
bacteriophora – imidacloprid, or H. bacteriophora – chlorantraniliprole
demonstrated synergistic effects in peanut fields, leading to reductions of grub lar-
vae and percent of injured legumes. Finally, cost–benefit analysis revealed that a
combination of H. bacteriophora – imidacloperid might be practical to field man-
agement of H. oblita in the peanut production.
13.7 Conclusion
IPM nowadays is one of the most important systems to control pest damages and to
produce high quality and healthy crops. Different methods are used to control
insects, in which biological and chemical control are most prominent techniques.
Although the sole use of these methods has given satisfactory results, IPM should
include all reasonable control methods to achieve best results at a minimum cost.
Since the use of biological control methods, with a focus on entomopathogens and
chemical insecticides, has its own disadvantages and benefits, it is advisable to cre-
ate a framework for their combined use. Such a framework requires laboratory and
field studies to examine positive or negative interactions when these two methods
are applied together. As shown in this chapter, some chemical compounds have
synergistic or additive effects on entomopathogens, while others cause some antag-
onistic responses. Therefore, before combining these two methods, screening
between conventional chemical insecticides to control a specific pest and EPN
should be done in both laboratory and field. Such an experiment involves the selec-
tion of insecticides from different classes, taking into account their diverse formula-
tions, spraying time and spray location. If these experiments are carried out carefully,
it seems that the proper combination of a low-risk insecticide with an entomopatho-
gen can successfully control the target pests, with lowest costs and optimal
outcomes.
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Chapter 14
Current State of Fungal Antagonists
with Special Emphasis on Indian Scenario
Purnima Das, Lakshmi Kanta Hazarika, Surajit Kalita, and Somnath Roy
Abstract The major objective of shifting from conventional to eco-friendly and

sustainable agriculture is to minimize the load of chemical inputs. The replacement
of chemical pesticides by biologicals is a prime step for achieving this objective.
Entomopathogenic fungi (EPF) can act as effective management tools for insect and
mite pest regulations without harming the biodiversity and environment. About 49
EPF species, predominantly from Deuteromycotina and Zygomycotina, have so far
been commercially exploited world-wide as biopesticides, among which the most
widely used species belong to genus Beauveria and Metarhizium. In India, a coun-
try of rich biodiversity, research on EPF has given rise to many products, out of
which about 638 EPF formulations have so far been registered. Despite of huge
resources and many advantages over conventional synthetic pesticides, EPF remain
relatively underutilized in Indian agriculture. Biopesticides represent only around
2–3% of the overall pesticide market in India, a share that is expected to increase
manifold in coming years. In this review, we provide an overview on promising EPF
as bio-control agents of the key agricultural pests and also the scope for future stud-
ies for their better utilization.
Keywords Entomopathogenic fungi · Pest control · Biodiversity · Fungal

antagonists
P. Das · S. Kalita
Department of Entomology, Assam Agricultural University, Jorhat, Assam, India
L. K. Hazarika
Assam Women’s University, Jorhat, Assam, India
S. Roy (*)
Division of Entomology, Tea Research Association, Jorhat, Assam, India

https://doi.org/10.1007/978-3-030-23045-6_14
366 P. Das et al.
14.1 Introduction
In nature, a pool of microorganisms including 1000 species of entomopathogenic

fungi (EPF) under phyla Entomopthoromycota, Blastocladiomycota, Mycosporidia,
Basidiomycota and Ascomycota (Vega et al. 2012; Wang and Wang 2017), 90 spe-
cies of bacteria, 1600 viruses (Moazami 2009) and 111 species of nematode (Hunt
and Nguyen 2016) regulate insect populations. However, about 49 EPF species pre-
dominantly from Deuteromycotina and Zygomycotina have so far been commer-
cially exploited as biopesticides representing numerically 44% of the antagonistic
microorganisms pool in number. The most widely used species is Bacillus thuringi-
ensis Berliner. Microorganisms exploited for biological control of insect pests
through introduction and augmentation contribute 1.4–2.5% of the total $28 billion
global pesticide market, amounting for $392–700 millions. Bacteria alone occupy
74% (Thakore 2006). Figure 14.1 shows the status of the remaining groups.
Moreover, EPF as alternatives to insecticides, or their combined applications
with insecticides could be very useful for managing resistant pests (Hoy 1999) as
well as for formulating sustainable integrated pest management strategies of various
crops and public health pests. Normally biopesticides are eco-safe alternatives and
inherently less harmful in compared to chemical pesticides. They are target specific,
effective in small quantities, easily degradable, do not leave problematic residues,
and their production cost is lower compared to synthetic pesticides. It is less likely
that target pests develop resistance. Another advantage is that unlike bacteria and
viruses, EPFs behave like contact insecticides.
However, main disadvantages are unavailability of quality products in sufficient
quantity in the local market, poor shelf life of majority of formulations, slow in
action, inconsistent or variable efficacy under field conditions due to the influences
of biotic and abiotic factors. In recent times, importance has been given to some
technical aspects of biopesticide development, such as mass production, formula-
tion, and selection of more virulent strains with consistent efficacy under field
80 74%
70
60
50
Share (%)
40
30
20
10% 8%
10 5% 3%
0
Bacteria Fungus Virus Predators Others
Biocontrol agents
Fig. 14.1 World’s biopesticide market as shared in percentage by different groups

14 Current State of Fungal Antagonists with Special Emphasis on Indian Scenario 367
c onditions. These are of course essential, but identification and modification of fun-
gal virulence determinants or genes must be undertaken to produce efficient
mycoinsecticides.
14.2 Historical Background
Earliest studies of EPF dated back to eighteenth century. While developing ways to
control diseases that devastated the silkworm industry in France, Agostino Bassi
(1773–1856) demonstrated that Beauveria bassiana (Bals.-Criv.) Vuill. was the
infectious agent causing the muscardine disease of silkworms. But the use of B.
bassiana for insect pest control was first studied by Audoin (1837). Subsequently,
many discoveries were made to isolate several EPFs, out of which the most impor-
tant fungi are Metarhizium anisopliae Sorokin, Nomuraea rileyi (Farl.) Samson,
Lecanicillium lecanii R. Zare & W. Gams, Hirsutella thompsonii Fisher and Isaria
fumosorosea (Wize) A.H.S. Br. & G. Sm. (Table 14.1).
B. bassiana isolated from a variety of insects worldwide is a filamentous imper-
fect fungus with high host specificity. Host range includes Leptinotarsa decemlin-
eata Say, Cydia pomonella Linnaeus, several genera of termites, and Helicoverpa
armigera Hubner (Thakur and Sandhu 2010), Dicladispa armigera Olivier
(Hazarika and Puzari 1997; Hazarika et al. 2005), Corcyra cephalonica Stainton
(Das 2015), Periplaneta americana L. (Mudoi et al. 2017) and several other vectors
of tropical infectious diseases such as Phlebotomus, Glossina morsitans and bugs of
genera Triatoma and Rhodnius.
Likewise, M. anisopliae is one of the commercially exploited EPFs against
(Sandhu et al. 2012) locusts, grasshoppers, cockroaches in both developed and
developing countries of Africa, America and Australia. It is formulated in oils, under
high humid conditions and applied, which resulted 90% kill of locusts within
7–21 days. It can be mass produced on rice grains. L. lecanii is another widely dis-
tributed EPF, which causes widespread epizootics in tropical and subtropical regions
(Nunez et al. 2008). Being a dimorphic hypomycete, N. rileyi can cause epizootics
in lepidopterans (noctuids) and coleopterans (Ignoffo 1981; Ignoffo et al. 1989;
Vargas et al. 2003). Its host specificity and eco-friendly nature encourage its use in
insect pest management. Limitations of EPFs are that they are unpredictable and
geographical locations and hosts play important roles in determining specificity and
virulence (Boucias et al. 1982; Vimaladevi et al. 2003).
In a review, Feng et al. (1994) summarized the progress and achievements made
in mass production of B. bassiana using different media and techniques by high-
lighting advantages and disadvantages of solid and liquid medium or submerged
culture. Methods were indicated to overcome problems associated with mechanical
production system. Solid- and liquid-state fermentation have been developed for
mass production of B. bassiana, based on diphasic and submerged techniques
(Rombach et al. 1988; Feng et al. 1994). Whatever the technique, it must result in
high productivity consuming less energy and produce low wastewater (Lonsane
368 P. Das et al.
Table 14.1 Entomopathogenic fungi isolated from insect pests

Stage of
Fungal spp. Insect spp. Order infection
Metarhizium Cabbage butterfly (Pieris brasicae) Lepidoptera Larvae
anisopliae White grub (Anomela spp, Apogonia Coleoptera Grub
spp)
Cutworm (Agrotis ipsilon) Lepidoptera Grub
Brinjal aphid (Myzus persicae) Hemiptera Nymph and
adult
Cowpea aphid (Aphis craccivora) Hemiptera Nymph and
adult
Termite (Odontotermes obesus) Isoptera All stages
Rhinoceros beetle (Oryctes Coleoptera Grub
rhinoceros)
Beauveria bassiana Sugarcane shot borer (Chilo Lepidoptera Larvae
infuscatellus)
Rice Hispa (Dicladispa armigera) Coleoptera All stages
White fly (Bemisia tabaci) Hemiptera Adult
Tea mosquito bug (Helopeltis Hemiptera Adult
theivora)
Dung beetle (Catharsius molossum) Coleoptera Adult
Black ant (Diacamma rugosum) Hymenoptera Adult
Rice case worm (Nymphula Lepidoptera Larvae
depunctalis)
Rice weevil (Sitophilus oryzae) Coleoptera Adult
Tiger moth (Creatonotos gamgis) Lepidoptera Larvae
Yellow tussock moth (Somenia Lepidoptera Larvae
scintillans)
Black fly (Aleurocanthus wouglumi) Hemiptera Nymph
Nomurea rileyii Cabbage looper (Spodoptera litura) Lepidoptera Larvae
Velvetbean moth (Anticarsia Lepidoptera Larvae
gemmatalis)
Cotton bollworm (Helicoverpa Lepidoptera Larvae
armigera)
Cabbage Looper (Trichoplusia ni) Lepidoptera Larvae
Clover leaf weevil (Hypera punctata) Coleoptera Adult
Black cutworm (Agrotis ipsilon) Lepidoptera Larvae
Armyworm moth (Mythimna Lepidoptera Larvae
unipuncta)
Verticillium lecanii Red spider mite (Oligonychus coffeae) Acarina Adult
Source: Hazarika et al. (2005), Roy and Muraleedharan (2014), Dutta et al. (2012), and Das (2015)
et al. 1992; Pandey et al. 2001). Many agricultural wastes were utilized for produc-
tion of EPF which include sugarcane bagasse, rice bran and rice husk (Mazumder
et al. 1995; Puzari et al. 1997). Besides wheat bran, soybean chunks, rice, maize and
oats were also utilized by many workers to mass produce B. bassiana and
M. anisopliae. Amongst the liquid medium, rice grewel, coconut water and potato
broths were the most common. Das et al. (2017) improved some of the media by
adding different carbon and nitrogen sources. It was also observed that addition of
crustacean chitin further improves the productivity of the liquid medium.
14.3 Mode of Action of Fungal Antagonists
The insect cuticle is the foremost barrier for any biological insecticides. It is mainly
composed of epicuticle, procuticle and epidermis. The outermost surface layer of
the epicuticle is the cement and lipid layer which are mostly resistant to enzymatic
degradation, and act as barrier to biotic and abiotic agents (Hadley 1981). Unless
physically disrupted, it can prevent entry of EPF. Recently, Wang and Wang (2017)
reviewed comprehensively the molecular mechanism of entomopathogenesis based
on researches conducted on species of Beauveria and Metarhizium. Different genes
mediating infection by an EPF have been functionally characterized and grouped
under adhesion, cuticle degradation, nutrient assimilation and stress management
(Butt et al. 2016). In general, spore adhesion, differentiation of infection structures,
detoxification, hemocoel adaptation, starvation and evasion of innate immunity are
different phases of an infection of a susceptible host by an EPF (Figs. 14.2 and
14.3a-d). Hydrophbin genes, hyd1 and hyd2, mediate adhesion of a B. bassiana
spore to lipid layer of the insect epicuticle (Boucias et al. 1988; Lord 2001; Goettel
et al. 2005; Zhang et al. 2011). Following this stage, the spore germinates in a high
humid and moderately hot environment as well as in presence of specific
Fig. 14.2 Schematic diagram of mode of action of entomopathogenic fungi

370 P. Das et al.
Fig. 14.3 Mode of action of B. bassiana on D. armigera. Conidia in contact with integument (a).
Germination of conidia (b). Appresoria formation and penetration of germ tube through the cuticu-
lar depression on prothorax (c). Penetration of germ tube through membranous cuticular depres-
sion. Scale bars: 10 μm (a, c, d); 1 μm (d). (d)
epicuticular hydrocarbons (Feng et al. 1994; Napolitano and Juarez 1997) to form a
germ tube (Wang and Wang 2017). Subsequently, an infection structure, the appres-
sorium, is formed (Hajek and St. Leger 1994; Wang and Wang 2017), which coin-
cides with secretion of aminopeptidases. Likewise, MAD 1 acts as hydrophobins of
Metarhizium. Long chain C18 cuticular fatty acids enhance the process (Bidochka
and Khachatourians 1991), while short chain fatty acids and aldehydes inhibit
growth of EPF (Smith and Grula 1982; Sosa-Gomez et al. 1997). However, it is
hypothesized that besides adhesion, these genes perform other functions, such as
cell surface hydrophobicity, virulence, and formation of rodlet layer (spore coat)
(Zhang et al. 2011). In addition to release of proteases and chitinases, the mother
spore translocates lipid droplets (LDs) to the appressorium. As a result of hydrolysis
of LDs, accumulation of high concentration of glycerol takes place, which gener-
ates a high turgor pressure to breach the epicuticle (Wang and Wang 2017) in pres-
ence of cyclic anti microbial peptides (cAMPs) (St. Leger et al. 1991).
Among the proteases found in EPF, the spore bound PR1 and PR2 endoproteases
have been well characterized from M. anisopliae infecting Calliphoran vomitoria
L. and Manduca sexta L. The secretion of PR1 and PR2 and their role in cuticle
invasion has also been established (St. Leger et al. 1994; Hussain et al. 2010).
Subsequently, an appressorium undergoes a series of morphological changes at

different layers such as infection pegs (epicuticle), penetrant hyphae and penetrant
plates (procuticle), and hyphal bodies or blastospores (hemocoel), in order to dis-
perse through the hemocoel (Hajek and St. Leger 1994; Bhattacharyya et al. 2004).
Though molecular mechanisms of spore adhesion and host recognition have been
understood to a great extent, host signals/ligands and transcriptional factors as acti-
vated by protein kinase signal pathways are yet to be researched.
Different fungal species obtain energy from their insect hosts through biotrophy
(nutrition derived only from living cells, which ceases once the cell dies), necrotro-
phy (killing and utilization of dead tissues), and hemibiotrophy (initially biotrophic
and then becoming necrotrophic). Generally deuteromycetes kill cells through the
release of toxins. Beauvericin, bassianolide, isarolides, and beauverolides are some
of the toxins isolated from B. bassiana (Hamill et al. 1969; Elsworth and Grove
1977), while destruxins (DTXs) A, B, C, D, E, F and cytochalasins are isolated from
M. anisopliae-infected hosts. DTXs depolarize the muscle membrane by activating
Ca+ channels and inhibit hemocytes (Bradfisch and Harmer 1990). Neuroactive
mycotoxins such as penitrem A, verruculogen and alfatrem appear to interact with
glycine- and GABA-receptors, thus reducing glycine production resulting in trem-
orgenic effects.
Evasion of host immunity in insects is an exciting field, less researched in India.
B. bassiana infected and killed adults, larvae and eggs of Dicladispa armigera
Olivier (Coleoptera: Chrysomelidae) as the process of pathogenesis have been stud-
ied and described (Figs. 14.2 and 14.3a–d) (Hazarika and Puzari 1990, 1995; Puzari
et al. 1994). In an interaction study between pathogen and hemocytes, it was
observed that B. bassiana colonized D. armigera hemocoel by evading host acute
immune system including hemocyte recognition, encapsulation, melanization and
phagocytosis as well as AMP production (Phukan et al. 2008). In this insect it was
revealed that granulocytes performed phagocytosis, encapsulation and nodulation
of blastospores (Phukan et al. 2008). However, cells react after a specific period of
infection time. Das (2015) reported that B. bassiana (Strain KR855715) at 1 × 109
conidia/ml caused 60% mortality of Corcyra cephalonica Stainton, whereas, the
same strain at 1 × 107 conidia/ml caused 64–72%, 52–68% and 16–36% mortality
in 3rd, 4th and 5th instars of Periplanta americana, respectively, within 10 days of
treatment (Mudoi et al. 2017).
Several studies showed that high hemocyte loads led to successful encapsulation
not only in Drosophilla melanogaster (Basset et al. 2000), but also in Helopeltis
theivora (Baruah and Hazarika 2006). When H. theivora hemocytes are challenged
by B. bassiana, cell disintegration followed by clumping, capsule and nodule for-
mation around B. bassiana took place (Baruah and Hazarika 2006). Hyphae of
Aspergilus flavus link are engulfed by immune system cells of Blatella (Kulshrestha
and Pathak 1997). However, cells react after a specific period of infection time
(Gray and Anderson 1983; Baruah and Hazarika 2006). When the object is big
enough to phagocytose, encapsulation occurs by forming multi-layered aggregates
(Lackie 1988). Similar to our observation, in response to B. bassiana inoculation,
hemocytes of Leptinotarsa decemlineata (Say) also disintegrate (Sirotina 1961).
372 P. Das et al.
Sewify and Hashem (2001) reported an increase in the hemocyte population of G.

mellonella following infection by Metarhizium up to 96 h.
Drosophila that is naturally infected by EPF exhibits an adapted response by
producing antifungal peptides like dorsomycin. Based on these findings and others
mainly by Schmid-Hempel (2005), a diagram is constructed to show how the fungus
and insect interacts (Fig. 14.2). Bangham et al. (2006) also developed a similar
scheme describing four immune responses of D. melanogaster against major chal-
lenges faced by insects, in which (1) differentiation of LA forms a melanized cap-
sule around the egg of a parasitoid, (2) triggering of Toll pathway occurs in response
to Gram+ bacteria and fungi, (3) Gram-bacteria trigger Imd pathway, and (4) viruses
trigger Jak-STAT pathway, resulting in transcription of antiviral genes.
14.4 Molecular Techniques for Species Characterization
Being ubiquitous, EPF may have a massive number of strains. In general, species
are separated based on morphological characteristics of spores, colonies growth or
nutrient requirements from earlier days, however, molecular identification tech-
niques are actually more appropriate. PCR-based tools have made it possible to
understand the phylogenetic characterization. A number of unspecific DNA based
methods have been used specially in Beauveria (Glare et al. 2008). DNA polymor-
phism using RAPD-PCR (Random Amplified Polymorphic DNA – Polymerase
Chain Reaction) is a widely used molecular technique in identification of this group
(Joshi and St Leger 1999). It was introduced in 1990 (Samsinakova et al. 1983) to
reveal polymorphism within completely unknown samples without probe hybrid-
ization or DNA sequencing, using short oligonucleotide primer (6–12 bases).
RAPD has already been used to estimate the diversity of a population, genotype
characterization or constructing the molecular phylogeny of closely related taxa
(Tigano-Milani et al. 1995). This is based on a product which may be a spectrum of
DNA fragments differing from each other in terms of length and nucleotide
sequence.
The application of RAPD markers is similar to those of other DNA polymor-
phism detection methods and can be used for characterization of a fungal isolate by
constructing a specific fingerprint, or for genetic stability testing of an individual
isolate. In India, using a 28S rDNA technique Uday et al. (2017) identified a potent
xylanase producing strain of A. niger Tiegh. (KP874102.1) growing in various sub-
strates such as beech wood xylan, oat spelt xylan and CM cellulose, which is a
potential candidate for enzymatic hydrolysis of orange peel.
Till now, 26 species of insect pathogens including 23 species of Ascomycete and
3 species of Entomopothoromycota have been sequenced (Wang and Wang 2017),
of which Metarhizium spp. predominate. This kind of molecular studies will help in
studying fungus-environment interaction that benefit agriculture, environment and
human health.
Universally primed (UP)-PCR has been used to separate genetically sympatric

isolates of Beauveria (Meyling and Eilenberg 2006). Thakur et al. (2005) studied 48
isolates of indigenous strains of B. bassiana employing protease zymography and
RAPD analysis, revealing high genetic and biochemical diversities amongst the
strains collected from lepidopterans and coleopterans. RFLPs (Restriction Fragment
Length Polymorphism), AFLPs (Amplified Fragment Length Polymorphism), ISSR
(Inter-Simple-Sequence Repeats), SSRs (Simple Sequence Repeats), or microsatel-
lites are other molecular techniques utilized for the characterization of both
Beauveria and Metarhizium (Sandhu et al. 2012). Recent development of microsat-
ellite markers (Rehner and Buckley 2003; Enkerli et al. 2005) linked B. bassiana to
plants as an endophyte (Arnold and Lewis 2005), as M. anisopliae, that was associ-
ated with the rhizosphere of plants (Hu and Leger 2002).
14.5 Commercialization of Entomopathogenic Fungi
Due to their variable performances under different environmental conditions, com-

mercialization and large scale application of biopesticides have been slowed down
during the last decade (Wang et al. 2003; Fravel 2005). The global biopesticide
market is continuously progressing, and it is estimated to be valued at $1.16 billion
in 2015. It is expected to reach $3.18 billion by 2021 at a Compound Annual Growth
Rate (CAGR) of 18.3% from 2016 to 2021. North America shares 44% of biopesti-
cides market, followed by the European Union (20%) and Oceania (20%), South
and Latin American countries (10%), India and other Asian countries (6%)
(Khachatourians 2009; Bailey et al. 2010). Out of about 1400 biopesticides avail-
able worldwide, 202 products including 102 microorganisms were registered in
USA (Chandler et al. 2011). H. thompsonii was the first mycoinsecticide registered
in the U.S. under the trade name of Mycar, used against spider mites (Kenneth et al.
1979). Amongst all the 171 products of EPF so far developed, B. bassiana-based
products dominate with 58 formulations representing 33.9% of the total, followed
by 49 products of L. lecanii and 11 products of M. anisopliae representing 33.9%,
with I. fumosorosea and B. brongniartii representing 5.8 and 4.1 per cent, respec-
tively (de Faria and Wraight 2007): They are used against insect pests of coffee,
bean, cabbage, corn, potato, and tomato and mosquitoes and flies (Florez 2002).
Some of the commercial products registered across the globe are listed in Table 14.2.
Compared to bacteria (285 species and strains), EPF registered in India are more
(638 species and strains) along with nuclear polyhedrosis viral (37), pheromone (2),
botanical products (1) and 7 others (Central Insecticide Board and Registration
committee, New Delhi, http://cibrc.nic.in/) (Fig. 14.4). Amongst the EPF antago-
nists registered for agricultural pest management, 108 products are based on B.
bassiana, followed by 95 with L. lecanii, 38 with M. anisopliae, 31 with P. lilaci-
nus, 5 with P. chlamydosporia and 1 with H. thompsonii (Table 14.3).
374 P. Das et al.
Table 14.2 Commercial microbials from various countries

Country Trade name Manufacturer
Beauveria bassiana France Ostrinil Natural Plant Protection (NPP)
India BioGuard Rich Plantrich Chemicals & Biofertilizers
Ltd.
India Bio-Power T. Stanes & Company Limited
India Racer Agri Life
India Daman International panacea Ltd.
India Beavera Jai Biotech Industries
India Brigade Kan Biosys Pvt Ltd.
India Bio-Be-Ba Microplex, Nagarjuna Agro
Chemicals
India Baba Multiplex Bio Tech Pvt. Ltd.
India Mycojaal Pest Control (India) Pvt. Ltd.
India Metabeave R. B. Herbal Agro
India Jas Beesi Shri Ram Solvent Extractions
India BBC Sri Biotech Laboratories India Ltd.
India Toxin Varsha Bio Science & Technology
Spain Trichobass-L & Trichodex S. A.
Trichobass-P
Africa Bb Plus & Bb Weevil Biological Control Products SA Ltd.
USA Balance Jabb of the Carolinesn Inc.
USA BotaniGuard & Laverlam International Corporation,
Mycotrol USA
USA CornGard Mycotech Corp., USA
USA Naturalis L Troy BiosciencesInc., USA
USA Naturalis H & G Troy BiosciencesInc., USA
USA Naturalis T & O Troy BiosciencesInc., USA
USA Organigard Emarald BioAgriculture Corp.,
Colombia Agronova Live Systems Technology S. A.,
Colombia
Lecanicillium UK Mycotal Koppert Biological Systems,
lecanii Netherlands
Russia Verticillin Biodron, Russia
Spain Trichovert Trichodex S. A., Spain
India Bio-Catch T. Stanes & Company Limited, India
India Biovert Rich Plantrich Chemicals & Biofertilizers
Ltd., India
India Mealikil Agri Life, India
India Vertimust Jai Biotech Industries
India Biogade-V Kan Biosys Pvt. Ltd.
India Vertifire-L International panacea Ltd.
India Cropfit Microplex
India Varsha Multiplex Bio Tech Pvt. Ltd.
India Biosar R. B. Herbal Agro
(continued)

Country Trade name Manufacturer
India Jasverti Shri Ram Solvent Extraction Pvt.
Ltd.
India Spider Sri Biotech Laboratories India Ltd.
India Shock Varsha Bioscience & Technology
Brazil Vertinat Natural Rural, Brazil
Colombia Ago Biocontrol Ago Biocontrol, Colombia
Holland Vertalec Koppert, Holland
Metarhizium Spain Trichomet Trichodex S. A., Spain
anisopliae
India Bio-Magic T. Stanes & Company Ltd., India
India Biomet Rich Plantrichs Chemicals &
Biofertilizers Ltd.
India Pacer Agri Life, India
India Kalichakra International panacea Ltd.
India Cropmet Microplex
India Metrocid Sri Biotech Laboratories India Ltd.
India Metaz Jai Biotech Industries
India Metarhizium Multiplex Bio Tech Pvt. Ltd.
India Jasmeta Shri Ram Solvent Extraction Pvt.
Ltd.
India Biostorm Varsha Bioscience & Technology
India Multiplex Multiplex Bio Tech Pvt. Ltd
Australia BioCane & Chafer Becker
Guard Underwood Inc., USA- Australia
USA Tacnure Novozymes Biologicals Inc., USA
USA Bio-BlastTM EcoScience, USA
Germany BIO 1020 Bayer, Germany
Source: Modified after Reddy et al. (2013)
Fig. 14.4 Microbial

product registered for use NPV
in India Bacterial 4%
30%
Fungus
66%
376 P. Das et al.
Table 14.3 Entomopathogenic fungal antagonists registered in India
Sl. No. Name of the entomopathogen Numbers of registered products

1 Beauveria bassiana 108
2 Lecanicillium (Verticillium) lecanii 95
3 Metarhizium anisopliae 38
4 Paecilomyces lilacinus 31
5 Pochonia chlamydosporia 5
6 Hirsutella thompsonii 1
14.6 Use of Fungal Antagonists in Pest Management
Ecology based pest-management strategies, biological control agents and

environment-friendly natural pesticides, of either indigenous origin or introduced,
may create a sustainable crop production system in which pest species densities are
maintained below the economic injury level.
Utilization of microorganisms or their by-products for the control of insect pest
species are of prime importance in this context. Beauveria and Metarhizium are the
two important genera that are commercially exploited targeting 700 species under
Lepidoptera, Hemiptera, Homoptera, Orthoptera, Diptera and Coleoptera (Moore
and Prior 1996; Moore et al. 1996; Hazarika and Puzari 1995, 1997; Hazarika et al.
2005; Khan and Ahmad 2015), of which, however, M. anisopliae is dominating (Li
et al. 2010; Dutta et al. 2012). They are found to be most effective against immature
stages of Hemiptera, Diptera, Coleoptera, Lepidoptera, Orthoptera and Hymenoptera
than vs the adult stages, while some others such as Aschersonia aleyrodis Webber
and M. rileyi Farlow have restricted host ranges of only whiteflies and lepidopteran
larvae respectively. Metarhizium (formerly Nomuraea) rileyi is mostly used against
Spodoptera litura F. (Ignoffo 1981), Trichoplusia ni Hubner, Heliothis zea Boddie,
Hypena scabra F. and Anticarsia gemmatalis Hubner (Mathew et al. 1998).
Lecanicillium. lecanii was found to be effective against whitefly and several aphids
species, including the green peach aphids (Myzus persicae Sulzer) in th greenhouse
chrysanthemums during the ‘70s (Hamlen 1979).
The application of B. bassiana to control the pine moth, Dendrolimus spp, in
China probably represents one of the largest uses of a biocontrol agent over 1 mil-
lion hectares of pine forest. Strain Bb-147 of B. bassiana is registered on maize for
controlling Ostrinia nubilalis Hübner and O. furnacalis Guenee. In addition to this,
the strain GHA is registered in US for controlling aphids, thrips, whitefly and
mealybugs. Strain ATCC 74040 is registered against many soft-bodied Homoptera,
Heteroptera and Coleoptera. Beauveria brongniartii Saccardo is registered on sug-
arcane and barley for controlling white grubs and cockchafers.
Various studies revealed that M. anisopliae (var. acridum) is effective against the
brown locust, Locustana pardalina Walker in Africa, Locusta migratoria L. in
Madagascar and the Australian plague locust, Chortoicetes terminifera Walker and
L. migratoria in Australia. M. flavoviride Gams and Roszypal has also been tested
against the tree locust, Anacridium melanorhodon Walker in Sudan, the rice
grasshopper, Hieroglyphus daganensis Krauss in Benin, Mali and Senegal and the
desert locust, Schistocerca gregaria Forsskal in Mauritania (Rosell et al. 2008).
The National Bureau of Agriculturally Important Insects (NBAII) in Bangalore
is maintaining 77 isolates of B. bassiana, 3 isolates of B. brongniartii, 39 of M.
anisopliae, 35 of Lecanicillium spp., 37 of N. rileyi, 3 of I. fumosorosea and 3 of I.
farinose, collected from various insect hosts from different geographical regions in
the country. Ramanujam et al. (2014) have identified several potential strains of B.
bassiana, M. anisopliae, Lecanicillium spp. and N. rileyi active against H. armig-
era, S. litura, Aphis craccivora Koch, Aphis gossypii Glover, Rhopalosiphum mai-
dis Fitch, Brevocoryne brassicae Linnaeus, Myzus persicae Sulzer and Bemisia
tabaci Gennadius across the country. Moreover, some promising strains of N. rileyi,
B. bassiana and M. anisopliae have been identified against pests of soybean,
groundnut and sugarcane (Table 14.4).
In order to have improved fungal antagonist formulations, technologies pertain-
ing to oil formulation of B. bassiana for management of pests of oilseed crops have
also been developed. Several technologies including B. bassiana based mycoinsec-
ticide for D. armigera management at Assam Agricultural University, Jorhat (Das
et al. 2017) and M. anisopliae-based formulation for Oryctes rhinoceros L. at
CPCRI, Kayangulam were developed (Ramanujam et al. 2003). B. bassiana (Strain
No. KR855715) at the rate of 1 × 109 conidia/ml showed effective results against
Table 14.4 Commercial products of entomopathogenic fungi and their target pests
Fungus Brand name Target pests Crop
B. bassiana Mycotrol Whiteflies/aphids/thrips Field crops
Naturalis Sucking insects Cotton, glasshouse crops
Conidia Coffee berry borer Coffee
Ostrinol Corn borer Maize
Myco-Jaal Diamond backback moth Cabbage
Biosoft Helocoverpa & sucking pests Several crops
Biowonder Rice pests Rice
B. brongniartii Betel Scarab beetle larvae Sugarcane
Engerlingspilz Scarab beetle larvae Pasture
Melocont Scarab beetle larvae Pasture
M. anisopliae BIO 1020 Black Vine weevil Glasshouse ornamental
Bio-BlastTM Termites House
Bio magic Brown plant hopper Rice
Multiplex Root grubs Several crops
M. flavoviride BioGreen Red-headed cockchafer Pasture/turf
V. lecanii Vertalec Aphids, whiteflies Glasshouse crops
Biocatch Whiteflies Cotton
Verticare Mealybugs & scales Citrus
P. fumosoroseus PFR-97TM Whiteflies/thrips Glasshouse crops
Prioroty Mites Wide range of crops
378 P. Das et al.
sixth instars of C. cephalonica, as reported by Das (2015). Wettable powder

formulation of B. bassiana at 10 g/kg of rice seeds showed the highest mortality of
C. cephalonica (75%) at 7 days of treatment. On the other hand, Mudoi (2016)
observed that the same B. bassiana (Strain No. KR855715) was also effective
against the 1st, 2nd and 3rd instars of P. americana, at a concentration of 1 ×
107 conidia/ml.
In the light of increasing awareness about adverse effects of pesticide residues in
food on human health, microbial product based pesticides play an important role in
India. Several works on using mycoinsecticides have been carried out in the country
for controlling different pests of rice, pulse, oilseeds, tea, sugarcane, coffee, coco-
nut, house hold pests and storage pests as well (Table 14.5).
Table 14.5 Biological Control of pests using entomopathogenic fungi in India

Fungus Crop Insect controlled Dose
B. bassiana Rice Rice Hisp, Dicladispa 1 × 107 spores/ml
armigera
Coffee Coffee berry borer, 1 × 107 spores/ml + 0.1% sunflower
Hypothenemus hampei oil + 0.1% wetting agent
Tea Tea looper, Buzura 2.5 g/l
suppressaria
Sunflower Head borer, Helicoverpa 200 mg/l
armigera
Green White grubs species 5 × 1013 conidia/ha
gram
Storage Rice moth, Corcyra 1 × 109 conidia/ml
cephalonica
Household Cockroach, Periplaneta 1 × 107 conidia/ml + Tween- 80
americana (0.023%)
B. Brongniarti Sugarcane White grub, Holotrichia 1 kg/acre
serrata
M. anisopliae Coconut Rhinoceros beetle, Oryctes 5 × 1011 spores/m3
rhinoceros
Sugarcane White grub species 1 × 1013 spores/ha
Pigeon pea Pod borer, H. armigera –
Potato White grub, Brahmina sp. 5 × 1013 conidia/ha
Soyabean White grub, Holotrichia 5 × 1013 conidia/ha
longipennis
L. Lecanii Coffee Green scale, Coccus viridis 16 × 106 spores/ml + Tween-80
Citrus Green scale, C. viridis 2 × 106 spores/ml + quinalphos
(0.005%) + Teepol (0.05%)
Mustard Mustard aphid, Lipaphis 1 × 106 spores/ml
erysimi
N. rileyi Castor Tobacco caterpillar, 10 × 1010 spores/ml
Spodoptera litura
Soybean S. litura, H. armigera, 2 × 108/ml
Thysonoplusia orichalcea
Source: Modified after Ramanujam et al. (2014), Das (2015), and Mudoi (2016)
14.7 Economics
For long-term positive effects of bio-pesticides use in sustainable pest management

program, there is a growing demand and awareness among farmers and policy mak-
ers. One of the strongest benefits of bio-pesticides is that they could be combined
with other pest management tactics. They are also relatively cheap to develop and
need to be re-developed less frequently, saving expenditures on research and devel-
opment. The efficacy of many of the bio-pesticides is equal to that of conventional
chemical pesticides. However, the mode of action is different. With many of the
bio-pesticides, the time from exposure to morbidity and death of the target insect
may be 2–10 days in general. Due to application of bio-pesticides in tea crops (70%
crop coverage) almost 9 kg of made tea increase could be achieved with a chemical
cost saving of Rs. 3750 (Hazarika et al. 2001). One of the multi-location field trial
studies against major pest of rice with B. bassiana and conventional insecticides
revealed that the mycoinsecticides were superior to monocrotophos in controlling
D. armigera, leading to an increased cost benefit ratio of 1:7.66 for B. bassiana
against 1:2.92 for monocrotophos (Hazarika and Puzari 1977).
14.8 Conclusion
Adverse effects of chemical pesticides on non-target organisms, food safety, envi-

ronmental issues and development of pest resistance have forced the scientific com-
munities to focus on the development of alternative eco-friendly measures. EPF
have a key role in sustainable pest management programs in many agricultural sys-
tems. However, the large-scale use of EPF as bio-pesticides can be hindered by a
number of issues. The major barriers include the inoculums short or poor shelf life
span, requiring a couple of weeks to kill the target pests, the need for a fungicide-
free environment, high relative humidity (> 80% RH), poor quality of some com-
mercial products, high-cost of commercial formulations and the possibilities of
contamination with mycotoxins (aflatoxins, trichothecenes, zearalenone, fumoni-
sins, citrinin, etc.). Hence, there is a need to promote R & D activities on EPF to
increase shelf-life of their formulation up to 12–18 months, by adding additives and
adjuvants including oil, sunscreen UV- and IR-protectants to make products more
active and durable in the field (wet land and dry land), better storage conditions
under different environments and ecosystems. Moreover, there is a tremendous
scope to utilize the biodiversity of Entomophthora, Zoophthora, Neozygites fungi,
belonging to the Entomophthorale group, which have a high potential for manage-
ment of sucking and lepidopteran pests.
Advanced techniques in molecular biology have the potential to manipulate
desirable traits of EPF to improve overall field activity. There is also the further need
for understanding the host-parasite interactions, in relation to crop ecosystem. This
is vital as every crop ecosystem is unique and provides different microclimatic
380 P. Das et al.
c onditions in which the target pests evolved a number of mechanisms to keep the
pathogen at bay. Though EPF have great potential, R & D activities for their com-
mercial exploitation as inundative, inoculative and classical biopesticides are not
adequate to meet the actual huge demand. In order to achieve a successful EPF-
based pest management strategy, a paradigm shift in the mindset of farmers is
needed. The Government and the industry jointly may develop a mission for the
popularization of EPF.
Acknowledgements The authors are grateful to Assam Agriculture University and Tocklai Tea
Research Institute, Jorahat, Assam, India, for the facilities provided for conducting the study.
Declaration Statements Ethics approval and consent to participate
This article does not contain any studies with human participants or animals performed by any
of the authors, so not applicable.
–– Consent for publication: Not applicable
Disclosure-All the experiments undertaken in this study comply with the current laws of the coun-
try where they were performed.
–– Availability of data and material: As it is review article it is not applicable
–– Competing interests: The authors declare that they have no competing interests.
–– Funding: As it is review it is not applicable
–– Authors’ contributions (Do not include any authors’ information)
LKH with PD and SR conceived the idea of the manuscript. PD, SK and SR participated in
preparing first draft of the manuscript. PD, SK and SR conducted literature surveys. All authors
read and approved the final manuscript.
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Index
A Anopheles aegypti, 170

Abacarus hystrix, 166 Anopheles gambiae, 87, 171
Abamectin, 310, 324, 346, 348, 358 Antagonists, 94, 173, 208, 342–359, 366–380
Acetamiprid, 309, 310, 329 Antheraea proylei, 72
Acetylcholine esterase, 333, 352 Anthocoridae, 262
Acrididae, 352 Anticarsia gemmatalis, 55, 64, 120, 164,
Actinedida, 263 368, 376
Acylamidase, 353 Antifeedant, 156, 307
Adalia bipunctata, 254, 255 Antioxidant, 190, 198–202, 285, 353
Adoxophyes honmai, 65, 76 Aphidius colemani, 306
Adoxophyes orana, 65, 69, 76, 279 Aphidoletes aphidimyza, 259, 262, 264
Adoxophyes orana GV (AdorGV), 69, 279, Aphis craccivora, 164, 166, 368, 377
280, 287, 288 Apidae, 54, 260, 261
Agrius convolvuli, 224 Apis mellifera, 54, 91, 130, 166, 257, 260,
Agroecosystem, 14, 24, 132, 134, 139, 290, 305, 306
334, 342, 343, 345, 346, 348, 354 Arabidopsis thaliana, 213
Agromyzidae, 261 Arabinose, 200
Agrotis ipsilon, 57, 65, 254, 255, 279, 368 Arabitol, 190
Agrotis segetum, 57, 59, 65, 128, 279, 287 Araneidae, 263
Aleurocanthus spiniferus, 165 Archaea, 77, 79, 88, 170, 212
Aleyrodes citri, 165 Archiascomycetes, 154
Alfa alfa looper, 44, 56 Arctiidae, 76
Allantonematidae, 249 Armadillidiidae, 262
Alphabaculovirus, 276 Armadillidium vulgare, 262
Alternaria alternata, 172 Aschersonia aleyrodis, 160, 166, 168, 189, 376
Amblyseius swirskii, 306 Aschersonia placenta, 189
American bollworm, 55, 161 Ascomycota, 24, 28, 31, 48, 90–92, 94,
Aminopeptidases, 156, 370 133–134, 152–173, 344, 366
Amsacta moorei, 71 Ascosphaera, 91, 133
Anagasta kuehniella, 26 Ascosphaera aphis, 91
Anagrapha falcifera, 56 Ascoviruses, 72
Anamorph, 33, 34, 92, 94, 96, 127, 133, 134, Asexual spore, 25, 89, 157, 186
138, 155, 161, 165, 186 AsGV, 279, 287
Annona squamosa, 332 Aspergillus, 133, 155, 162
Anomala cuprea, 307, 334 Aspergillus niger, 190, 372

https://doi.org/10.1007/978-3-030-23045-6
388 Index
Autographa californica, 56, 62, 64, 76, 304 Biopesticides, viii, 34–36, 44, 51, 78, 81, 82,
Autographa californica multiple 92, 116, 117, 128, 138, 139, 158, 163,
nucleopolyhedrovirus (AcMNPV), 58, 167, 276–278, 285, 286, 290, 334, 343,
62, 64, 73, 76, 278, 281 366, 373, 380
Avermectin, 121, 308, 309 Biotrophic, 25, 31, 34, 90, 371
Azadirachtin, 307–310, 358 Black vine weevil, 301, 377
Azinphos-ethyl, 348 Blaniulidae, 263
Azocyclotin, 355 Blaniulus guttulatus, 262
Azoxystrobin, 347 Blastocladiomycota, 24, 91, 92, 130–131, 366
Azygospore, 31, 131, 137 Blastopore, 135, 138, 158, 167, 301, 303, 344,
350, 371
Blattella germanica, 327, 349
B Blattodea, 33, 349
Bacillaceae, 77, 79, 88, 117, 120 Bombus spp., 260
Bacilli, 77, 79, 81 B. impatiens, 306
Bacillus spp., 26, 35, 77, 81–84, 88, 116, 117, B. terrestris, 257, 261, 306
120, 125, 299 Bombycidae, 54, 76
B. anthracis, 83–85 Bombyx mori, 26, 46, 54, 58, 63, 64, 76, 86,
B. larvae, 81 116, 164
B. licheniformis, 83 Bombyx mori nucleopolyhedrovirus
B. popilliae, 78, 79, 81, 82, 330 (BmNPV), 54, 64
B. sphaericus, 47, 78, 80, 81, 83, 119, 330 Boophilus annulatus, 262
B. thuringiensis, 12, 26, 27, 35, 46, 48, 51, Botrytis cinerea, 306
77–79, 81–87, 116, 117, 119, 125, 126, Brevibacillus, 88
167, 168, 289, 301, 321, 329, 330, 332, Brevibacillus laterosporus, 26, 27, 48,
344, 358, 366 120–121, 125
B. thuringiensis thompsoni, 78 Brevipalpus phoenicis, 166
B. weihenstephanensis, 84 Brown plant hopper, 308, 377
Bacteriophages, 78, 82 Bumble bees, 44, 260, 306
Baculoviridae, 55, 60–62, 276, 277
Baculovirus, 45, 54–58, 61–70, 72–75, 277,
278, 280, 283–286, 289, 290 C
Bagrada hilaris, 308 Cabbage moth, 55, 56
Banana weevil, 167 Caenorhabditis elegans, 87, 213
Basidiobolus ranarum, 96 Calacarus heveae, 166
Basidiomycota, 24, 28, 91, 92, 132, 154, 366 Calosoma granulatum, 256, 260
Bassianolide, 156, 159, 371 Carabidae, 254, 260
Beauveria, 31, 89, 90, 94, 128, 134, 138, 155, Carbamate, 2, 3, 12, 13, 247, 329–331, 342,
161, 167, 171, 172, 186, 187, 344, 369, 346, 357
372, 373, 376, 377 Carboxymethylcellulose, 191
Beauveria brongniartii, 92, 138, 153, 167, Carboxypeptidase, 157, 158
168, 329, 373, 376, 377 Catalase, 120, 190, 199, 200, 202, 333, 353
Beauveria pseudobassiana, 201 Cecidomyiidae, 262
Beauvericin, 156, 158, 371 Cellular immunity, 225, 301
Beauverolides, 156, 371 Cereal rust mite, 166
Bembidion properans, 255, 260 Chestnut weevil, 301
Bemisia tabaci, 12, 164, 308, 329, 331–333, Chilo spp.
350, 353, 368, 377 C. infuscatellus, 278, 279, 283, 287,
Benzoate, 346 288, 368
Benzylideneacetone (BZA), 222 C. iridescent, 71
Betabaculovirus, 276, 281 C. sacchariphagus, 278, 287
Bifenthrin, 309, 310 Chitinase, 123, 138, 156–159, 170, 231, 308,
Biodiversity, 24, 173, 335, 379 333, 344, 345, 353, 370
Index 389
Chlorantraniliprole, 352, 353, 358, 359 Cuticle, 31, 33, 49, 90, 92, 116, 135, 136, 138,
Chlorpyriphos, 2, 263, 347, 358 155–160, 169, 186, 198–200, 209–212,
Choristoneura fumerferana, 281 218, 229, 248, 302, 303, 308, 310, 324,
Chromobacterium substugae, 26 327, 328, 344, 351, 356, 369, 370
Chrysanthemum, 162, 264, 376 Cydia pomonella, 51, 55, 56, 69, 86, 128, 261,
Chrysoperla zastrowi sillemi, 253 279, 281, 286–289, 367
Chrysopidae, 253, 261 Cydia pomonella granulovirus (CpGV), 69,
Chymoelastase, 158 279, 281–283, 288, 289
Chymotrypsins, 157 Cyfluthrin, 327, 349
Chytridiomycota, 24, 28, 90–93, 130, 154 Cypermethrin, 2, 326, 328, 330, 347, 349,
Cicadellidae, 165 356, 357
Citrus white fly, 165 Cypovirus, 45, 54, 62, 63, 278
Clavicipitaceae, 33, 93, 97, 133, 134, 186 Cytolysin, 220, 223, 229
Clofentezin, 355 Cytoplasmic polyhedrosis virus (CPV), 45, 59,
Clostridium, 81 62, 63, 70, 278
Clostridium bifermentans, 26, 48, 121 Cytotoxic factors, viii, 208–232
Clothianidin, 325–328 Cytotoxins, 220, 223, 226, 227, 229
Cnephasia spp., 288
Cocci, 79
Coccinella undecimpunctata, 254 D
Coccinellid, 44, 302 Daktulosphaira vitifoliae, 152
Codling moth, 44, 56, 128, 152, 161, 261, Danio rerio, 213
286, 288 Dasyhelea obscura, 154
Coleomegilla maculata lengi, 302 Decomposers, 24, 133, 153
Coleoptera, 29, 32, 33, 51, 60, 77, 81, 83, 86, Deltabaculovirus, 276
87, 89, 120, 123, 124, 127, 155, 159, Deltamethrin, 2, 14, 326, 330, 347, 351,
160, 162, 164, 166, 208, 249, 252–260, 356–358
348, 349, 355–358, 368, 371, 376 Dendrolimus punctatus, 63
Colias eurytheme, 54 Dendrolimus spp., 161, 167, 376
Collembola, 263 Dermaptera, 33, 258, 263
Colorado potato beetle, 26, 123, 152, 161, Deuteromycota, 154
301, 348 Dextruxin, 158
Conidia, 30, 31, 33, 89, 132, 152, 186, 198, Diafenthiuron, 355
302, 319, 345, 371 Dialeurodis citri, 165, 350
Conidiobolus coronatus, 96 Diamondback moth, 55, 120, 122, 166, 168,
Conidiophores, 30, 132, 159, 302 253, 304
Coptotermes formosanus, 308 Diaphania nitidalis, 261
Cordyceps spp., 31, 33, 94–97, 127, 133, Diaprepes abbreviatus, 328, 331, 333
154, 165 Diatraea saccharalis, 69, 279, 301
C. bassiana, 33, 161 Dichloro-diphenyl-trichloroethane (DDT),
C. militaris, 95, 96 2–4, 6, 7, 10, 348
Cordycipitaceae, 33, 93, 133, 134, 161, 186 Difenoconazole, 347
Cosmopolites sordidus, 167, 357 Diglyphus begini, 256, 261, 264
Crioceris quatuordecimpunctata, 301 Dikarya, 91, 93, 154
Cry genes, 86, 119 Diptera, 29, 32, 33, 51, 56, 60, 62, 63, 70, 73,
Cry proteins, 27, 78, 81, 121, 126, 127 77, 81, 86, 87, 89, 120, 123, 127, 130,
Crypticola entomophaga, 90 160, 166, 208, 249, 253, 259, 261, 262,
Crystalliferous, 26 276, 350, 376
Culex nigripalpus, 70, 73 DNA-DNA hybridization, 52, 81, 84
Culex pipiens, 87, 164 DNA helicase, 73
Culicinomyces, 155 DNA hybridization, 73, 81
Curculio elephas, 301 DNA polymerase, 73
Curculionidae, 260, 348, 349, 357 DNA polymorphism, 372
390 Index
DNA sequencing, 74, 80, 372 Erythritol, 190

DNA viruses, 277 Erythrocytes, 223–227
Double-stranded DNA (dsDNA), 60, 71, Escherichia coli, 85, 224, 283
86, 277 Esterases, 156, 158, 170, 333, 351–353
Double-stranded RNA (dsRNA), 59, 277 Etoxazole, 355
Drosicha mangiferae, 253 Etridiazole, 311
Drosophila, 46, 62, 213–220, 372 Eubacteria, 79, 81, 88
Drosophila melanogaster, 123, 213, 214, Eukaryotes, 44, 79, 95, 208
220, 372 Eulophidae, 261
dsRNA viruses, 277 Eurotiomycetes, 91, 133
Duponchelia fovealis, 165 Eutectona machaeralis, 161, 163
Exomala orientalis, 325, 326, 331
E
Ecosystems, 2, 4, 7, 15, 33, 55, 89, 94, 153, F
155, 171, 208, 260, 276, 284, 290, 318, False spider mite, 166
323, 344, 379 Fenpropathrin, 309
Ectobiidae, 349 Filoviruses, 277
Edwardsiella tarda, 227 Fire ants, 171
Egg parasitoid, 253 Firmicutes, 77, 79, 117
Eicosanoids, 222, 224 Flonicamid, 309
Electron microscopy, 54, 202 Flupyradifurone, 309
Endochitinas, 157 Formicidae, 261, 263
Endophytes, 31, 82, 94, 97, 116, 125, 134, Frankliniella occidentalis, 302, 304, 306, 332
153, 373 Fructose, 200
Endoproteases, 156, 158, 370 Fungi imperfecti, 154
Enterobacteriaceae, 78, 79, 117, 122–123, Fusarium, 89
208, 224, 227, 248, 249
Entomophaga aulicae, 96
Entomophthorales, 24, 25, 28–34, 94, 96, 116, G
131, 134–138, 344 Galactose, 200
Entomophthora spp., 32, 48, 90, 94, 132, 299, Galleria mellonella, 86, 170, 171, 210, 217,
344, 345, 379 223, 224, 261, 303, 351, 356, 358, 372
E. anisopliae, 157 Gamacyhalothrin, 356, 357
E. virulenta, 167–169 Gammabaculovirus, 276
Entomophthoromycetes, 24, 29–31 Gamma-cyhalothrin, 347
Entomophthoromycota, 24, 28, 29, 90, 91, 93, Gelechiidae, 76
95, 96, 131–132 Genome sequencing, 63, 83, 84, 96
Entomopoxviruses (EPV), 45, 59, 62, 71, 278, Genomics, 52, 61–76, 80, 82–89, 94–97, 277,
319, 321, 322, 331, 334 278
Entomopthoromycotina, 24, 29–31 Genotypes, 57, 58, 62, 73, 372
Ephestia kuehniella, 253, 329 Geometridae, 76
Epicuticle, 31, 158, 159, 198, 200, 369, 371 Geranium carolinianum, 305
Epidermis, 218, 369 Gilpinia hercyniae, 55
Epithelial cells, 119, 224, 280 Glassy winged sharpshooter, 165
Epithelium, 27, 28, 48, 119, 120, 171, 208, Glomeromycota, 91, 92, 131
210, 224, 228, 229, 280 Glutaredoxins (grx), 199
Epizootics, 29, 31, 55, 56, 82, 92, 94, 120, Glycerol, 190, 370
162, 164, 186, 188, 276, 287, 288, 290, Glycosphingolipids, 217
318, 334, 345, 346, 367 Glyphosate, 347
ErelGV, 69, 279, 289 Golgi apparatus, 215–217, 219, 220
Erinnys ello, 279, 286, 289 Golgi complexes, 216
Erynia, 32, 132, 137, 299 Gracilicutes, 79
Index 391
Gram-negative, 78, 79, 87, 122, 123, 208, 221, H. bacteriophora, 51, 78, 208, 251,
223, 231, 248, 249 253–257, 259, 261–264, 301, 325, 326,
Gram-positive, 78, 79, 88, 117, 223, 229 330, 355, 358
Gram stain, 27, 79, 81 H. indica, 88, 251, 253, 257, 259, 261,
Granulocytes, 213, 214, 216–217, 225–227, 263, 301, 326, 356, 357
334, 371 H. taysearae, 252, 254
Granulosis, 277, 278, 280, 283, 289 Heterotermes tenuis, 331, 333
Granuloviruses (GVs), viii, 51, 54–56, 59, 61, Heterotrophic, 28, 89, 154
62, 72–74, 76, 276–291, 299 Hirsutella spp., 51, 94, 155, 161, 165–166,
Grape berry moth, 55 299, 344
Grape phylloxera, 152 H. gregis, 165
Greater wax moth, 210, 303, 351 H. kirchneri, 165
Green lacewing, 257, 261 H. necatrix, 165
Gryllidae, 263 H. nodulosa, 165
Gylpinia herciniae, 55 H. thompsonii, 51, 96, 128, 159, 165, 166,
Gypsy moth, 44, 303 168, 169, 307, 367, 373, 376
Histeridae, 254
Homalodisca coagulata, 165
H Homoptera, 29, 32, 78, 89, 127, 162, 376
Habrobracon hebetor, 303 Horizontal gene transfer, 58, 78, 82, 84, 97,
Haematopoietic tissues, 219 170
Haemocoel, 48, 156, 157, 159, 198, 208, Humoral immune responses, 212, 222
210–212, 220, 230, 248 Humoral response, 209, 217, 221–223
Haemocytes, viii, 47, 208–232 Hyadaphis foeniculi, 307
Haemolymph, 47, 155, 157–160, 170, 210, Hyblaeapara, 161
213, 216, 217, 220–222, 225, 230 Hydrocarbons, 198, 200, 202, 370
Haemolysin, 211, 212, 220, 223, 225–227, Hydrolytic enzymes, 158, 229, 249
229 Hylobius abietis, 301
Harmonia axyridis, 255, 302 Hypanthia cunea, 279, 289
Harposporium, 165 Hyperoxidant, 198, 201
Harrsinia billions, 76 Hypocreales, 24, 25, 28, 29, 31–34, 89–91, 93,
Heat-shock proteins (HSPs), 172, 190 116, 134–138, 165, 186, 344
Helicoverpa spp., 12, 55, 56
H. armigera, 11, 12, 66, 70, 121, 161, 164,
231, 279, 308, 367, 368, 377, 378 I
H. zea, 51, 66, 304, 305, 358 Ichneumonidae, 14, 261
Heliothis, 55, 62, 305, 329 Ichneumonids, 257, 261, 264
Heliothis virescens, 72, 86, 304, 329 Imidacloprid, 173, 307, 310, 322, 325–327,
Helopeltis spp., 162 330, 331, 333, 347–349, 355, 357, 358
Hemiascomycetes, 154 Immune reaction, 14, 213, 221–222
Hemibiotroph, 25, 33, 90, 371 Immune system, 10, 45, 53, 95, 158, 212, 217,
Hemiptera, 13, 14, 29, 32, 33, 51, 60, 90, 124, 221–229, 232, 333, 371
138, 160, 162, 165, 166, 252, 253, 258, Immunity, 95, 171, 212–220, 301, 369, 371
262, 349, 350, 352, 368, 376 Infective juveniles (IJs), 49, 209–211, 230,
Hemocoel, 27, 30, 31, 90, 92, 119, 120, 122, 248, 249, 254–257, 259–261, 263, 301,
123, 138, 170, 198, 343, 344, 354, 369, 302, 320, 321, 323, 324, 354–357
371 Inositol, 200
Hepadnaviruses, 277 Insect growth regulators (IGRs), 14, 307, 308,
Hepialidae, 76 334, 342
Herpesviruses, 74, 277 Integrated pest management (IPM), viii, 25,
Heterorhabditidae, 48, 78, 208, 248, 251 36, 152–173, 248, 276, 300, 307, 309,
Heterorhabditis spp., 49, 79, 88, 122, 311, 318, 343, 346, 347, 359, 366
208–210, 232, 249, 257, 261, 299 Ips sexdentatus, 302
392 Index
Isaria, 31, 94, 134, 156, 161, 164, 172, 186, Lufenuron, 308, 326, 327, 346, 355–358
188, 299, 344 Lycosidae, 263
Isaria fumosorosea, 51, 92, 153, 156, 166, Lygus hesperus, 303, 308, 309
168, 187–190, 200, 303, 310, 329, 331, Lymantria dispar, 51, 55, 63, 67, 285, 303
350, 367, 373, 377 Lymantriidae, 58, 76
Isarolides, 156, 371 Lysinibacillus, 88, 119
Isoptera, 89, 90, 208, 253, 368 Lysinibacillus sphaericus, 26, 48, 78, 81,
Ixodes scapularis, 263 82, 119
Ixodidae, 262, 263
M
J Mafanoxam, 311
Jassid, 302 Mahanarva posticata, 163
Junonia orithya, 164 Mahanarva spp., 167
Mallophaga, 78, 127
Mamestra brassicae, 55, 56, 67
K Manduca sexta, 86, 170, 171, 225, 227, 370
Kickxellomycotina, 24, 91, 131 Mannitol, 189, 190
Kinesin, 157 Mantodea, 33
Kinoprene, 355 Mastrus ridibundus, 257, 261
Mediterranean flour moth, 26, 77, 116
Melanin, 199, 217, 218, 220, 222
L Melanoplus sanguinipes, 71
Labidura riparia, 258, 263 Mermithidae, 49
Laboulbeniomycetes, 89, 91, 133, 154, 155 Messor himalayanus, 261
Lacanobia oleracea, 279, 283, 289 Metarhizium spp., 31, 89, 90, 94, 95, 97, 127,
Lactobacillaceae, 79 134, 138, 157–158, 161, 163–164, 167,
Lactose, 200, 290 169–172, 199, 299, 344, 369, 370, 372,
Lambda-cyhalothrin, 326, 327, 347 373, 376
Lasiocampidae, 76 M. acridum, 94, 170, 188–190, 201
Leafminer, 261, 264 M. album, 94, 97, 138
Lecanicillium, 31, 134, 155, 158, 166, 172, M. anisopliae, 48, 91, 92, 95, 128, 135,
299, 376, 377 138, 153, 157, 160–163, 166–168,
Lecanicillium aphanocladii, 189 170–172, 187–191, 199–201, 300–302,
Lepidiota negatoria, 310 304–307, 310, 311, 323, 327–330, 333,
Lepidoptera, 11, 12, 15, 29, 32, 33, 45, 51, 54, 334, 346–353, 367–371, 373, 375–378
56, 58, 60–65, 69, 72–74, 76, 77, 81, M. brunneum, 51, 95, 189, 201, 303, 304,
83, 86, 123, 124, 126, 127, 159, 160, 309, 311, 327, 331
162, 164–166, 168, 169, 208, 213–220, M. guizhouense, 94
249, 253, 254, 260, 261, 276, 278, 290, M. majus, 94
350, 351, 356, 358, 368, 376 M. robertsii, 95, 138, 170–172, 188–191,
Leptinotarsa decemlineata, 26–27, 87, 123, 200, 201, 301, 308, 347, 350
301, 302, 308, 348, 367, 371 Methomyl, 2, 329, 355
Leucoma salicis, 57, 58 Metoxyfenozide, 355
Light microscopy, 59, 216 Micrococcaceae, 79
Liotryphon caudatus, 257, 261 Miridae, 162
Lipases, 138, 156, 157, 159, 211, 220 Mithymna unipuncta, 218
Lipopolysaccharides (LPS), 221, 223, 224 Mitochondrial DNA (mtDNA), 52, 154
Liriomyza trifolii, 261, 264 Monophagous, 343
Liviidae, 350 Monosporella unicuspidata, 154
Loculoascomycetes, 154 Monoxygenase, 352, 353
Locusta migratoria, 307, 352, 353, 376 Musca domestica, 87, 120, 137, 307, 350
Locustana pardalina, 376 Muscidae, 350
Luciaphorus perniciosus, 253 Mutations, 11, 50, 52, 58, 82
Index 393
Mycelium, 121, 155, 160, 162, 320, 323 Occlusion-derived virus (ODV), 58, 277,
Mycoinsecticides, 31, 152, 153, 163, 166–169, 278, 280
171, 172, 198–202, 367, 373, 377–379 Odonata, 33, 60
Mycoparasites, 31, 97, 131, 134, 158 Oenocytoids, 213–215, 217, 218, 220
Mycorrhizae, 153 Onychiuridae, 263
Mycosporidia, 28, 366 Onychiurus armatus, 263
Mycotoxicity, 158, 173, 332, 379 Oomycetes, 90, 128
Myriangiales, 133, 154 Oomycota, 90, 128–129
Myzus persicae, 13, 162, 166, 170, 302, 368, Ophiocordyceps, 90, 127
376, 377 Ophiocordyceps sinensis, 94–96
Ophiocordycipitaceae, 33, 93, 133, 134
Ophiostoma, 89
N Organochlorines, 2, 3, 6, 7, 10, 15, 342, 346
N-acetyl-D-glucosamine, 218 Organophosphates, 2, 3, 10, 12, 13, 247
N-acetyl glucosamine, 158 Organophosphous, 324
Necrotrophy, 90, 371 Orgyia pseudotsugata, 55, 57, 58, 65
Neisseriaceae, 79, 117, 122, 124 Oribatida, 263
Neocallimastigomycota, 91, 92 Orius albidipennis, 258, 262
Neodiprion lecontei, 70, 72 Orthomyxoviruses, 277
Neodiprion sertifer, 62, 70 Orthoptera, 29, 33, 51, 60, 78, 123, 127, 160,
Neolecta, 154 162, 166, 208, 253, 352, 376
Neonicotinoid, 325, 331, 332, 342 Oryctes rhinoceros, 62, 128, 164, 368,
Neoscona theisi, 259, 263 377, 378
Neoseiulus cucumeris, 303, 306 Ostrinia nubilalis, 86, 128, 161, 376
Neozygites, 299 Otiorhynchus sulcatus, 301
Neozygites tanajoae, 303, 310 Oxamyl, 355
Nephotettix spp., 161 Oxidative stress, vii, 160, 171, 190, 198–202
Neuroptera, 253, 257, 261 Oxindole, 222
Nilaparvata lugens, 308, 323, 329
Noctuidae, 11, 12, 55, 76, 224, 254, 260, 350,
356, 358 P
Nomuraea, 94, 155, 159, 161, 162, 164, Paecilomyces spp., 94, 155, 164, 310
344, 376 P. farinosus, 161
Nomuraea rileyi, 159, 160, 164, 168, 169, 186, P. fumosoroseus, 164, 166, 167, 187, 346,
367, 368, 377, 378 347, 351, 377
Non-crystalliferous, 26 P. lilacinus, 35, 51, 167, 169, 373, 376
Non-spore-forming, 48, 79 Paenibacillaceae, 79, 117, 120–121
Nosema, 129, 299 Paenibacillus spp., 82, 88, 120, 308
Notodontidae, 76 P. lentimorbus, 26, 47, 120
Nucelocapsids, 59 P. popilliae, 26, 47, 51, 78–79, 82,
Nucleopolyhedrosis, 277, 278 120, 125
Nucleopolyhedrovirus (NPVs), 51, 54–57, 59, Pandora, 32, 94, 132, 137, 299, 344
61, 62, 64, 65, 68–70, 72–74, 76, Paralobesia viteana, 55
276–278, 280, 281, 283, 287, 299, 304 Parasporal crystals, 27, 47, 117, 126
Nudiviruses, 280 Parvoviruses, 277
Nutrient agar, 27 Periplaneta americana, 367, 371, 378
Nutrient broth, 27 Peroxidase, 199, 202, 333, 353
Nymphalidae, 76 Peroxisome, 198, 200
Pezizomycotina, 24, 31–34, 133, 154
Phaenositylenchidae, 249
O Phagocytosis, 47, 199, 209, 213–216, 221,
Occlusion bodies (OBs), 54, 56, 59, 61, 70, 228, 229, 334, 371
72, 73, 277, 280, 285, 288, 290 Phasmatodea, 33
394 Index
Phenoloxidase, 222, 223, 333, 352, 353 Pseudomonadaceae, 79, 88, 122, 123
Phorate, 2, 5, 347 Pseudomonas spp., 81, 123, 321
Photorhabdus spp., 48, 49, 79, 81, 88, 117, P. entomophila, 48, 88, 123, 226
208, 209, 220–223, 225, 227, 228, 230, P. putida, 88, 123, 125
232, 248, 249, 252, 253, 354 Pseudoplusia, 164
P. asymbiotica, 88, 226, 251 Pseudopodia, 215, 216, 219, 220
P. luminescens, 78, 86, 88, 89, 122, Pterostichus cupreus, 255, 260
225–227, 230, 251, 253 Pucciniomycotina, 91
P. temperata, 88, 89 Pyralidae, 54, 76, 224, 253, 261, 351
Phthorimaea operculella, 55, 70, 278, 279, Pyrenomycetes, 154
286, 287 Pyrethrins, 3, 308, 309
Phyllocoptruta oleivora, 128, 159 Pyrethroids, 2, 3, 11, 12, 14, 329–331, 347,
Phylogeny, vii, 44–98, 133, 277, 372 357, 358
Phytoalexines, 332 Pyrilla purpusilla, 163
Picornaviruses, 277 Pyriproxyfen, 347, 355
Pieridae, 54, 76, 253, 290
Pieris brassicae, 54, 253, 278, 279, 290
Pine weevil, 301 R
Piperonyl-butoxide, 352, 355 Rachiplusia nu, 350
Plasma membranes, 126, 217, 226, 280 Rachiplusia ou, 65, 76
Plasmatocytes, 14, 213–216, 218–220, 224, Randomly amplified polymorphic DNA
225, 227, 334 (RAPD), 52, 372, 373
Plasmids, 46, 78, 82–84, 86, 230 Reactive oxygen species (ROS), 171, 172,
Plathypena scabra, 164 190, 198, 200–202
Pleosporales, 133, 154 Restriction fragment length polymorphism
Plutella xylostella, 32, 55, 56, 65, 86, 120, (RFLP), 52, 83
166, 200, 253, 278, 279, 285, 304, 333 Retroviruses, 277
Plutellidae, 76, 253 Rhabditida, 78
Pollinators, 14, 44, 260, 305 Rhizoctonia solani, 201
Polydnavirus (PDV), 45, 280 Rhizosphere, 81, 94, 135, 373
Polymerase Chain Reaction, 372 RNA viruses, 45, 59, 62
Popillia japonica, 51, 79, 83, 87, 120, 128, Rphopalosiphum rufiabdominale, 308
323, 325–328, 331 Rubber tree mite, 165–166
Porcellionidae, 262
Porcellio scaber, 262
Potato dextrose broth, 319 S
Potato tuber moth, 55 Sabouraud Dextrose Agar, 188
Poxvirus, 71, 277 Sabouraud Dextrose Broth (SDB), 188
Profenofos, 346 Sabouraud Maltose Agar (SMA), 188
Prohaemocytes, 214, 215, 219 Saccharomyces cerevisiae, 153
Prokaryotes, 44, 77, 87, 88 Saccharomycotina, 133, 154
Propamocard, 311 Salmonella enterica, 224
Propiconazole, 310, 347 Saturniidae, 76
Prostephanus truncatus, 302 Scelionidae, 263
Proteases, 27, 28, 123, 126, 138, 156–158, Schistocerca gregaria, 163, 377
170, 212, 220, 223, 224, 344, 370, 373 Schizosaccharomyces, 154
Proteinases, 159, 225 Sclerotia, 201
Proteobacteria, 78, 117, 122, 223 Sclerotinia minor, 201
Proteus mirabilis, 224, 226, 227 Sclerotinia sclerotiorum, 201
Protoplasts, 25, 31, 34, 90, 137, 173 Sclerotium rolfsii, 201
Protozoa, 13, 24, 44, 49, 78, 127, 129, Scutigerella immaculata, 262
213, 229 Semiochemicals, 304–305
Pseudaletia unipuncta, 70, 224, 279, 281 Septobasidiobasidiaceae, 91
Index 395
Serotype, 78, 81 Tebufenpyrad, 355, 356

Serratia spp., 81, 82, 88, 122, 125, 299 Teleomorph, 33, 34, 94, 133, 138, 155, 161
S. entomophila, 26, 48, 87, 88, 122, 125, Tenebrio molitor, 202, 311, 328
301 Tenericutes, 79, 117
S. marcescens, 48, 81, 87, 122, 224, 227, Teretriosoma nigrescens, 302
301, 308, 321, 323, 329, 330, 332 Tetradonematidae, 249
S. proteamaculans, 87, 122, 125 Thanasimus formicarius, 302
Sexual spores, 25, 89, 128, 133, 153 Thermotolerance, vii, 172, 186–191, 199
Simplicillium lanosoniveum, 189 Thiamethoxam, 308–310, 323, 325, 326,
Sminthuridae, 263 328–330, 333, 347
Soil-borne bacteria, 24–36 Thiolutin, 223
Soil-borne entomopathogen, 24–36 Thiomethoxam, 331, 332
Solenopsis invicta, 171 Thioredoxins (trx), 199
Sordariomycetes, 24, 31–34, 91, 133 Thrips, 136, 158, 160, 163, 167, 168, 172,
Sorosporella, 155 262, 263, 302, 304, 326, 328, 332,
Spherulocytes, 213–215, 217–218 376, 377
Sphingidae, 76, 224 Thysanoplusia orichalcea, 65, 76, 378
Spinosad, 326, 329, 331, 346, 350, 356, 357 Thysanoptera, 33, 51, 249, 252, 253
Spirilla, 79 Tipula paludosa, 62
Spodoptera exigua MNPV (SeMNPV), 58, Togaviruses, 277
59, 68 Tolypocladium cylindrosporum, 189
Spodoptera spp. Tolypocladium inflatum, 96, 189
S. exigua, 51, 55, 58, 68, 86, 125, 200, 224, Torrubiella, 165
279 Tortricidae, 76, 261
S. frugiperda, 12, 68, 70, 72, 86, 260, 279, Trehalase, 157, 158, 170
281, 289, 329, 331, 356, 357 Trialeurodes spp., 162, 164
S. littoralis, 69, 86, 210, 227, 301, Trialeurodes vaporariorum, 162, 311
329, 331 Tribolium castaneum, 12, 202, 310, 332, 356
Sporangium, 27 Trichogramma chilonis, 253
Spore-forming bacteria, 46 Trichogrammatidae, 253
Sporulation, 25, 47, 48, 54, 90, 117, 119, 120, Trichoplusia ni, 63, 69, 70, 72, 73, 76, 86, 164,
137, 158, 171, 187, 198, 320, 323, 327, 279, 281, 301, 368, 376
328, 346, 347 Trifloxystrobin, 347
ssDNA viruses, 277 Triflumizolet, 311
Staphylinidae, 254, 263 Triterpenoids, 332
Steinernema spp., 49, 79, 208–211, 250, 257, Trypsins, 157, 345
260, 264, 299 Tuber magnatum, 153
S. carpocapsae, 51, 208, 250, 254–257, Typhlodromalus aripo, 303
259–264, 326, 330, 355–358 Tyrosine, 223
S. kraussei, 250, 257, 261, 301, 358
Sugarcane spittlebugs, 167
Superoxide dismutase (SOD), 160, 171, 199, U
202, 333, 353 Ultraviolet (UV) radiation, 169, 171, 172, 189,
Symbionts, 77–79, 116, 117, 208–212, 198, 248, 266, 286, 345
220–232, 248–254, 319, 354
Symbiotic bacteria, 48, 86, 209–212, 247–267
Synergy, 173, 301, 308 V
Vairimorpha, 49, 299
Varroa destructor, 166
T Verticillium spp., 155, 158, 161, 162, 166, 368,
Taphrina, 154 376
Taphrinomycotina, 133, 154 V. lacanii, 162
Tebufenozide, 355 Vibro, 79
396 Index
W Y
White grubs, 128, 138, 167, 168, 323, 331, Yellow muscardine, 164
333, 355, 358, 368, 376, 378 Yersinia, 88, 123, 226, 299
White truffle, 152–153 Yersinia enterocolitica, 123, 226, 230
Wiseana cervinata, 76
Z
X Zinc-chelator, 223
Xenorhabdus spp., 48, 49, 79, 86, 88, 117, Zoophthora, 32, 94, 132, 304, 344, 379
208–211, 220–226, 228–232, 248–250, Zoophthora radicans, 304
253, 354 Zoospore, 91, 128, 130
X. bovienii, 86, 250 Zygomycetes, 28, 93, 131
X. nematophila, 86, 88, 211, 222, 224, 225, Zygomycota, 24, 94, 131, 154, 344
227, 229, 231, 250 Zygomycotina, 366
Xestia c-nigrum, 70, 72, 76, 279, 281
Xylella fastidiosa, 165

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Sustainability in Plant and Crop Protection

Md. Aslam Khan

Microbes for Sustainable

ISSN 2567-9805 ISSN 2567-9821 (electronic)

© Springer Nature Switzerland AG 2019

Director, Kailash Chandra

It is generally acknowledged that agricultural yields must be increased in the com-

Jazan, Saudi Arabia Md. Aslam Khan

1 Synthetic Chemical Insecticides: Environmental and Agro

10 Granuloviruses in Insect Pest Management.......................................... 275

Index ................................................................................................................. 387

Md. Aslam Khan and Wasim Ahmad

Abstract Synthetic pesticides are indispensable in intensive agricultural produc-

Keywords Synthetic pesticides · Environment · Contaminant · Non target

© Springer Nature Switzerland AG 2019 1

1.2 Background and History

Monocrotophos, phorate, phosphamidon, methyl parathion and dimethoate are

1.3 Environmental Contaminants

Pesticides hold a unique position among environmental contaminants due to their

1.4 Impact on Human Health

Fig. 1.3 Different pathways of pesticides exposure

poisonings is much higher, as Richter (2002) reports 26 million human pesticide

that acute intoxication by organophosphates may be responsible for chronic impair-

(reductions in acetylcholinesterase enzyme [AChE] due to contamination by chemi-

1.5 Resistance Among Insect Pests

resistances, leaving no effective protective options available. The intensive use of

1.6 Effects on Non -Target Organisms

1.7 Effects on Beneficial Arthropods

present in agroecosystems, including the natural enemies of agricultural insect pests

1.8 Pesticides Degradation

Microbial degradation of pesticides applied to soil is the main mechanism which

markets, with the pioneer research on biodegradation of 2,4-D (Audus 1949). In a

Tan Li Peng, Samsuddin Ahmad Syazwan, and Seng Hua Lee

© Springer Nature Switzerland AG 2019 23

Keywords Soil-borne · Entomopathogens · Bacteria · Fungi · Bio-insecticides

Soil, the unconsolidated mineral or organic material on the immediate surface of

Detailed life-cycles of entomopathogenic fungus belonging to Entomophthorales

review on the different types of soil-borne entomopathogenic bacteria and fungi,

2.2 Soil-Borne Entomopathogenic Bacteria

Most of the discovered entomopathogenic bacteria are soil-borne aoecies such as

2.2.2 Mechanism of Action

The insecticidal mechanism of Bacillus thuringiensis is well known by the scientific

The specificity of entomopathogenic bacteria towards hosts depends on the pres-

2.3 Soil-Borne Entomopathogenic Fungi

stable microclimate conducive to continual fungal activity, even during extreme

2.3.1 Entomophthorales (Entomopthoromycotina:

Entomophthorales (Schröter, 1893) is an order now treated under

2.3.2 Hypocreales (Pezizomycotina: Sordariomycetes)

The order Hypocreales (Lindau, 1897) classified in Sordariomycetes, one of the

Aphids, other Homoptera

Meristacruma Nematodes Gryganskyi et al. (2013)

Plutella xylostella, other

Table 2.2 Differences between Entomophthorales and Hypocreales

2.4 Current Status as Biopesticides

Table 2.3 Example of commercially available biopesticides derived from entomopathogenic

Gangwar, R. K. (2017). Role of biological control agents in integrated pest management

Mudasir Gani, Taskeena Hassan, Pawan Saini, Rakesh Kumar Gupta,

Abstract Insects, like other organisms, are susceptible to a variety of diseases

Jazan, Saudi Arabia Md. Aslam Khan

1 Synthetic Chemical Insecticides: Environmental and Agro

10 Granuloviruses in Insect Pest Management.......................................... 275

1.2 Background and History

1.3 Environmental Contaminants

1.4 Impact on Human Health

1.5 Resistance Among Insect Pests

1.6 Effects on Non -Target Organisms

1.7 Effects on Beneficial Arthropods

1.8 Pesticides Degradation

2.2 Soil-Borne Entomopathogenic Bacteria

2.2.2 Mechanism of Action

2.3 Soil-Borne Entomopathogenic Fungi

2.3.1 Entomophthorales (Entomopthoromycotina:

2.3.2 Hypocreales (Pezizomycotina: Sordariomycetes)

2.4 Current Status as Biopesticides

3.2 Molecular Phylogenetics and its Importance

3.3 Entomopathogenic Viruses

3.3.1 Origin, Natural History and Geographical Distribution

3.3.2 Taxonomy and Evolution

3.3.3 Genomics and Phylogeny

3.4 Entomopathogenic Bacteria (EB)

3.4.1 Origin, Natural History and Geographical Distribution

3.4.2 Taxonomy and Evolution

3.4.3 Genomics and Phylogeny

3.5 Entomopathogenic Fungi (EF)

3.5.1 Origin, Natural History and Geographical Distribution

3.5.2 Taxonomy and Evolution

3.5.3 Genomics and Phylogeny

4.2 Bacterial History and Diversity

4.2.1 Gram Positive: Firmicutes and Actinobacteria

4.2.2 Gram-Negative: Proteobacteria

4.3 Soil Habitat and Pathogenesis of Bacteria

4.4 Fungal History and Diversity