Agriculturally Important Microorganisms: Harikesh Bahadur Singh Birinchi Kumar Sarma Chetan Keswani Editors
Agriculturally Important Microorganisms: Harikesh Bahadur Singh Birinchi Kumar Sarma Chetan Keswani Editors
Agriculturally Important Microorganisms: Harikesh Bahadur Singh Birinchi Kumar Sarma Chetan Keswani Editors
Agriculturally
Important
Microorganisms
Commercialization and Regulatory
Requirements in Asia
Agriculturally Important Microorganisms
Harikesh Bahadur Singh
Birinchi Kumar Sarma • Chetan Keswani
Editors
Agriculturally Important
Microorganisms
Commercialization and Regulatory
Requirements in Asia
Editors
Harikesh Bahadur Singh Birinchi Kumar Sarma
Department of Mycology and Plant Department of Mycology and Plant
Pathology Pathology
Institute of Agricultural Sciences Institute of Agricultural Sciences
Banaras Hindu University Banaras Hindu University
Varanasi, Uttar Pradesh, India Varanasi, Uttar Pradesh, India
Chetan Keswani
Department of Mycology and Plant
Pathology
Institute of Agricultural Sciences
Banaras Hindu University
Varanasi, Uttar Pradesh, India
During the last five decades, application of chemicals in agriculture has helped in
managing many pests and diseases, thereby reducing yield losses in crops. However,
concerns are often expressed with regard to pesticide residues in food stuff, environ-
mental pollution, imbalance of ecological equilibrium and resurgence of minor
pests and pathogens. In sustainable intensification of agriculture through green
economy, biopesticides have gained immense significance. Despite some progress
in biopesticide production and supply, the scale of its use in India still remains rela-
tively small in comparison to chemical pesticides. Much of the production goes to
government agencies for distribution to farmers in integrated pest management
(IPM) programmes. The distribution system for biopesticides is underdeveloped in
many areas.
This volume on commercial use of agriculturally important microbes in the form
of biopesticides includes contributions from vastly experienced Asian experts in a
comprehensive manner describing most recent facts and extended case studies. I
address the vital issues pertaining to translation of biopesticide research from lab to
land. Further, commercialization and regulatory issues concerning biopesticides
have also been discussed in a manner that will be invaluable for academicians, sci-
entists, researchers and policymakers.
I congratulate the editors for this useful effort.
v
Preface
The past century has witnessed a slow but steady emergence of biopesticides and
biofertilizers as potential supplementary and eco-friendly inputs in comparison to
their chemical counterparts. Unfortunately, despite considerable research and devel-
opment efforts, biopesticide use has remained limited to only 2.5 % of the total
chemical use in most of the Asian countries. Several technological constraints in
these countries have been responsible for the limited adoption which is exemplified
by: lack of knowledge, poor availability of standard stable products and situation-
specific packages, inconsistent establishment and performance in different crop and
agroclimatic domains, inadequate information on safety, and reluctance on the part
of big industries for promotion.
The main focus of this book is to review the current status of research, develop-
ment, and use of these bioinputs in agro-based clusters in developing Asian coun-
tries and develop a strategy for addressing critical issues such as policy support,
quality control, regulatory management, and public-private participation in imple-
mentation of biopesticides in routine agriculture.
The first section will give an overview of the book and will try to develop a con-
sensus on issues of quality requirements, quality control, regulatory management,
commercialization, and marketing of agriculturally important microorganisms.
Despite the global progress in establishing a biopesticide supply, the scale of
biopesticide use remains relatively small in comparison to chemical pesticides.
Thus, the second section will deal with commercialization aspects for implementa-
tion of biological control in routine agricultural practices and includes expert views
on topics like licensing and enforcing intellectual property rights on hybrid PGPR
strains; innovating plant protection strategies in organic farming; identifying and
resolving constraints in commercialization of biopesticides; exposing spurious
biopesticide trade; and expanding the consortium model in biopesticide research.
vii
Contents
Part I Introduction
1 Synthesis of Policy Support, Quality Control,
and Regulatory Management of Biopesticides
in Sustainable Agriculture....................................................................... 3
Chetan Keswani, Birinchi Kumar Sarma,
and Harikesh Bahadur Singh
ix
x Contents
xiii
xiv Contributors
xvii
Part I
Introduction
Synthesis of Policy Support, Quality
Control, and Regulatory Management 1
of Biopesticides in Sustainable
Agriculture
Abstract
Growing awareness of organic food production throughout the globe has called
for green and sustainable agricultural practices. Agriculturally important micro-
organisms offer significant benefits in increasing crop yield and improving crop
health under both biotic and abiotic stresses. Broad range of microorganisms
have been registered and commercialized as biopesticides and biofertilizers all
over the world. Analyzing the fact that popularity of biopesticides and their enor-
mous potential are growing, a strong framework for regulation, registration, and
quality control for microorganism-based products on global scale is urgently
required. Scientists, government regulatory bodies, and industrial representatives
must discuss on strategies and future prospects of policy support for ensuring
quality of biopesticides in their respective countries.
Keywords
Biopesticides • Policy support • Quality control • Regulatory issues
1.1 Introduction
The Food and Agricultural Organization (FAO) has predicted an increase in the
demand of world food production by 70 %. In order to catch up with the supply
demand for growing population which is expected to reach 10 billion by 2050,
agro-based economies are employing various strategies for improving crop produc-
tion (UN 2011). Currently there is an urgent need to enhance current food
production and livelihood chances from ever-reducing per capita available arable
land. In order to meet the food and feed requirements, green revolution has been
very impressive, but unfortunately the inputs used in intensive agriculture have led
to the several objectionable effects on the human and environmental health.
Production of more food from less available arable land is only a phase of chal-
lenges; actually the original challenge lies in the fact that crop production should be
through safe and sustainable practices.
The global biopesticide market is estimated to reach US$ 6.6 billion by 2020
with expected growth at CAGR of 18.8 % from 2015 to 2020. In India, biopesticide
market is forecasted to show high growth with projected compounded annual rate of
19 % over the 2015–2020 period (https://www.kenresearch.com/agriculture-and-
animal-care/crop-protection/india-biopesticides-market-research-report/669-104.
html). Asia-Pacific region represented largest consumption and demand for bioin-
secticides and shared 27.7 % and 38 % of volume and value, respectively, of the
global market in 2013. Biopesticide market in this region is projected to attain fast-
est growth from 2015 to 2020 with CAGR of 17.8 % (http://www.mordorintelli-
gence.com/industry-reports/asia-pacific-biopesticides-market-industry ).
Consumption of pesticides in countries like India, China, and other developing
countries of the region is booming in order to fulfill the demand of the growing
population. However, reduced availability of arable land per person in India, China,
and other countries in this region is a matter of concern especially in Southeast Asia
where per person availability of arable land is decreasing by 0.1 ha per person.
Increasing demand for pesticide-free food and organic products is another key
driver in the biopesticide market growth that encourages sustainable farming prac-
tices. The market of biopesticide however is mostly constrained by the farmer’s
poor awareness on application of microbial agents and availability of the products.
Additionally, low shelf life of formulated products forces farmers to frequent the
application of biopesticides, thus resulting in increased costs.
Most of the biopesticide products undergo various abiotic stresses during produc-
tion, and application stages resulted in their inconsistence field performance and
poor shelf life. Likewise, the microorganisms released in the fields through formu-
lated product must be acclimatized to local environment. All these performance
issues depend upon the selection of superior strains of bioagents. Thus, the purity
1 Synthesis of Policy Support, Quality Control, and Regulatory Management… 5
and viability of their culture must be protected and ensured irrespective of labora-
tory practices to minimize the farmer’s risk. Guarantee on the performance ability
of parent culture in any given environment must be assured for consumer confi-
dence in the product.
Microbial consortium-based products also possess multiple benefits (Jain et al.
2013). QA for such microbial consortium product needs careful inspection in terms
of cultural methods, mass production, and microbial composition in the product.
Addition of various survival factors for suppression of microbe’s metabolism is
an important aspect for improving shelf life of the formulated products. Information
regarding the microbe survival and multiplication in favorable environments must
be mentioned. A quality product with maximum satisfaction of the consumers has
to be the watchword of any production system. Quality must, therefore, be ensured
at all costs (Parmar 2010).
any. It also requires information regarding the source of origin, habitat and morpho-
logical description of microorganism used in the formulation, test methods, qualita-
tive analysis, and shelf-life claims.
Chemical composition of the formulated product, cfu/g of the product, percent
content of the biocontrol organism in the formulation, and the nature of biomass are
required. Physical characteristics such as percentage of carrier/filler, wetting/dis-
pending agent, stabilizers/emulsifiers, contaminants/impurities, and moisture con-
tent of the product are also required. Information related to the process of
manufacturing and production including type of fermentation, biological end prod-
ucts, and methods of mass multiplication is necessary for registration.
to even provide them more data. It is also possible that regulating authority using the
registration model of chemical pesticide requests inappropriate information. Some
regulatory authorities in the UK, for example, have acknowledged that basing the
regulatory system for biopesticides on a chemical pesticide model has been a barrier
to biopesticide commercialization (ACP 2004). A key question is whether the regu-
lator, having recognized a problem, is able to do something about it. Social science
theory indicates that government regulators and other bureaucratic organizations are
vulnerable to “goal displacement,” during which they turn their focus away from
achieving outcomes and instead concentrate more on internal processes. This can
lead to systemic problems and stand in the way of introducing innovations into the
regulatory system. This is not to say that regulatory innovation is not possible, and
where there is sound evidence that a particular group of biopesticides presents mini-
mal risk, the regulators have modified the data requirements. For example, the
OECD regards semiochemicals used for arthropod control as presenting minimal
hazard, with straight chain lepidopteran pheromones that form the majority of
semiochemical-based biopesticides being thought sufficiently safe as to justify
“substantial reductions in health and environmental data requirements” (OECD
2001).
Most of the research in the field of agriculture in Asia has been mainly fundamental.
Green revolution has resulted in onset of applied and advance research in various
agricultural disciplines and other applied areas where scientists realized the impor-
tance of effective research to reach to the farmers and the ease with which it could
enhance the economic status of the farmers. In Indian subcontinent, fertilization and
plant disease management practices were mostly conventional such as the use of
cow dung, leftovers of the previous crops, and rouging and burning of diseased
plants. But it was only after the emergence of chemical fungicides on global com-
mercial scale that the production of crops escalated and changed the agricultural
practice scenario forever. Now same is the situation with natural antagonistic organ-
isms. The scientists from all over the world have screened, selected, and tried the
most effective isolates against wide range of plant pathogens.
In this sequence the next rational step is to commercialize these biocontrol prod-
ucts. Commercialization of these eco-friendly biocontrol agents is necessary to
lower the harmful effects caused by injudicious used of chemical pesticides. In
many cases developing countries are continuously applying various harmful chemi-
cals which have been banned in other developed countries. In such scenario it is
well anticipated that development, production, and successful commercialization of
biocontrol agents will effectively reduce and eliminate the use of hazardous chemi-
cals and ultimately reduce their deteriorating impact on human and environment
health. In developing countries, it is very tough to convince the large-scale farmers
to incorporate biocontrol products in their disease management practices as their
illiteracy and overdependency on chemical pesticides is a generation-old practice.
8 C. Keswani et al.
In India where major portion are farmers, it is roughly estimated that only 1.5–2.0
% of plant protection market is occupied by biocontrol products (Annon 2011).
It has been assumed that research related to the biocontrol is very fascinating and
successful at the initial scale and showed very good results in vivo but failed to
replicate its nature in large-scale or infield condition. Additionally there are very
few products which have been commercialized. Globally, about 80 biocontrol prod-
ucts have been commercialized. These products are constrained in their application
as they can manage limited number of plant pathogens and have not been tested on
various major crops before being commercialized. Furthermore, relatively very lit-
tle investment has been made in the production of commercial formulation of bio-
control agents probably due to the cost of production, testing, registration, and
marketing (Singh et al. 2002, 2004). The various efforts made in commercialization
of biocontrol agents may be classified in three groups based on level of difficulty
and potential of repeat sales. The first group includes direct application of the bio-
control agents precisely on the infection area when and where needed. This process
is carried to prevent the pathogen movement by applying large inoculums of bio-
control agents as in case of seed treatment with antagonistic microorganism for
protection of seeds against soilborne pathogens. Biocontrol agents commercialized
to date mostly come under this group except Gliocladium virens GL-21 which is
applied only in close vicinity of seed or plant roots in order to facilitate rapid
colonization.
Plant growth-promoting rhizobacteria (PGPR) constitutes the second category
and is applied at one place, e.g., on seed surface with the viewpoint that they will
colonize the plant roots and protect them from various plant pathogens. This group
exemplifies the strategy of augmentative application where the antagonists persist
with plant throughout the life of host and rapidly increase the population in the rhi-
zosphere (Cook 1993). This category presents a more challenging approach to bio-
control as the bioagents are subjected to greater competition under environmental
conditions.
The third class belongs to the biocontrol agents, which are applied on the infec-
tion area once or several times. Most of the successful examples of this category are
biocontrol agents of insects and perennial weeds. The reason behind the success of
this group of biocontrol agents is that they persist on the host for a longer period for
their survival and multiplication. However, despite many success stories of effective
control, their commercialization have been relatively slow, and therefore applica-
tion is mostly restricted to greenhouses.
Major reasons for failure of the biocontrol and problems associated with their
commercialization are as follows:
large-scale production technology is required for biocontrol agents that do not pro-
duce enough spores in liquid media. Unfortunately mass production technologies
based on solid substrates are not commonly available (Connick et al. 1990).
Selection of the cost-effective technology for the mass production viable and propa-
gules is a matter of concern. One of the major objectives in biopesticide production
is to maintain the viability of the active ingredient possibly for 2 years. The main
challenge faced by producers is maintaining the shelf life of microorganism during
the storage period. If the product carries less numbers of active ingredients due to
shorter shelf life, the overall performance of the formulation will be affected.
We have to find a way out of this quagmire to assume that our commercialized
biocontrol products have the effectiveness and safety as their chief traits. Only then
commercialization of biocontrol products can be assumed to be successful. For bio-
control to be more acceptable, the concept that the disease should be managed rather
than completely controlled is useful and has to be instilled in the mind of end users.
Besides, the market size, inconsistency, methods of production, formulation, and
distribution have made commercial companies reluctant to support the sustained
efforts in biocontrol research. Recent advances in genomics, transcriptomics,
proteomics, and metabolomics can guide to the development of next-generation
production with increased shelf life.
1.8 Conclusion
To shape the future of biocontrol of plant diseases at a global level, the big challenge
is how the modern technology can be translated from lab to land. Although, after so
many years of intensive research and despite all the success stories of biocontrol, it
is evident that the number of commercially available biocontrol products is far
lower than chemical counterparts. It can be stated that the commercialization of
biocontrol products is far behind due to the inappropriate policies and tedious
1 Synthesis of Policy Support, Quality Control, and Regulatory Management… 11
registration process worldwide. Thus, it is vital for the policy maker to facilitate the
registration process and at the same time be stringent enough to regulate the spuri-
ous products in the market. Moreover, biocontrol research will only be blue-sky
research if trust and awareness among the farmers are lacking. Thus, government,
industries, and academia should join hands in sensitizing the farmers to adopt eco-
friendly and sustainable farming practice and agri-inputs.
References
Advisory Committee on Pesticides (2004) Final report of the sub-group of the advisory committee
on pesticides on: alternatives to conventional pest control techniques in the UK: a scoping
study of the potential for their wider use. Advisory Committee on Pesticides, York. See http://
www.pesticides.gov.uk/uploadedfiles/Web_Assets/ACP/ACP_alternatives_web_subgrp_
report.pdf. Accessed 1 Apr 2016
Alam G (2000) A study of biopesticides and biofertilizers in Haryana, India, Gatekeeper series no.
93. IIED, London
Anonymous (2011) Fifty years of agrochemicals and India’s march towards food and nutritional
security. Dhanuka Agritech Limited, Gurgaon, Haryana, India, p 94
Arora NK, Khare E, Maheshwari DK (2010) Plant growth promoting rhizobacteria: constraints in
bioformulation, commercialization, and future strategies. In: Maheshwari DK (ed) Plant
growth and health promoting bacteria. Springer, Berlin, pp 97–116
Cok RJ, Baker KF (1983) The nature and practice of biological control of plant pathogens.
American Phytopathology Society Press, St. Paul
Connick Jr WJ, Lewis JA Quimby Jr PC (1990) Formulation of biocontrol agents for use in plant
pathology. In UCLA symposia on molecular and cellular biology (USA)
Cook RJ (1993) Making greater use of microbial inoculants in agriculture. Ann Rev Phytopathol
31:53–80
http://www.mordorintelligence.com/industry-reports/asia-pacific-biopesticides-market-industry.
Accessed 21 Mar 2016
https://www.kenresearch.com/agriculture-and-animal-care/crop-protection/india-biopesticides-
market-research-report/669-104.html. Accessed 15 Apr 2016
Ignacimuthu S, Sen A (eds) (2001) Microbials in insect pest management. Science Publishers Inc.,
Enfield, p 174
Jain A, Singh A, Singh BN, Singh S, Upadhyay RS, Sarma BK, Singh HB (2013) Biotic stress man-
agement in agricultural crops using microbial consortium. In: Maheshwari DK (ed) Bacteria in
agrobiology: disease management, vol 5. Springer-Verlag, Berlin/Heidelberg, pp 427–448
Kennedy JS, Rabindra RJ, Sathiah N, Grzywacz D (1999) The role of standardisation and quality
control in the successful promotion of NPV insecticides. In: Ignacimuthu S (ed) Biopesticides
in insect pest management. Phoenix Publishing House, New Delhi, pp 170–174
Keswani C (2015) Proteomics studies of thermotolerant strain of Trichoderma spp. Ph.D. thesis,
Banaras Hindu University, Varanasi, India
Kulshrestha S (2004) The status of regulatory norms for biopesticides in India. In: Kaushik E (ed)
Biopesticides for sustainable agriculture: prospects and constraints. TERI Press, New Delhi, pp
67–72
Organisation for Economic Co-operation and Development (2001) Series on pesticides no. 12.
Guidance for registration requirements for pheromones and other semiochemicals used for
arthropod pest control. See http://www.oecd.org/dataoecd/44/31/33650707.pdf. Accessed 28
Mar 2016
Organisation for Economic Co-operation and Development (2003) Series on pesticides no. 18.
Guidance for registration requirements for microbial pesticides. See http://www.oecd.org/
dataoecd/4/23/28888446.pdf. Accessed 15 Apr 2016
12 C. Keswani et al.
Abstract
Plants have been inoculated with plant growth-promoting microorganisms to
enhance crop yield and performance over four decades. The two central aspects
for success of inoculation are the effectiveness of the bacterial strain and the
application technology. This chapter discusses characteristics of ideal carriers
for bacterial inoculants [plant growth-promoting bacteria (PGPB) and plant
growth-promoting rhizobacteria (PGPR)] and focuses on superior formulations
for the future, mainly polymeric and encapsulated formulations and new emerg-
ing ideas in the field of inoculation. Future research avenues are highlighted.
Keywords
Inoculants • Plant growth-promoting bacteria • PGPR • PGPB • Rhizobia
2.1 Introduction
peat, other materials have been proposed, including bagasse, animal manure, alfalfa
powder, coir dust (coco peat), biochar, perlite, rock phosphate, charcoal, and a range
of coals, lignite, talc, and inorganic soil fractions, mainly clays (Bashan 1998;
Stephens and Rask 2000; Bashan et al. 2014).
The five categories of desirable general characteristics for a good formulation are
listed here (Bashan et al. 2014):
Naturally, no single inoculant can have all these capacities at top-end quality.
However, a good inoculant should have as many of these characteristics at a reason-
ably good quality. Synthesizing “super-inoculants” or finding a polymer used in
more expensive industries, such as pharmaceuticals, nanotechnology, or cosmetics
to accommodate all the desired features is theoretically feasible (John et al. 2011;
Schoebitz et al. 2013b). So far, these formulations are produced in laboratories and
have not employed in commercial products. So far, no effort to synthesize a carrier
with defined superior characteristics for agricultural and environmental purposes
has been reported, presumably because of high cost.
20 Y. Bashan et al.
The old method of inoculating seeds and plants with bacterial culture suspension, as
done since the pioneering times of plant inoculation four decades ago, still prevails
today. It is a common practice among researchers because it is the least laborious
and most described method in the literature.
Without doubt, peat is the main carrier for rhizobia in North and South America,
Europe, and Australia and the main ingredient of inoculants that is sold in large
volumes. It is also suitable for most other PGPB/PGPR. Yet, peat is rarely available
and expensive in most of Asia and Africa. All other carriers proposed for PGPB/
PGPR–rhizobia are compared with the standard peat carrier. Performance of peat-
based inoculants and its shortcomings were intensively and continuously reviewed,
as well as details of production of variants. Currently, technical details of the basic
peat-based inoculant such as grain size, pH, optimal moisture, other amendments,
quality of inoculants, quality control standards, and occupational health and safety
are common knowledge (Stephens and Rask 2000; Catroux et al. 2001; Date 2001;
Deaker et al. 2004, 2011; Xavier et al. 2004).
2 Superior Polymeric Formulations and Emerging Innovative Products of Bacterial… 21
Inorganic inoculants can be made from natural inorganic materials, natural poly-
mers, or synthetic materials. Apart from polymeric inoculants, inorganic inoculants
are the oldest version of inoculants (Bashan 1998), and a few such as clay and bio-
char (300 °C burnt carbon) are experimentally used for reforestation in semiarid
zones and as a potential carrier for PGPB/PGPR and carrier for processing polluted
water with bacteria (Hale et al. 2014; Schoebitz et al. 2014; Stelting et al. 2014).
While most of these inoculants are used on a small scale for crop production, all
polymeric inoculants, as far as we know, are experimental. Yet, because they open a
new approach to formulation with endless industrial variations, these inoculants are
described and discussed in detail in this chapter.
Table 2.1 A sample of polymeric formulations used for producing inoculants of plant growth-
promoting bacteria for plants from 1998 to 2015
Additives or Plant species
Formulation treatment Microorganisms used or substrate References
Alginate None Azospirillum Tomato Bashan et al.
brasilense; (2002), Yabur
Azospirillum combined et al. (2007), and
with Methylobacterium Joe et al. (2014)
sp.
Alginate None Azospirillum Several desert Bashan et al.
brasilense trees (2009a, b, 2012)
Alginate Organic olive Azospirillum Pinus Mengual et al.
residue brasilense and Pantoea halepensis (2014)
dispersa
Alginate None Azospirillum Wheat; maize Bashan and
brasilense; A. Gonzalez (1999),
lipoferum; Bacilio et al.
Pseudomonas (2004), El-Komy
fluorescens; Bacillus (2005), Bashan
megaterium; Serratia et al. (2006),
marcescens; Schoebitz et al.
Enterobacter sp. (2013a, c), Ben
Farhat et al.
(2014), and
El-Gamal et al.
(2015)
Alginate None Agaricus bisporus Agaricus Friel and
(champignon) bisporus McLoughlin
(1999)
Alginate None Chlorella vulgaris, C. Tertiary Gonzalez and
sorokiniana together wastewater Bashan (2000),
with Azospirillum treatment Gonzalez-Bashan
brasilense, Bacillus et al. (2000),
pumilus, or Lebsky et al.
Phyllobacterium (2001), de-Bashan
myrsinacearum; et al. (2002, 2004),
Synechococcus (2005), (2008),
elongatus together de-Bashan and
with A. brasilense Bashan (2004,
2008), Hernandez
et al. (2009),
Perez-Garcia et al.
(2010),
Covarrubias et al.
(2012), Cruz et al.
(2013), and
Ruiz-Güereca and
Sánchez-Saavedra
(2016)
(continued)
2 Superior Polymeric Formulations and Emerging Innovative Products of Bacterial… 23
(1) Nontoxic in nature and free of harmful preservatives that affect bacteria within
the inoculant and later the inoculated plants
(2) Slowly degradable in the soil by soil microorganisms, thereby gradually releas-
ing the bacteria in needed quantities, usually at the time of seed germination
and emergence of seedlings, leaving no secondary pollution
(3) Provide significant physical protection for the bacteria from soil competitors
and many environmental stresses (Zohar-Perez et al. 2003; Covarrubias et al.
2012; Cruz et al. 2013)
(4) Hold sufficient moisture for survival of the bacteria
(5) Dispersible in water to allow movement of the bacteria from the polymer to the
plants, when necessary
2 Superior Polymeric Formulations and Emerging Innovative Products of Bacterial… 25
More beneficial features are that these polymeric inoculants (1) can be stored
dried at various ambient temperatures for extended time without refrigeration, (2)
offer a consistent batch quality for manufacturing and at the same time a better
environment for the bacteria, (3) can be industrially manipulated to fit the needs of
specific PGPB/PGPR, and (4) can be further amended with nutrients to improve
short-term survival of the bacteria upon inoculation, which enhances the success of
the inoculation process. This last feature is especially essential for associative PGPB
competing in the rhizosphere with native microbes.
The major drawback of polymeric inoculants is that the raw materials for most
polymers are relatively expensive, compared to peat, soil, and organic inoculants,
and require further expensive handling by the industry at costs similar to those in the
fermentation industry. As a direct result, so far no commercial polymeric inoculants
are currently available. Yet, these inoculants may represent the future technology. A
positive aspect of polymeric inoculants is that they are still the domain of research
laboratories and does not have proprietary protection of private companies; hence,
relatively more information is available in the scientific literature.
the beads occurs when the polymer is slowly degraded by the native soil microor-
ganisms and in the process releasing the PGPB–rhizobia to the soil where plants
that need inoculation are growing.
Through 2015, alginate derivatives are the preferred polymer for immobilization of
microorganisms. The alginate formulations are used for various purposes: applica-
tion of biocontrol agents and mycoherbicides, immobilization of cellular organisms
and enzymes, increase of stability of recombinant plasmids in host cells, bacterial
chemotaxis research, and primary polymer in formulations of drugs. Alginate is a
naturally occurring polymer available worldwide, mainly from different marine
macroalgae in large and sustainable quantities (Draget et al. 2002; Yabur et al.
2007), as well as from several bacteria (Sabra et al. 2001; Trujillo-Roldan et al.
2003). The preparation of beads containing PGPB/PGPR is fairly easy and straight-
forward and involves a multistep procedure at room temperature with minimal
amounts of additional chemicals and equipment; thus, it is very popular in research.
Procedures to formulate PGPB/PGPR in alginate are available (Bashan et al. 2002;
de-Bashan et al. 2004, 2015; de-Bashan and Bashan 2010; Bashan and de-Bashan
2015). Occasionally, biomass of the immobilized PGPB/PGPR strain is low for the
specific application; thus, a second multiplication of the immobilized PGPB/PGPR
in the already-formed beads is necessary. This step is fairly simple (Bashan 1986).
The advantages of alginate formulations are their nontoxic nature, biodegradability,
availability at low costs (in 2015, US$ 2 per kg: Chinese product), slow release of
the entrapped microorganisms into the soil that can be designed and controlled by
variation in the polymeric structure, and approval for human use by the US Federal
Food and Drug Administration (Bashan et al. 2002; Zohar-Perez et al. 2002).
encapsulated bacteria was demonstrated (Young et al. 2006). The success of this
particular immobilization technique was attributed to the dual benefits of humic
acid for the microbes and plant and the chemical properties of the humic acid,
including easy mixing with alginate without interfering in the formation of the algi-
nate gel beads. One benefit of humic acid in the structure of alginate bead is that it
serves as a carbon source for encapsulated Pseudomonas putida and B. subtilis,
which promotes survival of the encapsulated microorganisms during storage (Rekha
et al. 2007). Immobilization of an encapsulated phosphate-solubilizing bacteria
Serratia sp. was superior to a non-immobilized inoculant of the same strain on
wheat plants (Schoebitz et al. 2013c). The effectiveness of free and encapsulated
PGPR Raoultella planticola in promoting cotton growth under saline stress demon-
strates that encapsulated inoculants have more positive effects on cotton seedlings
than free cells (Wu et al. 2014). The efficacy of wet macrobeads of an alginate/
starch mix was demonstrated when several Streptomyces spp. were inoculated onto
a perennial shrub under revegetation conditions in the field in semiarid environment
(Mengual et al. 2016).
An extended survival time in dry alginate bead was demonstrated when two
PGPB, Azospirillum brasilense and Pseudomonas fluorescens, immobilized in two
types of alginate bead inoculants recovered after being dried and stored at ambient
temperature for 14 years. Although the populations in the beads had decreased,
significant numbers survived (105–106 CFU g−1 beads). After inoculating wheat
plants in a growth chamber, both species colonized and enhanced growth equal to
those that had not been stored (Bashan and Gonzalez 1999). Vassilev et al. (2001)
demonstrated that tomato plants inoculated with an AM fungus (Glomus desertic-
ola) and phosphate-solubilizing yeast (Yarrowia lipolytica) that were co-immobilized
in alginate are a useful technique for establishing plants in nutrient-deficient soils.
Several plant growth parameters were equal in treatments whether the tomatoes
were inoculated with the free AM fungus or alginate-entrapped fungus, but inocula-
tion with the fungus and the yeast produced better results.
White mushroom (Agaricus bisporus) cultivation was improved with wet algi-
nate inoculant applied to spawn, providing a shorter adaptation (lag) period and
higher growth rate in pasteurized compost, compared to liquid spawn and conven-
tional commercial grain spawn. Superiority of this delivery system is attributed to
high biomass loading capacity of the beads, protection of the mycelia in the bead
microenvironment, and even spatial distribution of beads in the compost (Friel and
McLoughlin 1999).
Protection against high temperatures can be provided by alginates. Alginate for-
mulation supported high populations and survival of the phosphate-solubilizing
bacteria Pseudomonas striata and Bacillus polymyxa at storage temperatures of 40
°C (Viveganandan and Jauhri 2000). Several macrobead alginate formulations of B.
subtilis and Pseudomonas corrugata were found superior over liquid inoculants or
charcoal-based inoculants for improving maize growth under low temperatures in
the Indian Himalayas (Trivedi et al. 2005). Survival of the rhizobacteria Raoultella
terrigena and A. brasilense during encapsulation can be improved by incorporating
28 Y. Bashan et al.
Fig. 2.1 Immobilization of microorganisms in alginate inoculants. (a, b) Wet macrobead inocu-
lant and (c, d) dry microbead inoculant. (a) Inoculants of (from left) Azospirillum brasilense,
Chlorella vulgaris , C. vulgaris (in alginate obtained from Sargassum ), C. sorokiniana .
( b ) Macrobeads containing an association between A. brasilense and C. sorokiniana. (c, d) Dry
alginate microbeads of A. brasilense (arrow)
microorganisms and ability to colonize sugar beet were measured after 1 year.
Although dried alginate beads reduced the quantity of viable bacteria, the micro-
beads provided a satisfactory level of root colonization and protection against two
fungal pathogens Pythium ultimum and Rhizoctonia solani. The capability of the
immobilized bacteria to produce the antifungal metabolite 2,4-diacetylphloroglu-
cinol was not affected after storage for 12 months (Russo et al. 2001). (4) Alginate
beads coated with peanut oil, containing the entomopathogenic fungus Beauveria
bassiana, were used against the red fire ant Solenopsis invicta. Broadcast applications
and individual mound treatments with this inoculant reduced activity of the ant
populations (Bextine and Thorvilson 2002).
Ironically, although commercial alginate preparations are not yet available for
PGPB/PGPR, several other polymers that are used in industrial and environmental
microbiology may be considered as substitutes when the microorganisms fail to
adapt to alginate preparations. Even though all materials are still experimental, it is
worth mentioning them to encourage further research on these potential carriers.
Earlier cases are listed in Bashan (1998) and Bashan et al. (2014).
32 Y. Bashan et al.
Formulation using chitosan as a carrier for several PGPR was mixed with soilless
growth medium for successful biocontrol against cucumber mosaic virus in tomato
(Murphy et al. 2003). Five PGPB were prepared into several formulations of poly-
mers composed of carboxymethyl cellulose–starch. These formulations maintained
the bacterial strains in high numbers during 60–120 days of storage. The formula-
tions were effective in promoting the growth of sugarcane cuttings (da Silva et al.
2012). Rhizobia that were formulated with the same inoculants kept their popula-
tions inside the inoculants during 165 days of storage and were still capable of
promoting growth of cowpeas (Júnior et al. 2009). P. fluorescens was formulated
with three polymers: commercial film-forming “methacrylic acid copolymer”
(Evonik Industries, Darmstadt, Germany), ethyl cellulose, and a modified starch.
The best performer was the commercial polymer, where bacteria survived for 1
year. Survival of bacteria was related to the microspheres’ residual moisture; the
highest survival of bacteria occurred when the residual moisture was ~25 %. This
inoculant was not tested on plants (Amiet Charpentier et al. 1998). With limited
information on these carriers, it is impossible to predict their future as bacterial
inoculants.
(Zohar-Perez et al. 2002). Dry seed coats of various formulations of alginate, either
alone or with bran and chitin additives, but not containing a PGPB, did not affect the
viability or percent of germination of seeds of wheat, basil, cabbage, and radish
(Sarrocco et al. 2004), but Na alginate supplied as an additive to liquid formulation
without polymerization significantly extended the shelf life of Sinorhizobium meli-
loti (Rouissi et al. 2014). Dry beads (by hot temperature) made of alginate–benton-
ite were very efficient in slow release of the PGPR Raoultella planticola, but this
formulation was not tested on plants (He et al. 2015). Other combinations of algi-
nate with pea protein of B. subtilis protect the cells in the soil; the PGPR was capa-
ble of colonizing two model plants (Gagné-Bourque et al. 2015).
The inherent limitations of live cell-based inoculants are still a strong bottleneck in
widespread application of bioproducts and continue to delay commercialization and
use of PGPB/PGPR on a larger scale. Evidently, there is growing interest to produce
and market bioproducts that are not affected by soil and weather conditions and are
capable of consistent performance. “Inoculants” made of active fragments and
metabolites of PGPB/PGPR in the absence of living cells are emerging. Many elici-
tors of plant defenses and secondary metabolites of PGPB/PGPR are documented.
These microbial products, if produced commercially, have potential as “inoculants”
2 Superior Polymeric Formulations and Emerging Innovative Products of Bacterial… 35
for reducing agrochemical use (Compant et al. 2013). There are growing numbers
of commercial, cell-free microbial products based on metabolites, which can be
integrated with growers’ standard practices and are generally not affected by soil or
weather conditions. These products are based on the assumption that all beneficial
microbes act on plants through bioactive molecules (Mabood et al. 2008; Zhuang
et al. 2013). Microorganisms have been the source of some highly active biomole-
cules of significant commercial importance, and, due to their chemical complexity,
biological fermentation processes remain the best means of production (Warrior
2000).
Several natural products from microbes, such as harpin proteins, have found
commercial applications in improving crop health (Copping and Duke 2007).
Lipochitooligosaccharides are nodulation factors secreted by rhizobia that induce
nodules on the roots of legumes (Truchet et al. 1991). Although lipochitooligosac-
charides are important signal molecules for plant–symbiont interactions, they can
directly impact general nonlegume plant growth and development (Prithiviraj et al.
2003; Tanaka et al. 2015). Potential commercial lipochitooligosaccharide products
for seed and foliar applications in legumes and nonlegumes, such as corn, are cur-
rently available in North America. Concentrated metabolites containing lipochi-
tooligosaccharides, exopolysaccharides, and hormones produced from rhizobia
have a shelf life of 24 months. This product improved yields of soybean and corn
and is currently registered for use in Brazil (Marks et al. 2013).
Lipochitooligosaccharides are known to improve plant symbiosis with vesicular–
arbuscular mycorrhizal fungi (Xie et al. 1995). More practical uses of this chemical
are expected in coming years.
Even though the prevailing opinion in many new companies using PGPB/PGPR is
that formulation is a side issue and quick to develop once the right PGPB/PGPR is
selected, realistically, developing new and effective formulations for inoculation of
PGPB/PGPR and rhizobia is a very slow process and consumes resources. Creation
of new formulations is a challenge in practical microbiology, and shortcuts usually
lead to failure of the inoculant in the field. Improvements in formulations are key to
the development of enhanced high-end inoculants. Literature surveys show that the
identification of new isolates having PGPB/PGPR capacities is often not difficult,
and many are identified annually. Yet, development of most PGPB/PGPR strains
stops there, without ever reaching the formulation stage.
While several thousand articles in the literature describe inoculation of plants
with PGPB/PGPR, only a handful focused on delivery systems. Mostly, formula-
tions and application techniques, if described at all, are hidden within the Materials
and Methods section of publications on other topics. Grant applications in the devel-
oped countries regarding formulations are rarely successful. This topic, which spans
fundamental microbiology and industrial technologies done simultaneously in
36 Y. Bashan et al.
research facilities and commercial agricultural fields, is mostly neglected. For the
future of plant inoculation, this topic cannot be ignored.
The following research topics should be top priorities of research on new or
improved delivery systems for PGPB/PGPR and rhizobia, excluding peat formula-
tions, which have already reached their peak:
volume of the inoculant is small and growth conditions are easier to control.
Hence, more research and transfer of technologies should be targeted to nursery-
grown plants. For example, seedling roots dipped in microbial inoculants with-
out formulation are effective and easy for microbial inoculation of transplanted
rice (Choudhury and Kennedy 2004). This treatment needs to be modified and
applied to other transplanted crops.
• A number of PGPR produce lipopeptides with biosurfactant activity (Raaijmakers
et al. 2010) that directly inhibit pathogens and provide systemic-induced resis-
tance in crops (Ongena et al. 2007; Ongena and Jacques 2008). There are pros-
pects of developing cell-free lipopeptide product biofungicides where part of the
efficiency of the product is due to lipopeptides supplied along with live cells.
More exploration of microbial metabolites is required to obtain more benefits
from such PGPB/PGPR.
• Microbiome studies produced so far only fundamental knowledge regarding the
complexity of the microbial world. Beneficial microbial strains capable of ben-
eficially changing soil microbial community structure have been isolated (de-
Bashan et al. 2010a, b; Kang et al. 2013; Lopez et al. 2013). However, this
fundamental information is yet to serve as a basis for the next generation of
inoculants (Berg et al. 2013, 2014; Massart et al. 2015).
• Information regarding formulation and application techniques should be pre-
cisely described in the literature. Specifically, each new manuscript should con-
tain the precise formulation of the inoculants in quantitative details, including all
non-active materials and active supplements. In cases where the inoculant is pro-
prietary or is intellectual property, the serial registration number of the patent or
the intellectual property and the country of registration should be disclosed. All
bacterial species and variants, not just the genus, should be disclosed and be
available from microbial collections that are available to the public. When strains
are intellectual property of an organization, the name of the organization holding
the rights should be disclosed. If specific sequence(s) of a strain is publically
known, this should be disclosed as the definitive identification of the strain. All
microbial strains in a consortium must be listed. Inoculation techniques should
be described in detail sufficient to allow repetition of the experiment (Bashan
et al. 2016).
Acknowledgments We thank Ira Fogel at CIBNOR, Mexico, for editorial and English improve-
ments. This review was supported by The Bashan Institute of Science, USA (contribution
2016–008).
2 Superior Polymeric Formulations and Emerging Innovative Products of Bacterial… 39
Dedication This chapter is dedicated to the memory of the Israeli soil microbiologist Prof. Yigal
Henis (1926–2010) of the faculty of Agriculture, the Hebrew University of Jerusalem in Rehovot,
Israel, one of the pioneers of studies of inoculants in Israel.
References
Adesemoye AO, Kloepper JW (2009) Plant–microbes interactions in enhanced fertilizer-use effi-
ciency. Appl Microbiol Biotechnol 85:1–12
Albareda M, Rodriguez-Navarro DN, Camacho M, Temprano FJ (2008) Alternatives to peat as a
carrier for rhizobia inoculant: solid and liquid formulations. Soil Biol Biochem
40:2771–2779
Amer GA, Utkhede RS (2000) Developments of formulations of biological agents for management
of root rot of lettuce and cucumber. Can J Microbiol 46:809–816
Amiet-Charpentier C, Gadille P, Digat B, Benoit JP (1998) Microencapsulation of rhizobacteria by
spray-drying: formulation and survival studies. J Microencapsul 15:639–659
Andrews M, James EK, Cummings SP, Zavalin AA, Vinogradova LV, McKenzie BA (2003) Use of
nitrogen fixing bacteria inoculants as a substitute for nitrogen fertilizer for dryland
Graminaceous crops: progress made, mechanisms of action and future potential. Symbiosis
35:209–229
Atieno M, Herrmann L, Okalebo R, Lesueur D (2012) Efficiency of different formulations of
Bradyrhizobium japonicum and effect of co-inoculation of Bacillus subtilis with two different
strains of Bradyrhizobium japonicum. World J Microbiol Biotechnol 28:2541–2550
Bacilio M, Rodriguez H, Moreno M, Hernandez J-P, Bashan Y (2004) Mitigation of salt stress in
wheat seedlings by a gfp-tagged Azospirillum lipoferum. Biol Fertil Soils 40:188–193
Bashan Y (1986) Alginate beads as synthetic inoculant carriers for the slow release of bacteria that
affect plant growth. Appl Environ Microbiol 51:1089–1098
Bashan Y (1998) Inoculants of plant growth-promoting bacteria for use in agriculture. Biotechnol
Adv 16:729–770
Bashan Y, de-Bashan LE (2010) How the plant growth-promoting bacterium Azospirillum pro-
motes plant growth – a critical assessment. Adv Agron 108:77–136
Bashan Y, de-Bashan LE (2015) Inoculants for Azospirillum. In: Cassán FD, Okon Y, Creus CM
(eds) Handbook for Azospirillum. Technical issues and protocols. Springer International
Publishing, Cham, pp 469–485
Bashan Y, Gonzalez LE (1999) Long-term survival of the plant-growth-promoting bacteria
Azospirillum brasilense and Pseudomonas fluorescens in dry alginate inoculant. Appl Microbiol
Biotechnol 51:262–266
Bashan Y, Holguin G (1994) Root-to-root travel of the beneficial bacterium Azospirillum brasi-
lense. Appl Environ Microbiol 60:2120–2131
Bashan Y, Levanony H (1987) Horizontal and vertical movement of Azospirillum brasilense Cd in
the soil and along the rhizosphere of wheat and weeds in controlled and field environments.
J Gen Microbiol 133:3473–3480
Bashan Y, Hernandez JP, Leyva LA, Bacilio M (2002) Alginate microbeads as inoculant carrier for
plant growth-promoting bacteria. Biol Fertil Soils 35:359–368
Bashan Y, Holguin G, de-Bashan LE (2004) Azospirillum-plant relationships: physiological,
molecular, agricultural, and environmental advances (1997–2003). Can J Microbiol
50:521–577
Bashan Y, Bustillos JJ, Leyva LA, Hernandez JP, Bacilio M (2006) Increase in auxiliary photopro-
tective photosynthetic pigments in wheat seedlings induced by Azospirillum brasilense. Biol
Fertil Soils 42:279–285
Bashan Y, Salazar B, Puente ME (2009a) Responses of native legume desert trees used for refores-
tation in the Sonoran Desert to plant growth-promoting microorganisms in screen house. Biol
Fertil Soils 45:655–662
40 Y. Bashan et al.
Bashan Y, Salazar B, Puente ME, Bacilio M, Linderman RG (2009b) Enhanced establishment and
growth of giant cardon cactus in an eroded field in the Sonoran Desert using native legume
trees as nurse plants aided by plant growth-promoting microorganisms and compost. Biol Fertil
Soils 45:585–594
Bashan Y, Salazar B, Moreno M, Lopez BR, Linderman RG (2012) Reforestation of eroded desert
soil with native trees: effects of inoculation with plant growth-promoting microorganisms, lim-
ited amounts of compost and water and plant density. J Environ Manage 102:26–36
Bashan Y, de-Bashan LE, Prabhu SR, Hernandez J-P (2014) Advances in plant growth-promoting
bacterial inoculant technology: formulations and practical perspectives (1998–2013). Plant
Soil 378:1–33
Bashan Y, Kloepper JW, de-Bashan LE, Nannipieri P (2016) A need for disclosure of the identity
of microorganisms, constituents, and application methods when reporting tests with microbe-
based or pesticide-based products. Biol Fertil Soils 52(3):283–284
Ben Farhat M, Taktek S, Chouayekh H (2014) Encapsulation in alginate enhanced the plant growth
promoting activities of two phosphate solubilizing bacteria isolated from the phosphate mine
of Gafsa. Net J Agric Sci 2:131–139
Ben Rebah F, Prevost D, Yezza A, Tyagi RD (2007) Agro-industrial waste material and wastewater
sludge for rhizobial inoculant production: a review. Bioresour Technol 98:3535–3546
Berg G (2009) Plant–microbe interactions promoting plant growth and health: perspectives for
controlled use of microorganisms in agriculture. Appl Microbiol Biotechnol 84:11–18
Berg G, Zachow C, Müller H, Philipps J, Tilcher R (2013) Next-generation bio-products sowing
the seeds of success for sustainable agriculture. Agronomy 3:648–656
Berg G, Grube M, Schloter M, Smalla K (2014) Unraveling the plant microbiome: looking back
and future perspectives. Front Microbiol 5:148. doi:10.3389/fmicb.2014.00148
Bextine BR, Thorvilson HG (2002) Field applications of bait-formulated Beauveria bassiana algi-
nate pellets for biological control of the red imported fire ant (Hymenoptera: Formicidae).
Environ Entomol 31:746–752
Brockwell J (1977) Application of legume seed inoculants. In: Hardy RWF, Gibson AH (eds) A
treatise on dinitrogen fixation, vol 4, Agronomy and ecology. Wiley, New York, pp 277–309
Calvo P, Nelson L, Kloepper JW (2014) Agricultural uses of plant biostimulants. Plant Soil
383:3–41
Campos DC, Acevedo F, Morales E, Aravena J, Amiard V, Jorquera MA, Inostroza NG, Rubilar M
(2014) Microencapsulation by spray drying of nitrogen-fixing bacteria associated with lupin
nodules. World J Microbiol Biotechnol 30:2371–2378
Catroux G, Hartmann A, Revellin C (2001) Trends in rhizobial inoculant production and use. Plant
Soil 230:21–30
Chang SJ, Lee CH, Hsu CY, Wang YJ (2002) Biocompatible microcapsules with enhanced
mechanical strength. J Biomed Mater Res 59:118–126
Choudhury ATMA, Kennedy IR (2004) Prospects and potentials for systems of biological nitrogen
fixation in sustainable rice production. Biol Fertil Soils 39:219–227
Compant S, Brader G, Muzammil S, Sessitsch A, Lebrihi A, Mathieu F (2013) Use of beneficial
bacteria and their secondary metabolites to control grapevine pathogen diseases. BioControl
58:435–455
Cong PT, Dung TD, Hien TM, Hien NT, Choudhury ATMA, Kecskés KL, Kennedy IR (2009)
Inoculant plant growth-promoting microorganisms enhance utilisation of urea-N and grain
yield of paddy rice in southern Vietnam. Eur J Soil Biol 45:52–61
Copping LG, Duke SO (2007) Natural products that have been used commercially as crop protec-
tion agents. Pest Manage Sci 63:524–554
Cortés-Patiño S, Bonilla RR (2015) Polymers selection for a liquid inoculant of Azospirillum
brasilense based on the Arrhenius thermodynamic model. Afr J Biotechnol 14:2547–2553
Covarrubias SA, de-Bashan LE, Moreno M, Bashan Y (2012) Alginate beads provide a beneficial
physical barrier against native microorganisms in wastewater treated with immobilized bacteria
and microalgae. Appl Microbiol Biotechnol 93:2669–2680
2 Superior Polymeric Formulations and Emerging Innovative Products of Bacterial… 41
El-Komy HMA (2005) Coimmobilization of Azospirillum lipoferum and Bacillus megaterium for
successful phosphorus and nitrogen nutrition of wheat plants. Food Technol Biotech
43:19–27
Escalante FME, Cortés-Jiménez D, Tapia-Reyes G, Suárez R (2015) Immobilized microalgae and
bacteria improve salt tolerance of tomato seedlings grown hydroponically. J Appl Phycol
27:1923–1933
Forestier S, Alvarado G, Badjel SB, Lesueur D (2001) Effect of Rhizobium inoculation methodolo-
gies on nodulation and growth of Leucaena leucocephala. World J Microbiol Biotechnol
17:359–362
Fox JL (2015) Agricultural probiotics enter spotlight. Nat Biotechnol 33:122. doi:10.1038/
nbt0215-122
Friel LT, McLoughlin AJ (1999) Immobilisation as a strategy to increase the ecological compe-
tence of liquid cultures of Agaricus bisporus in pasteurised compost. FEMS Microbiol Ecol
30:39–46
Fuentes-Ramirez LE, Caballero-Mellado J (2005) Bacterial biofertilizers. In: Siddiqui ZA (ed)
PGPR: biocontrol and biofertilization. Springer, Dordrecht, pp 143–172
Gagné-Bourque F, Xu M, Dumont M-J, Jabaji S (2015) Pea protein alginate encapsulated Bacillus
subtilis B26, a plant biostimulant, provides controlled release and increased storage survival.
J Fertil Pestic 6:2
Gaumann A, Laudes M, Jacob B, Pommersheim R, Laue C, Vogt W, Schrezenmeir J (2000) Effect
of media composition on long-term in vitro stability of barium alginate and polyacrylic acid
multilayer microcapsules. Biomaterials 21:1911–1917
Gibbs BF, Kermasha S, Alli I, Mulligan CN (1999) Encapsulation in the food industry: a review.
Int J Food Sci Nutr 50:213–224
Gonzalez LE, Bashan Y (2000) Increased growth of the microalga Chlorella vulgaris when coim-
mobilized and cocultured in alginate beads with the plant growth-promoting bacterium
Azospirillum brasilense. Appl Environ Microbiol 66:1527–1531
Gonzalez-Bashan LE, Lebsky V, Hernandez JP, Bustillos JJ, Bashan Y (2000) Changes in the
metabolism of the microalgae Chlorella vulgaris when coimmobilized in alginate with the
nitrogen-fixing Phyllobacterium myrsinacearum. Can J Microbiol 46:653–659
Gurley HG, Zdor RE (2005) Differential rhizosphere establishment and cyanide production by
alginate-formulated weed-deleterious rhizobacteria. Curr Microbiol 50:167–171
Hale L, Luth M, Kenney R, Crowley D (2014) Evaluation of pinewood biochar as a carrier of
bacterial strain Enterobacter cloacae UW5 for soil inoculation. Appl Soil Ecol 84:192–199
Hartmann A, Bashan Y (2009) Ecology and application of Azospirillum and other plant growth-
promoting bacteria (PGPB) - special issue. Eur J Soil Biol 45:1–2
He Y, Wu Z, Tu L, Han Y, Zhang G, Li C (2015) Encapsulation and characterization of slow-release
microbial fertilizer from the composites of bentonite and alginate. Appl Clay Sci
109–110:68–75
Hernandez JP, de-Bashan LE, Bashan Y (2006) Starvation enhances phosphorus removal from
wastewater by the microalga Chlorella spp. co-immobilized with Azospirillum brasilense.
Enzym Microb Tech 38:190–198
Hernandez JP, de-Bashan LE, Rodriguez DJ, Rodriguez Y, Bashan Y (2009) Growth promotion of
the freshwater microalga Chlorella vulgaris by the nitrogen-fixing, plant growth-promoting
bacterium Bacillus pumilus from arid zone soils. Eur J Soil Biol 45:88–93
Herridge DF (2007) Inoculation technology for legumes. In: Dilworth MJ, James EK, Sprent JI,
Newton WE (eds) Nitrogen-fixing leguminous symbioses, vol 7. Springer, Dordrecht,
pp 77–115
Herrmann L, Lesueur D (2013) Challenges of formulation and quality of biofertilizers for success-
ful inoculation. Appl Microbiol Biotechnol 97:8859–8873
Joe MM, Saravanan VS, Islam MR, Sa T (2014) Development of alginate-based aggregate inocu-
lants of Methylobacterium sp. and Azospirillum brasilense tested under in vitro conditions to
promote plant growth. J Appl Microbiol 116:408–423
2 Superior Polymeric Formulations and Emerging Innovative Products of Bacterial… 43
John RP, Tyagi RD, Brar SK, Surampalli RY, Prevost D (2011) Bio-encapsulation of microbial
cells for targeted agricultural delivery. Crit Rev Biotechnol 31:211–226
Johri BN, Sharma A, Virdi JS (2003) Rhizobacterial diversity in India and its influence on soil and
plant health. Adv Biochem Eng Biotechnol 84:49–89
Júnior PIF, Rohr TG, de Oliveira PJ, Xavier GR, Rumjanek NG (2009) Polymers as carriers for
rhizobial inoculant formulations. Pesq Agropec Brasileira 44:1184–1190
Kang Y, Shen M, Wang H, Zhao Q (2013) A possible mechanism of action of plant growth-
promoting rhizobacteria (PGPR) strain Bacillus pumilus WP8 via regulation of soil bacterial
community structure. J Gen Appl Microbiol 59:267–277
Keswani C, Mishra S, Sarma BK, Singh SP, Singh HB (2014) Unraveling the efficient application
of secondary metabolites of various Trichoderma. Appl Microbiol Biotechnol 98:533–544
Kong HJ, Mooney DJ (2003) The effects of poly(ethyleneimine) (PEI) molecular weight on rein-
forcement of alginate hydrogels. Cell Transplant 12:779–785
Kourkoutas Y, Bekatorou A, Banat IM, Marchant R, Koutinas AA (2004) Immobilization technolo-
gies and support materials suitable in alcohol beverages production: a review. Food Microbiol
21:377–397
Lebsky VK, Gonzalez-Bashan LE, Bashan Y (2001) Ultrastructure of interaction in alginate beads
between the microalga Chlorella vulgaris with its natural associative bacterium Phyllobacterium
myrsinacearum and with the plant growth-promoting bacterium Azospirillum brasilense. Can
J Microbiol 47:1–8
Lodewyckx C, Vangronsveld J, Porteous F, Moore ERB, Taghavi S, Mezgeay M, van der Lelie D
(2002) Endophytic bacteria and their potential applications. Crit Rev Plant Sci 21:583–606
Lopez BR, Bashan Y, Trejo A, de-Bashan LE (2013) Amendment of degraded desert soil with
wastewater debris containing immobilized Chlorella sorokiniana and Azospirillum brasilense
significantly modifies soil bacterial community structure, diversity, and richness. Biol Fertil
Soils 49:1053–1063
Lucy M, Reed E, Glick BR (2004) Applications of free living plant growth-promoting rhizobacte-
ria. A Van Leeuw 86:1–25
Mabood F, Jung WJ, Smith DL (2008) Signals in the underground: microbial signaling and plant
productivity. In: Nautiyal CS, Dion PE (eds) Molecular mechanisms of plant and microbe
coexistence. Springer, Berlin/Heidelberg, pp 291–318
Marks BB, Megías M, Nogueira MA, Hungria M (2013) Biotechnological potential of rhizobial
metabolites to enhance the performance of Bradyrhizobium spp. and Azospirillum brasilense
inoculants with soybean and maize. AMB Express 3:21
Massart S, Martinez-Medina M, Jijakli MH (2015) Biological control in the microbiome era: chal-
lenges and opportunities. Biol Control 89:98–108
Mathre DE, Cook RJ, Callan NW (1999) From discovery to use. Traversing the world of commer-
cializing biocontrol agents for plant disease control. Plant Dis 83:972–983
Mathu S, Herrmann L, Pypers P, Matiru V, Mwirichia R, Lesueur D (2012) Potential of indigenous
bradyrhizobia versus commercial inoculants to improve cowpea (Vigna unguiculata L. walp.)
and green gram (Vigna radiata L. wilczek.) yields in Kenya. Soil Sci Plant Nutr 58:750–763
Mengual C, Roldán A, Caravaca F, Schoebitz M (2014) Advantages of inoculation with immobi-
lized rhizobacteria versus amendment with olive-mill waste in the afforestation of a semiarid
area with Pinus halepensis Mill. Ecol Eng 73:1–8
Mengual C, Schoebitz M, Caravaca F, Roldán A (2016) Assessment of the potential role of
Streptomyces strains in the revegetation of semiarid sites: the relative incidence of strain origin
and plantation site on plant performance and soil quality indicators. Biol Fertil Soils
52:53–64
Morsy EM (2015) Efficiency of free and encapsulated yeast strains as PGRs producers on faba
bean plants. Middle East J Appl Sci 5:534–542
Murakata T, Honma H, Nakazato S, Kuroda C, Sato S (2001) Control of particle size of calcium
alginate gel bead by application of electric field to interface between aqueous and organic
phases. J Chem Eng Jpn 34:299–305
44 Y. Bashan et al.
Murphy JF, Reddy MS, Ryu C-M, Kloepper JW, Li R (2003) Rhizobacteria-mediated growth
promotion of tomato leads to protection against Cucumber mosaic virus. Phytopathology
93:1301–1307
Nguyen HT, Deaker R, Kennedy IR, Roughley RJ (2003) The positive yield response of field-
grown rice to inoculation with multi-strain biofertilizer in the Hanoi area, Vietnam. Symbiosis
35:231–245
Nussinovitch A (2010) Polymer macro- and micro-gel beads: fundamentals and applications.
Springer, Berlin, p 303
Olson S (2015) An analysis of the biopesticide market now and where it is going. Outlooks Pest
Manag 26:203–206
Ongena M, Jacques P (2008) Bacillus lipopeptides: versatile weapons for plant disease biocontrol.
Trends Microbiol 16:115–125
Ongena M, Jourdan E, Adam A, Paquot M, Brans A, Joris B, Arpigny JL, Thonart P (2007)
Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resis-
tance in plants. Environ Microbiol 9:1084–1090
Pereg L, de-Bashan LE, Bashan Y (2016) Assessment of affinity and specificity of Azospirillum for
plants. Plant Soil 399(1–2):389–414. doi:10.1007/s11104-015-2778-9
Perez-Garcia O, de-Bashan LE, Hernandez JP, Bashan Y (2010) Efficiency of growth and nutrient
uptake from wastewater by heterotrophic, autotrophic, and mixotrophic cultivation of Chlorella
vulgaris immobilized with Azospirillum brasilense. J Phycol 46:800–812
Polyak B, Geresh S, Marks RS (2004) Synthesis and characterization of a biotin-alginate conju-
gate and its application in a biosensor construction. Macromolecules 5:389–396
Prithiviraj B, Zhou X, Souleimanov A, Khan WK, Smith DL (2003) A host specific bacteria-to-
plant signal molecule (Nod factor) enhances germination and early growth of diverse crop
plants. Planta 216:437–445
Raaijmakers JM, De Bruijn I, Nybroe O, Ongena M (2010) Natural functions of lipopeptides from
Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol Rev
34:1037–1062
Reddy CA, Saravanan RS (2013) Polymicrobial multi-functional approach for enhancement of
crop productivity. Adv Appl Microbiol 82:53–113
Reetha D, Kumaresan G, John Milton D (2014) Studies to improve the shelf life of Azospirillum
lipoferum immobilized in alginate beads. Int J Recent Scientific Res 5:2178–2182
Rekha PD, Lai WA, Arun AB, Young CC (2007) Effect of free and encapsulated Pseudomonas
putida CC-FR2-4 and Bacillus subtilis CC-pg104 on plant growth under gnotobiotic condi-
tions. Bioresour Technol 98:447–451
Rivera D, Obando M, Barbosa H, Rojas Tapias D, Bonilla Buitrago R (2014) Evaluation of poly-
mers for the liquid rhizobial formulation and their influence in the Rhizobium-cowpea interac-
tion. Univ Sci 19:265–275
Rizvi PQ, Choudhury RA, Ali A (2009) Recent advances in biopesticides. In: Khan MS, Zaidi A,
Musarrat J (eds) Microbial strategies for crop improvement. Springer Verlag, Berlin/Heidelberg,
pp 185–203
Rojas-Tapias D, Ortega-Sierra O, Rivera Botía D, Bonilla R (2015) Preservation of Azotobacter
chroococcum vegetative cells in dry polymers. Univ Sci 20:201–207
Rouissi T, Tyagi RD, Brar SK, Prevost D (2014) Development of efficient suspension formulation
of starch industry wastewater grown Sinorhizobium meliloti for agricultural use. Int J Agric
Innov Res 3:1083–1093
Roy M, Saha S, Das J, Srivastava RC (2015) Technologies of microbial inoculation in rice – a
review. Agric Rev 36:125–132
Ruiz-Güereca DA, Sánchez-Saavedra MP (2016) Growth and phosphorus removal by
Synechococcus elongatus co-immobilized in alginate beads with Azospirillum brasilense.
J Appl Phycol. doi:10.1007/s10811-015-0728-9
Russo A, Basaglia M, Tola E, Casella S (2001) Survival, root colonisation and biocontrol capaci-
ties of Pseudomonas fluorescens F113 LacZY in dry alginate microbeads. J Ind Microbiol
Biotechnol 27:337–342
2 Superior Polymeric Formulations and Emerging Innovative Products of Bacterial… 45
Sabaratnam S, Traquair JA (2002) Formulation of a Streptomyces biocontrol agent for the suppres-
sion of Rhizoctonia damping-off in tomato transplants. Biol Control 23:245–253
Sabra W, Zeng AP, Deckwer WD (2001) Bacterial alginate: physiology, product quality and pro-
cess aspects. Appl Microbiol Biotechnol 56:315–325
Sarrocco S, Raeta R, Vannacci G (2004) Seeds encapsulation in calcium alginate pellets. Seed Sci
Technol 32:649–661
Schoebitz M, Simonin H, Poncelet D (2012) Starch filler and osmoprotectants improve the survival
of rhizobacteria in dried alginate beads. J Microencapsul 29:532–538
Schoebitz M, Ceballos C, Ciampi L (2013a) Effect of immobilized phosphate solubilizing bacteria
on wheat growth and phosphate uptake. J Soil Sci Plant Nut 13:1–10
Schoebitz M, López MD, Roldán A (2013b) Bioencapsulation of microbial inoculants for better
soil–plant fertilization. A review. Agron Sustain Develop 33(4):751–765. doi:10.1007/
s13593-013-0142-0 (In press)
Schoebitz M, Osman J, Ciampi L (2013c) Effect of immobilized Serratia sp. by spray-drying
technology on plant growth and phosphate uptake. Chilean J Agric Anim Sci 29:111–119
Schoebitz M, Mengual C, Roldán A (2014) Combined effects of clay immobilized Azospirillum
brasilense and Pantoea dispersa and organic olive residue on plant performance and soil prop-
erties in the re-vegetation of a semiarid area. Sci Total Environ 466–467:67–73
Selvamukilan B, Rengalakshmi S, Tamizoli P, Nair S (2006) Village-level production and use of
biocontrol agents and biofertilizers. In: Uphoff N, Ball AS, Fernades E, Herren H, Husson O,
Laing M, Palm C, Pretty J, Sanchez P, Sanginga N, Thies J (eds) Biological approaches to
sustainable soil systems. CRC Press, Boca Raton, pp 647–653
Singleton P, Keyser H, Sande E (2002) Development and evaluation of liquid inoculants. In:
Herridge D (ed) Inoculants and nitrogen fixation of legumes in Vietnam. ACIAR Proceedings
109e. pp 52–66
Sivakumar PK, Parthasarthi R, Lakshmipriya VP (2014) Encapsulation of plant growth promoting
inoculant in bacterial alginate beads enriched with humic acid. Int J Curr Microbiol App Sci
3:415–422
Stelting SA, Burns RG, Sunna A, Bunt CR (2014) Survival in sterile soil and atrazine degradation
of Pseudomonas sp. strain ADP immobilized on zeolite. Bioremediat J 18:309–316
Stephens JHG, Rask HM (2000) Inoculant production and formulation. Field Crops Res
65:249–258
Tanaka K, Cho SH, Lee H, Pham AQ, Batek JM, Cui S, Qiu J, Khan SM, Joshi T, Zhang ZJ, Xu D,
Stacey G (2015) Effect of lipo-chitooligosaccharide on early growth of C4 grass seedlings.
J Exp Bot. doi:10.1093/jxb/erv260p.erv260
Trejo A, de-Bashan LE, Hartmann A, Hernandez JP, Rothballer M, Schmid M, Bashan Y (2012)
Recycling waste debris of immobilized microalgae and plant growth-promoting bacteria from
wastewater treatment as a resource to improve fertility of eroded desert soil. Environ Exp Bot
75:65–73
Trivedi P, Pandey A (2008) Recovery of plant growth-promoting rhizobacteria from sodium algi-
nate beads after 3 years following storage at 4 degrees. J Ind Microbiol Biotechnol
35:205–209
Trivedi P, Pandey A, Palni LMS (2005) Carrier-based preparations of plant growth-promoting
bacterial inoculants suitable for use in cooler regions. World J Microbiol Biotechnol
21:941–945
Truchet G, Roche P, Lerouge P, Vasse J, Camut S, de Billy F, Prome J-C, Denarie J (1991)
Sulphated lipo-oligosaccharide signals of Rhizobium meliloti elicit root nodule organogenesis
in alfalfa. Nature 351:670–673
Trujillo-Roldán MA, Moreno S, Segura D, Galindo E, Espín G (2003) Alginate production by an
Azotobacter vinelandii mutant unable to produce alginate lyase. Appl Microbiol Biotechnol
60:733–737
Tu L, He Y, Yang H, Wu Z, Yi L (2015) Preparation and characterization of alginate–gelatin
microencapsulated Bacillus subtilis SL-13 by emulsification/internal gelation. J Biomat Sci-
Polym E 26:735–749
46 Y. Bashan et al.
Abstract
The symbiotic agreement of rhizobia with leguminous plants is making a valu-
able contribution to agriculture primarily as nitrogen fixers and secondarily as
plant growth promoters by their key role as phosphate solubilizers, growth hor-
mone producers, abiotic and biotic stress relievers, and host-plant resistance
enhancer. In the so far identified 14 genera and 105 species of rhizobia, a huge
number of research reports were reported in various aspects. Genetically modi-
fied rhizobia with desirable traits have also been surfed to a large extent. Besides
their potentiality, the commercial success of rhizobia as a bio-inoculant is poor,
because most of the inoculants produced worldwide are of poor or suboptimal
quality. Though voluminous data and better understanding are available on vari-
ous formulation technologies, longevity and efficacy of the final product are
loosed at the farmer’s end. This book chapter is focused to address various types
of formulations applicable to rhizobia, quality control for longevity, gaps in
knowledge on bringing the native potential of rhizobia during formulation, and
critical control points to be considered during its development. The chapter also
shares ICRISAT’s experience in its rhizobial collection, formulation develop-
ments, and efficacy testing.
Keywords
Rhizobia • Legumes • Asia • Formulations • Peat • Legislations
3.1 Introduction
Rhizobia are primarily considered for nitrogen fixation. Still the research on SNF in
relation to rhizobia is ongoing including genetically modified rhizobia (Lindström
and Mousavi 2010; Okazaki et al. 2016). After the concept of plant growth-
promoting rhizobacteria by Kloepper, rhizobia have also been surfed to a large
extent for its plant growth-promoting (PGP) properties (Kloepper and Schroth
1978). Hence, a developed rhizobial inoculum will provide additional plant and soil
health benefits besides fixing nitrogen. PGP properties of rhizobia have been
reviewed previously by Gopalakrishnan et al. (2014) and Naveed et al. (2015). The
representatives of rhizobia with PGP traits have been given here.
Phytohormones are the essential substances for plant growth stimulation. They
include indole-3-acetic acid (IAA), cytokinin, and gibberellins. IAA is the foremost
phytohormone and plays a role in cell division and differentiation and also in nodule
formation. Rhizobia strains are also reported to produce IAA via indole-3-pyruvic
acid and indole-3-acetaldehyde pathway (Camerini et al. 2008). Similarly rhizobia
have been reported to produce cytokinins which are involved in root development
and root hair formation (Senthilkumar et al. 2009). Gibberellins which are respon-
sible for stem elongation and leaf expansion are also reported in Rhizobium (Boiero
et al. 2007). Some reports are there for production of abscisic acid which stimulates
stomatal closure, induces seeds to store proteins, and induces gene transcription for
protease inhibitors (Dobbelaere et al. 2003).
The stress of the plant depends on host-plant reaction which can be influenced by
rhizobia and the symbiosis (Yang et al. 2009). Several reviews periodically docu-
mented the stress tolerance of Rhizobium and Bradyrhizobium against soil salinity,
acidity, alkalinity, osmotic stress, and temperature fluctuations (Graham 1992;
Kulkarni and Nautiyal 2000; Grover et al 2010).
3 Formulation and Commercialization of Rhizobia: Asian Scenario 51
Peat
Compost
Organic
Animal waste
Sludge/industrial waste
Broth culture
Soil: mineral/clay
Inoculant type
Talc
Agar culture Inorganic
Perlite
Dried culture
Vermiculite
Carrier
Alginate
Polymeric
Modified starch
Chitosan
900000 14000000
12199899 12054666
800000 775510
12000000
700000
Pulses (Tonnes)
10000000
Fertilizers (Tonnes)
600000
7907265
500000 8000000
400000 6000000
289716 289282 277345
300000
218302 4000000
200000
895208 2000000
100000
0 0
India China China Canada Russian
Mainland Fedaration
Fig. 3.2 Top pulse producers and fertilizer consumers of the world (Note: Top seed producer
(based on average data of 1993–2013) on the left axis ▲ Top fertilizer consumers (based on aver-
age data of 2006–2009) on the right axis)
Effective rhizobial strain is the central core for developing an inoculant which is
necessitated in order to provide rhizobia for new legume cultivars and species and
extend and optimize the legume cultivation under fluctuating environmental condi-
tions. Brockwell et al. (1995) have listed a set of essential and desirable characters
for inoculant strains including host specificity, competence with native rhizobia
population and also with agrochemicals, genetic stability, etc. Asian countries
including India (Ansari et al. 2014), China (Jiao et al. 2015), Nepal (Adhikari et al.
2012), and Myanmar (Htwe et al. 2015) have been reported with vast diversity of
nodulating rhizobia. Recent reports on diversity analysis of rhizobia under hostile
environments such as soils with acidity (Mishra et al. 2014), alkalinity (Singh et al.
2016), and micronutrient deficiency (Unno et al. 2015) indicate the research initia-
tives on the exploration of Asian rhizobial strains. The large genetic diversity noticed
on soybean native rhizobia of Asian countries further supports the phenomenon
(Biate et al. 2014). Reeve et al. (2015) captured the phylogenetic and biogeographic
diversity of root nodule bacteria across the world through two genome sequencing
reports, which has only 7 entries for rhizobia from Asian origin among the 107
selected strains. However, these 7 entries include 3 among the total of 13 type strains
and 1 among the total of 14 elite strains with commercial significance, indicating
that the complete characterization and exploration of rhizobial biodiversity of Asian
countries will pave way for inoculant development.
Besides the native flora, genetic modification has also been done in rhizobia, mainly
to compete with the indigenous strains and to improve its efficacy to form nodules
and to fix nitrogen.
number of strains occupied the nodules than the control strains. It appears to be an
efficient method of nodulation, but on the yield of crop after 3 years of experimenta-
tion, they were all equal in inoculated and un-inoculated plants. This informs that
inoculant strains will improve in nodulation only when indigenous competing popu-
lation is less efficient which might not be frequent.
Among the inoculants are the primitive types such as broth culture, agar culture, and
dried/lyophilized cells. These types of inoculants could not be promoted to practical
technology, though it is least laborious and has proved records at research centers,
because of impractical application at large scales and its failure to meet economic
and commercial needs (Bashan et al. 2014). Hence, a carrier is necessary for the
development of a successful inoculant.
The major markets, such as Europe and Australia, supply the inoculants in solid
carriers, most commonly peat, for seed application (Catroux et al. 2001; Singleton
et al. 2002). However, in North and South America, the inoculants supplied are
clay- and peat-based granular and liquid inoculants (Singleton et al. 2002; Xavier
et al. 2004). The Asian market also depends on peat for its inoculants because of its
potential in holding high numbers of rhizobia (greater than 108 cells/g) during the
storage. Unlikely, they do not have enough peatlands due to the lack of harmonized
policies related to the management of peatlands besides their presence in Indonesia,
India, Malaysia, Myanmar, the Philippines, Singapore, Thailand, and Vietnam. In
the last few years, a forward look for its sustainable management has arisen. The
projects ASEAN Peat l and Forests Project (APFP) and SEApeat were aimed in
reducing deforestation and degradation of peatland forests and to strengthen the
policies for its management. On the other end, a large area of peatlands in Vietnam
has been designated as protected area and national parks (http://www.aseanpeat.
net/).
It should also be considered that whether the peat belongs to these regions is
original peat. Thomas et al. (1974) have evaluated the physicochemical characters
of peat obtained from Nilgiri reserves of India and concluded that the material was
58 R. Vijayabharathi et al.
not an original peat as it lacks the main traits like water-holding capacity and organic
carbon content. It is noticed that the Indian peat has 20–50 % organic carbon,
whereas Australian and American peat has 65 and 86 % organic carbon content,
respectively (NIIR 2004). Conservation policies for peatland management by
Europe, Australia, and America have become stringent as they have key roles in
biodiversity, carbon sequestration, and fuel-related application. This indirectly leads
to the unavailability and high export cost for other countries (Joosten 2015).
As an alternative to peat, other organic carriers such as lignite and charcoal can
be used which have also proved to be efficient in carrying rhizobia with the shelf life
of 4–6 months (Argal et al. 2015; Gao et al. 2015). Research on alternate carrier was
started more than four decades ago on carriers such as lignite and coal, clays and
mineral soils, compost, farmyard manure, pressmud, agricultural waste, and inor-
ganic materials like vermiculite, perlite, ground rock phosphate, calcium sulfate,
polyacrylamide gels, and alginate (Kandasamy and Prasad 1971; Dube et al. 1980;
Chao and Alexander 1984; Iswaran et al. 1972; Philip and Jauhri 1984; Sadasivam
et al. 1986; Sparrow and Ham 1983; Dommergues et al. 1979; Jung et al. 1982).
There are numerous reports on research and development of successful rhizobial
formulations which were tested in fields of various research stations. However,
there are very few number of coordinated network projects on large-scale evaluation
in Asian countries. On the contrary, the International Network of Legume Inoculation
Trials (INLIT) funded by the US Agency for International Development (USAID)
in the University of Hawaii’s NifTAL project assessed the need for inoculation in
tropical agricultural systems by conducting 228 trials on various legumes such as
green gram, soybean, black gram, groundnut, cowpea, chickpea, lentil, pigeon pea,
and common bean. Worldwide Rhizobial Ecology Network (WREN), the follow-up
program of NifTAL, evaluated the factors contributing to variations in inoculation
response including a number of infective rhizobia, edaphic characteristics, crop
fixed-N demand, and soil fixed-N supply (Singleton et al. 1992).
Effective regulatory quality control (QC) program has key role in the successful
production of rhizobial inoculants. This may be supported by appropriate legislation
as in Canada, Uruguay, and France or may be voluntary as in Australia, Thailand,
New Zealand, and South Africa. Contrarily, in the USA, regulatory control and
independent testing are considered unnecessary, with manufacturers conducting
their own internal QC. Irrespective of the QC nature, all QC programs should moni-
tor the numbers and quality of the strains in the inoculants along with the contami-
nating microorganisms. In Asia, 90 % of inoculants sampled had <108 rhizobia g−1
carrier and most of samples were contaminated (Thompson 1992).
Besides these barriers, many Asian countries commercialized rhizobia inocu-
lants. This includes the following: (1) Pakistan, Fasloon Ka Jarasimi Teeka (AARI),
BioPower (NIBGE), Biozote (NARC), and Rhizogold (ISES, UAF), consists of
Rhizobium sp. (Naveed et al. 2015), and (2) Japan, the Tokachi Federation of
Agricultural Cooperative (TFAC), produces Mamezo (rhizobia are mixed with peat
and the natural organic matters), R-processing seeds (leguminous seeds inoculated
with rhizobia), and hyper-coating seeds (leguminous grass seed coated by rhizobia
within the capsule of calcium carbonate) (Yokoyama and Ohyama 2007).
Table 3.2 Viability and longevity of five different formulations of six different rhizobia
Colony-forming unit (CFU/ml) of different carrier materials in months
0 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th 13th
Chickpea
IC-59
T1 4.5 × 109 3.0 × 109 2.4 × 109 2.2 × 109 2.0 × 108 1.5 × 108 1.3 × 108 1.1 × 108 9.1 × 107 5.7 × 107 2.3 × 107 1.2 × 107 8.6 × 106 3.0 × 106
T2 2.0 × 109 1.8 × 109 1.4 × 109 1.2 × 109 8.9 × 108 7.5 × 108 5.4 × 108 4.3 × 108 1.2 × 108 9.0 × 106 4.2 × 106 1.2 × 106 7.5 × 105 4.5 × 106
T3 2.9 × 109 2.0 × 109 2.1 × 109 1.7 × 108 1.2 × 108 8.9 × 107 6.3 × 107 4.8 × 107 2.3 × 107 1.1 × 107 9.7 × 106 5.6 × 106 2.3 × 106 1.1 × 106
T4 7.5 × 108 4.7 × 108 5.5 × 108 4.2 × 108 1.0 × 108 9.3 × 107 7.1 × 107 5.6 × 107 2.8 × 107 9.0 × 106 7.0 × 106 1.1 × 106 7.3 × 105 2.6 × 105
T5 1.9 × 109 1.1 × 109 1.4 × 109 1.1 × 109 8.4 × 108 8.5 × 107 6.7 × 107 4.5 × 107 2.7 × 107 1.7 × 107 9.3 × 106 4.8 × 106 2.5 × 106 1.2 × 106
IC-76
T1 3.9 × 109 4.3 × 109 2.15 × 109 1.75 × 109 1.3 × 109 1.1 × 109 1 × 109 9.6 × 108 5.1 × 108 4.3 × 108 1.2 × 108 1.0 × 108 9.3 × 107 2.0 × 107
T2 3.3 × 109 1.8 × 109 9.5 × 108 5.5 × 108 1.1 × 108 9.9 × 107 7.1 × 107 5.0 × 107 3.4 × 107 2.1 × 107 1.1 × 107 7.6 × 106 2.5 × 106 1.0 × 106
T3 5.0 × 109 2.0 × 109 1.1 × 109 1.1 × 109 6.6 × 108 1.1 × 108 8.9 × 107 6.1 × 107 4.9 × 107 3.1 × 107 1.2 × 107 5.6 × 106 3.1 × 106 1.2 × 106
T4 7.0 × 108 4.0 × 108 2.5 × 108 1.5 × 108 1.1 × 108 7.6 × 107 4.3 × 107 2.1 × 107 8.1 × 106 6.1 × 106 3.2 × 106 1.1 × 106 5.5 × 105 2.1 × 105
T5 2.3 × 109 1.1 × 109 5.5 × 108 4.0 × 108 1.0 × 108 6.6 × 107 3.3 × 107 1.1 × 107 9.3 × 106 3.7 × 106 2.3 × 106 1.1 × 106 7.5 × 105 3.5 × 105
Pigeon pea
IC-3195
T1 7.6 × 109 5.9 × 109 3.2 × 109 1.7 × 109 1.0 × 109 9.0 × 108 8.3 × 108 8.1 × 108 7.6 × 108 6.3 × 108 2.1 × 108 1.2 × 108 4.1 × 107 1.4 × 107
T2 4.7 × 109 2.6 × 109 1.4 × 109 7.0 × 108 5.9 × 108 2.5 × 108 1.3 × 108 8.7 × 107 7.0 × 107 5.6 × 107 1.1 × 107 8.3 × 106 3.7 × 106 1.3 × 106
T3 6.8 × 109 1.6 × 109 9.0 × 108 5.0 × 108 4.0 × 108 1.7 × 108 9.7 × 107 5.3 × 107 2.1 × 107 1.8 × 107 9.3 × 106 7.6 × 106 5.0 × 106 1.5 × 106
T4 9.5 × 109 4.0 × 109 2.1 × 109 1.1 × 109 7.0 × 108 2.3 × 108 8.9 × 107 7.1 × 107 6.5 × 107 4.0 × 107 1.3 × 107 8.9 × 106 4.2 × 106 1.7 × 106
T5 8.7 × 109 3.4 × 109 1.9 × 109 1.0 × 109 8.0 × 108 5.6 × 108 2.3 × 108 1.0 × 108 9.6 × 107 7.0 × 107 3.0 × 107 9.1 × 106 5.0 × 106 2.1 × 106
IC-4062
T1 9.9 × 109 8.5 × 109 6.3 × 109 2.85 × 109 2.3 × 109 1.6 × 109 8.6 × 108 4.7 × 108 2.5 × 108 1.8 × 109 1.1 × 109 6.6 × 107 4.6 × 107 2.1 × 107
T2 3.8 × 109 2.1 × 109 1.6 × 109 9.0 × 108 4.2 × 108 3.2 × 108 1.3 × 108 1.0 × 108 9.1 × 107 7.0 × 107 3.0 × 107 8.6 × 106 4.3 × 106 1.2 × 106
T3 2.1 × 109 7.9 × 109 6.3 × 109 3.6 × 109 1.6 × 109 1.1 × 109 8.8 × 108 4.7 × 108 3.1 × 108 2.4 × 108 1.1 × 108 7.8 × 107 5.2 × 107 1.6 × 107
(continued)
Table 3.2 (continued)
Colony-forming unit (CFU/ml) of different carrier materials in months
0 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th 13th
9 9 9 8 8
T4 2.0 × 10 1.7 × 10 1.3 × 10 8.0 × 10 4.0 × 10 9.8 × 107 7.9 × 107 5.5 × 107 3.2 × 107 1.0 × 107 6.9 × 106 3.3 × 106 1.1 × 106 6.8 × 105
9 9 9 9 8
T5 2.0 × 10 2.3 × 10 1.8 × 10 1.0 × 10 6.6 × 10 9.1 × 107 6.3 × 107 4.3 × 107 2.7 × 107 1.9 × 107 9.4 × 106 7.8 × 106 3.3 × 106 1.1 × 106
Groundnut
IC-7001
T1 9.4 × 109 8.0 × 109 7.0 × 109 6.3 × 109 1.9 × 109 1.0 × 109 9.2 × 108 8.3 × 108 6.0 × 108 5.6 × 108 2.4 × 108 1.1 × 108 7.9 × 107 3.4 × 107
9 9 9
T2 8.4 × 10 5.8 × 10 4.9 × 10 3.6 × 109 1.5 × 109 8.8 × 108 5.4 × 108 4.8 × 108 3.1 × 108 2.2 × 108 1.1 × 107 7.7 × 107 3.5 × 107 9.1 × 106
9 9 9
T3 7.3 × 10 4.0 × 10 3.6 × 10 3.1 × 109 1.8 × 109 5.2 × 108 4.2 × 108 3.7 × 108 2.5 × 108 2.5 × 108 2.1 × 107 1.1 × 108 6.7 × 107 3.0 × 107
9 9 9
T4 8.1 × 10 5.6 × 10 4.8 × 10 3.9 × 109 1.3 × 109 1.1 × 109 7.3 × 108 4.2 × 108 3.1 × 108 1.4 × 108 9.0 × 107 3.1 × 107 5.6 × 106 3.1 × 106
9 9 9
T5 10 × 10 9.1 × 10 8.1 × 10 5.8 × 109 2.8 × 109 9.3 × 108 5.2 × 108 3.1 × 108 1.5 × 108 8.0 × 107 4.8 × 107 1.6 × 107 7.9 × 106 2.9 × 106
IC-7113
T1 8.3 × 109 7.9 × 109 4.7 × 109 2.65 × 109 2.1 × 109 8.8 × 108 5 × 108 4.5 × 108 3.7 × 108 2.2 × 108 1.2 × 108 8.6 × 107 3.9 × 107 1.8 × 107
9 9 8
T2 5.1 × 10 1.4 × 10 9.1 × 10 4.2 × 108 1.1 × 108 6.1 × 107 4.8 × 107 3.4 × 107 2.5 × 107 1.3 × 107 1.1 × 107 9.0 × 106 4.8 × 106 1.2 × 106
T3 4.2 × 109 1.4 × 109 5.0 × 108 1.2 × 108 9.0 × 107 4.0 × 107 2.9 × 107 2.3 × 107 2.1 × 107 1.6 × 107 8.8 × 106 4.3 × 106 1.2 × 106 6.1 × 105
T4 1.8 × 109 9.9 × 108 4.0 × 108 1.5 × 108 1.0 × 108 9.8 × 107 6.6 × 107 4.8 × 107 2.3 × 107 1.5 × 107 8.9 × 106 5.8 × 106 2.1 × 106 4.5 × 105
T5 2.8 × 109 1.4 × 109 8.8 × 109 4.6 × 109 2.2 × 109 6.3 × 107 5.4 × 107 3.3 × 107 2.5 × 107 1.9 × 107 7.6 × 106 3.7 × 106 1.2 × 106 5.3 × 105
TI, peat; T2, talc; T3, talc + starch; T4, charcoal; and T5, charcoal + sugarcane powder
3 Formulation and Commercialization of Rhizobia: Asian Scenario 61
International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), based
at Patancheru, Hyderabad, India, has been using peat-based rhizobial formulation
for its mandate crops chickpea, pigeon pea, and groundnut. In order to find an effi-
cient alternative carrier material, a total of six rhizobia (two specific for chickpea,
IC-59, IC-76; two specific for pigeon pea, IC-3195, IC-4062; two specific for
groundnut, IC-7001, IC-7113) were formulated as five different inoculants using
peat, talc, talc amended with starch, charcoal, and charcoal amended with sugarcane
powder, and shelf life was evaluated for a period of 13 months (Table 3.2). Among
the carrier materials, peat was found to be the best as it holds 107 rhizobia for IC-76,
IC-3195, IC-4062, IC-7001, and IC-7113 and 106 for IC-59 even after 13 months of
storage. On the whole, the shelf life maintenance was observed in the order of
peat > talc amended with starch > talc > charcoal amended with sugar cane pow-
der > charcoal. The results also suggest that the use of proper additives to the inocu-
lants can tremendously enhance the shelf life of the product.
3.5 Conclusions
From the literature survey, it is observed that legume inoculants gained more atten-
tion in developed countries with successful stories like soybean in Brazil, pea and
lentil in Canada, and subterranean clover in Australia. In Asia, though there is a
considerable interest in rhizobial inoculant development, still many factors such as
undisturbed supply of good-quality carrier material, well-developed technology,
quality control legislations, well-defined good manufacturing practices, training
programs, well-planned field demonstrations, and governmental support for small-
scale industries are creating constraints for further development. Unification of all
these sectors can lead to the development of a low cost, high shelf life, and highly
effective rhizobial inoculants.
References
Adhikari D, Kaneto M, Itoh K, Suyama K, Pokharel BB, Gaihre YK (2012) Genetic diversity of
soybean-nodulating rhizobia in Nepal in relation to climate and soil properties. Plant Soil
357(1–2):131–145
Afzal A, Bano A (2008) Rhizobium and phosphate solubilizing bacteria improve the yield and
phosphorus uptake in wheat (Triticum aestivum L.). Int J Agric Biol 10:85–88
Albareda M, Rodriguez-Navarro DN, Camacho M, Temprano FJ (2008) Alternatives to peat as a
carrier for rhizobia inoculant: solid and liquid formulations. Soil Biol Biochem
40:2771–2779
Albareda M, Rodriguez-Navarro DN, Temprano FJ (2009) Use of Sinorhizobium (Ensifer) fredii
for soybean inoculants in south Spain. Eur J Agron 30:205–211
Allen EK, Allen ON (1950) Biochemical and symbiotic properties of the rhizobia. Bacteriol Rev
14:273–330
62 R. Vijayabharathi et al.
Amarger N, Hirsch P, Klingmüller W (2001) Assessing the risks involved in the release of geneti-
cally manipulated microorganisms. In: Kessler C, Economidis I (eds) EC-sponsored research
on safety of genetically modified organisms. A review of results. European Communities,
Luxembourg, pp 62–64
Ansari PG, Rao DLN, Pal KK (2014) Diversity and phylogeny of soybean rhizobia in central India.
Ann Microbiol 64(4):1553–1565
Argal MS, Rawat AK, Aher SB, Rajput PS (2015) Bioefficacy and shelf life of Rhizobium legumi-
nosarum loaded on different carriers. Appl Biol Res 17(2):1–7
Arora NK, Kang SC, Maheshwari DK (2001) Isolation of siderophore producing strains of
Rhizobium meliloti and their biocontrol potential against Macrophomina phaseolina that
causes charcoal rot of groundnut. Curr Sci 81:673–677
Arora NK, Khare E, Naraian R, Maheshwar DK (2008) Sawdust as a superior carrier for produc-
tion of multipurpose bioinoculant using plant growth-promoting rhizobial and pseudomonad
strains and their impact on productivity of Trifolium repens. Curr Sci 95:90–94
Bashan Y, de-Bashan LE, Prabhu SR, Hernandez JP (2014) Advances in plant growth-promoting
bacterial inoculant technology: formulations and practical perspectives (1998–2013). Plant
Soil 378(1–2):1–33
Ben Rebah F, Tyagi RD, Prevost D (2002a) Wastewater sludge as a substrate for growth and carrier
for rhizobia: the effect of storage conditions on survival of Sinorhizobium meliloti. Bioresour
Technol 83:145–151
Ben Rebah F, Tyagi RD, Prevost D, Surampalli RY (2002b) Wastewater sludge as a new medium
for rhizobial growth. Water Qual Res J Can 37:353–370
Bhattacharyya PN, Jha DK (2012) Plant growth-promoting rhizobacteria (PGPR): emergence in
agriculture. World J Microbiol Biotechnol 28:1327–1350
Biate DL, Kumar LV, Ramadoss D, Kumari A, Naik S, Reddy KK, Annapurna K (2014) Genetic
diversity of soybean root nodulating bacteria. In: Maheshwari DK (ed) Bacterial diversity in
sustainable agriculture. Springer, Cham, pp 131–145
Boiero L, Perrig D, Masciarelli O, Penna C, Cassan F, Luna V (2007) Phytohormone production
by three strains of Bradyrhizobium japonicum and possible physiological and technological
implications. Appl Microbiol Biotechnol 74:874–880
Bosworth AH, Williams MK, Albrecht KA, Kwiatkowski R, Beynon J, Hankinson TR, Ronson
CW, Cannon F, Wacek TJ, Triplett EW (1994) Alfalfa yield response to inoculation with
recombinant strains of Rhizobium meliloti with an extra copy of dctABD and/or modified nifA
expression. Appl Environ Microbiol 60:3815–3832
Bramley RGV, Ellis N, Nable RO, Garside AL (1996) Changes in soil chemical properties under
long-term sugar cane monoculture and their possible role in sugar yield decline. Aust J Soil Sci
34:967–984
Brockwell J, Bottomley PJ, Thies JE (1995) Manipulation of rhizobia microflora for improving
legume productivity and soil fertility: a critical assessment. Plant Soil 174:143–180
Bullard GK, Roughley RJ, Pulsford DJ (2005) The legume inoculant industry and inoculant qual-
ity control in Australia: 1953–2003. Aust J Exp Agric 45:127–140
Burton JC (1979) Rhizobium species. In: Peppler HJ, Perlman D (eds) Microbial technology:
Microbial processes. Academic, New York, pp 29–58
Camerini S, Senatore B, Lonardo E, Imperlini E, Bianco C, Moschetti G, Rotino GL, Campion B,
Defez R (2008) Introduction of a novel pathway for IAA biosynthesis to rhizobia alters vetch
root nodule development. Arch Microbiol 190:67–77
Carson KC, Meyer JM, Dilworth MJ (2000) Hydroxamate siderophore of root nodule bacteria.
Soil Biol Biochem 32:11–21
Catroux G, Hartmann A, Revellin C (2001) Trends in rhizobial inoculant production and use. Plant
Soil 230(1):21–30
Chandra S, Choure K, Dubey RC, Maheshwari DK (2007) Rhizosphere competent Mesorhizobium
loti MP6 induces root hair curling, inhibits Sclerotinia sclerotiorum and enhances growth of
Indian mustard (Brassica campestris). Braz J Microbiol 38:128–130
Chao WL, Alexander M (1984) Mineral soils as carriers for Rhizobium inoculants. Appl Environ
Microbiol 47:94–97
3 Formulation and Commercialization of Rhizobia: Asian Scenario 63
Clayton GW, Rice WA, Lupwayi NZ, Johnston AM, Lafond GP, Grant CA, Walley F (2004a)
Inoculant formulation and fertilizer nitrogen effects on field pea: crop yield and seed quality.
Can J Plant Sci 84:89–96
Clayton GW, Rice WA, Lupwayi NZ, Johnston AM, Lafond GR, Grant CA, Walley F (2004b)
Inoculant formulation and fertilizer nitrogen effects on field pea: Nodulation, N2 fixation and
nitrogen partitioning. Can J Plant Sci 84:79–88
Crawford SL, Berryhill DL (1983) Survival of Rhizobium phaseoli in coal-based legume inocu-
lants applied to seeds. Appl Environ Microbiol 45:703–705
Daza A, Santamaria C, Rodriguez-Navarro DN, Camacho M, Orive R, Temprano F (2000) Perlite
as a carrier for bacterial inoculants. Soil Biol Biochem 32:567–572
Deaker R, Roughley RJ, Kennedy IR (2004) Legume seed inoculation technology – a review. Soil
Biol Biochem 36:1275–1288
Deshwal VK, Pandey P, Kang SC, Maheshwari DK (2003) Rhizobia as a biological control agent
against soil borne plant pathogenic fungi. Ind J Exp Biol 41:1160–1164
Dillewijn P, Soto MJ, Villadas PJ, Toro N (2001) Construction and environmental release of a
Sinorhizobium meliloti strain genetically modified to be more competitive for alfalfa nodula-
tion. Appl Environ Microbiol 67:3860–3865
Diouf D, Forestier S, Neyra M, Lesueur D (2003) Optimisation of inoculation of Leucaena leuco-
cephala and Acacia mangium with Rhizobium under greenhouse conditions. Ann For Sci
60:379–384
Dobbelaere S, Vanderleyden J, Okon Y (2003) Plant growth-promoting effects of diazotrophs in
the rhizosphere. Crit Rev Plant Sci 22:107–149
Dommergues YR (1995) Nitrogen fixation by trees in relation to soil nitrogen economy. Fertil Res
42(1):215–230
Dommergues YR, Diem HG, Divies C (1979) Polyacrylamide entrapped Rhizobium as an inocu-
lant for legumes. Appl Environ Microbiol 37:779–981
Drahos DJ, Hemming BC, McPherson S (1986) Tracking recombinant organisms in the environ-
ment: beta-galactosidase as a selectable non-antibiotic marker for fluorescent pseudomonads.
Biotechnology 4:439–444
Dube JN, Mahere DP, Bawat AF (1980) Development of coal as a carrier for rhizobial inoculants.
Sci Cult 46:304
FAOSTAT Data (2016) Food and Agriculture Organization of the United Nations. Statistical data-
base. Accessed at http://faostat3.fao.org/browse/Q/QC/E and, http://faostat3.fao.org/browse/R/
RF/E on 8 Mar 2016
Fink CR, Waggoner PE, Ausubel F (1999) Nitrogen fertilizer: retrospect and prospect. Proc Natl
Acad Sci USA 96:1175–1180
Forestier S, Alvarado G, Badjel SB, Lesueur D (2001) Effect of Rhizobium inoculation methodolo-
gies on nodulation and growth of Leucaena leucocephala. World J Microbiol Biotechnol
17:359–362
Fred EB, Baldwin IL, McCoy E (1932) Root nodule bacteria and leguminous plants. University of
Wisconsin Press, Madison
Gao TG, Xu YY, Jiang F, Li BZ, Yang JS, Wang ET, Yuan HL (2015) Nodulation characterization
and proteomic profiling of Bradyrhizobium liaoningense CCBAU05525 in response to water-
soluble humic materials. Scientific reports, 5
Gopalakrishnan S, Sathya A, Vijayabharathi R, Srinivas V (2016) Formulations of plant growth-
promoting microbes for field applications. In: Singh DP, Singh HB, Prabha R (eds) Microbial
inoculants in sustainable agricultural productivity. Springer, New Delhi.
doi:10.1007/978-81-322-2644-4_15
Gopalakrishnan S, Sathya A, Vijayabharathi R, Varshney RK, Gowda CLL, Krishnamurthy L
(2014) Plant growth-promoting rhizobia: Challenges and opportunities. 3 Biotech. doi:10.1007/
s13205-014-0241-x
Graham PH (1963) Vitamin requirements of root-nodule bacteria. J Gen Microbiol 30:245–248
Graham PH (1992) Stress tolerance in Rhizobium and Bradyrhizobium and nodulation under
adverse soil conditions. Can J Microbiol 38:475–484
64 R. Vijayabharathi et al.
Graham-Weiss L, Bennett ML, Alan SP (1987) Production of bacterial inoculants by direct fer-
mentation on nutrient-supplemented vermiculite. Appl Environ Microbiol 53(9):2138–2140
Grover M, Ali SZ, Sandhya V, Rasul A, Venkateswarlu B (2010) Role of microorganisms in adap-
tation of agriculture crops to abiotic stresses. World J Microbiol Biotechnol 27:1231–1240
Guthrie FB (1896) Inoculation of soil for leguminous crops. Agric Gaz NSW 7:690–694
Halder AK, Chakrabarty PK (1993) Solubilization of inorganic phosphate by Rhizobium. Folia
Microbiol 38:325–330
Halder AK, Mishra AK, Bhattacharya P, Chakrabarthy PK (1990) Solubilization of rock phosphate
by Rhizobium and Bradyrhizobium. J Gen Appl Microbiol 36:1–92
Hamaoui B, Abbadi JM, Burdman S, Rashid A, Sarig S, Okon Y (2001) Effects of inoculation with
Azospirillum brasilense on chickpeas (Cicer arietinum) and faba beans (Vicia faba) under dif-
ferent growth conditions. Agronomie 21:553–560
Herridge DF, Peoples MB, Boddey RM (2008) Global inputs of biological nitrogen fixation in
agricultural systems. Plant Soil 311(1):1–18
Hirsch PR (1997) Acquisition of genes from indigenous bacteria by inoculant strains at long-term
release sites. In: Hoeveler A, Cresti M (eds) Biotechnology programme (1992–1994), final
report. European Commission, Luxembourg, pp 231–234
Hirsch PR, Spokes JD (1994) Survival and dispersion of genetically modified rhizobia in the field
and genetic interactions with native strains. FEMS Microbiol Ecol 15:147–159
Htwe AZ, Yamakawa T, Sarr PS, Sakata T (2015) Diversity and distribution of soybean-nodulating
bradyrhizobia isolated from major soybean-growing regions in Myanmar. Afr J Microbiol Res
9(43):2183–2196
Huang J, Rozelle S (1995) Environmental stress and grain yields in China. Am J Agric Econ
77:853–864
Hungria M, Campo RJ, Souza EM, Pedrosa FO (2010) Inoculation with selected strains of
Azospirillum brasilense and A. lipoferum improves yields of maize and wheat in Brazil. Plant
Soil 331:413–425
Hynes RK, Jans DC, Bremer E, Lupwayi NZ, Rice WA, Clayton GW, Collins MM (2001)
Rhizobium population dynamics in the pea rhizosphere of rhizobial inoculant strain applied in
different formulations. Can J Microbiol 47:595–600
Iswaran V, Sen A, Apte R (1972) Plant compost as a substitute for peat for legume inoculants. Curr
Sci 41:299
Jiao YS, Liu YH, Yan H, Wang ET, Tian CF, Chen WX, Guo BL, Chen WF (2015) Rhizobial diver-
sity and nodulation characteristics of the extremely promiscuous legume Sophora flavescens.
Mol Plant Microbe Interact 28(12):1338–1352
Jiao J, Wu LJ, Zhang B, Hu Y, Li Y, Zhang XX, Guo HJ, Liu LX, Chen W, Zhang Z, Tian CF (2016)
MucR is required for transcriptional activation of conserved ion transporters to support nitro-
gen fixation of Sinorhizobium fredii in soybean nodules. Mol Plant Microbe Interact. doi:http://
dx.doi.org/10.1094/MPMI-01-16-0019-R
Joosten H (2015) Peatlands, climate change mitigation and biodiversity conservation: An issue
brief on the importance of peatlands for carbon and biodiversity conservation and the role of
drained peatlands as greenhouse gas emission hotspots, vol 2015727. Nordic Council of
Ministers, Denmark
Jordan DC (1984) Family III Rhizobiaceae. In: Kneg NR, Holt JG (eds) Bergey’s manual of sys-
tematic bacteriology. Williams & Wilkins, Baltimore, pp 234–244
Jung G, Mugnier J, Diem HG, Dommergues YR (1982) Polymer-entrapped Rhizobium as an inoc-
ulant for legumes. Plant Soil 65(2):219–231
Kandasamy R, Prasad NN (1971) Lignite as a carrier of rhizobia. Curr Sci 40:496
Keyser HH, Somasegaran P, Bohlool BB (1993) Rhizobial ecology and technology. In: Metting EB
(ed) Soil microbial ecology: applications in agricultural and environmental management.
Marcel Dekker, New York, pp 205–226
Khalid A, Arshad M, Zahir ZA (2004) Screening plant growth-promoting rhizobacteria for improv-
ing growth and yield of wheat. J Appl Microbiol 96:473–480
Kinzig AP, Socolow RH (1995) Is nitrogen fertilizer use nearing a balance? Phys Today
47:24–35
3 Formulation and Commercialization of Rhizobia: Asian Scenario 65
Revellin C, Meunier G, Giraud J-J, Sommer G, Wadoux P, Catroux G (2000) Changes in the physi-
ological and agricultural characteristics of peat-based Bradyrhizobium japonicum inoculants
after long-term storage. Appl Microbiol Biotechnol 54:206–211
Robleto EA, Kmiecik K, Oplinger ES, Nienhuis J, Triplett EW (1998) Trifolitoxin production
increases nodulation competitiveness of Rhizobium etli CE3 under agricultural conditions.
Appl Environ Microbiol 64:2630–2633
Ronson CW, Bosworth A, Genova M, Gusbrandsen S, Hankinson T, Kwiatkowski R, Robie C,
Sweeney P, Szeto W, Williams M, Zablotowitcz R (1990) Field release of genetically-
engineered Rhizobium meliloti and Bradyrhizobium japonicum strains. In: Gressoff PM, Roth
LE, Stacey G, Newton WE (eds) Nitrogen fixation: achievements and perspectives. Chapman
and Hall, New York/London, pp 397–403
Rozelle S, Veeck G, Huang JK (1997) The impact of environmental degradation on grain produc-
tion in China 1975–1990. Econ Geog 73:44–66
Sadasivam KV, Tyagi RK, Ramarethinam S (1986) Evaluation of some agricultural wastes as car-
riers for bacterial inoculants. Agric Wastes 17:301–306
Savant NK, Datnoff LE, Snyder GH (1997) Depletion of plant-available silicon in soils: A possible
cause of declining rice yields. Comm Soil Sci Plant Nutr 28:1245–1252
Scupham AJ, Bosworth AH, Ellis WR, Wacek TJ, Albrecht KA, Triplett EW (1996) Inoculation
with Sinorhizobium meliloti RMBPC-2 increases alfalfa yield compared with inoculation with
a non-engineered wild-type strain. Appl Environ Microbiol 62:4260–4262
Senthilkumar M, Madhaiyan M, Sundaram SP, Kannaiyan S (2009) Intercellular colonization and
growth promoting effects of Methylobacterium sp. with plant-growth regulators on rice (Oryza
sativa L. CvCO-43). Microbiol Res 164:92–104
Singh RP, Manchanda G, Singh RN, Srivastava AK, Dubey RC (2016) Selection of alkalotolerant
and symbiotically efficient chickpea nodulating rhizobia from North‐West Indo Gangetic
Plains. J Basic Microbiol 56(1):14–25
Singleton PW, Bohlool BB, Nakao PL (1992) Legume response to rhizobial inoculation in the
tropics: Myths and realities. In: Lal R, Sanchez PA (eds) Myths and science of soils of the trop-
ics. Soil Science Society of America and American Society of Agronomy Special Publication,
Madison, pp 135–155
Singleton P, Keyser H, Sande E (2002) Development and evaluation of liquid inoculants. In:
Herridge D (ed) Inoculants and nitrogen fixation of legumes in Vietnam. ACIAR Proceedings,
Canberra, pp 52–66
Sparrow SD, Ham GE (1983) Nodulation, N2 fixation, and seed yield of navy beans as influenced
by inoculant rate and inoculant carrier. Agron J 75:20–24
Temprano FJ, Albareda M, Camacho M, Daza A, Santamaría C, Rodríguez-Navarro DN (2002)
Survival of several Rhizobium/Bradyrhizobium strains on different inoculant formulations and
inoculated seeds. Int Microbiol 5:81–86
Thomas PK, Venkataramanan C, Vasu K (1974) Quality and quantity of peat material reserves in
the Nilgiris. Proc Ind Natl Sci Acad 40:608
Thompson JA (1992) Consultant report to UNFAO. IND/86/003. FAO, Rome
Unno Y, Shinano T, Minamisawa K, Ikeda S (2015) Bacterial community shifts associated with
high abundance of Rhizobium spp. in potato roots under macronutrient-deficient conditions.
Soil Biol Biochem 80:232–236
Van Elsas JD, Heijnen CE (1990) Methods for the introduction of bacteria into soil: a review. Biol
Fert Soils 10(2):127–133
Van Nieuwenhove C, Van Holm L, Kulasooriya SA, Vlassak K (2000) Establishment of
Azorhizobium caulinodans in the rhizosphere of wetland rice (Oryza sativa L.). Biol Fert Soils
31:143–149
Vance CP (1998) Legume symbiotic nitrogen fixation: Agronomic aspects. In: Spaink HP,
Kondorosi A, Hooykaas PJJ (eds) The Rhizobiaceae, molecular biology of model plant-
associated bacteria. Kluwer Academic Publishers, Dordrecht, pp 509–530
3 Formulation and Commercialization of Rhizobia: Asian Scenario 67
Vessey JK (2004) Benefits of inoculating legume crops with rhizobia in the northern Great Plains.
Retrieved 2/2005, from http://www.plantmanagementnetwork.org/pub/cm/review/2004/
inoculant/
Vessey JK, Pawlowski K, Bergman B (2004) Root-based N2-fixing symbioses: Legumes, actino-
rhizal plants, Parasponia sp. and cycads. Plant Soil 266:205–230
Vessey KJ (2003) Plant growth-promoting rhizobacteria as biofertilizers. Plant Soil 255:571–586
Vincent JM (1962) Influence of calcium and magnesium on the growth of Rhizobium. J Gen
Microbiol 28:653–663
Vitousek PM, Aber JD, Howarth RW, Likens GE, Matson PA, Schindler DW, Schlesinger WH,
Tilman DG (1997) Human alteration of the global nitrogen cycle: sources and consequences.
Ecol Appl 7:737–750
Walley F, Clayton G, Gan Y, Lafond G (2004) Performance of rhizobial inoculant formulations in
the field. Retrieved 2/2005, from http://www.plantmanagementnetwork.org/pub/cm/
review/2004/inoculant/
Wani PA, Khan MS, Zaidi A (2007) Effect of metal tolerant plant growth promoting Bradyrhizobium
sp. (vigna) on growth, symbiosis, seed yield and metal uptake by green gram plants.
Chemosphere 70:36–45
Xavier IJ, Holloway G, Leggett M (2004) Development of rhizobial inoculant formulations. Crop
Manage 3(1)
Yang J, Kloepper JW, Ryu CM (2009) Rhizosphere bacteria help plants tolerate abiotic stress.
Trends Plant Sci 14(1):1–4
Yokoyama T, Ohyama T (2007) Current status and future direction of commercial production and
use of bio-fertilizers in Japan. Food and Fertilizer Technology Center (FFTC), Taipei
Regulatory Issues in Commercialization
of Bacillus thuringiensis-Based 4
Biopesticides
Estibaliz Sansinenea
Abstract
The utility of biopesticides, as a component of integrated pest management
(IPM), has won acceptance over the world. An entomopathogenic organism
should be highly specific and effective against the target pest and should be suc-
cessfully produced. Bacillus thuringiensis (Bt) was discovered as a soil bacte-
rium, which fulfills all these requirements and is being used as a biopesticide in
agriculture, forestry, and mosquito control. In spite, biopesticides have many
advantages as green pesticides and their use has had a slow growth, mainly
because the farmers are less confident in selecting biopesticides over the synthet-
ics. However, the global biopesticide market is substantially growing every year.
The regulations about the pesticides have many concerns which do not apply to
biopesticides, and these issues have been made difficult to introduce them to the
market.
Keywords
Biopesticides • Bacillus thuringiensis • Integrated pest management • Green
pesticide • Entomopathogenic
E. Sansinenea (*)
RoyanoFacultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla,
CP. 72570 Pue, Puebla, Mexico
e-mail: [email protected]
4.1 Introduction
A pest is an animal or insect that causes problems for people especially by damag-
ing crops. Each species of crop plant is affected by different pest, which varies
according to country and region. The natural selection has developed the different
mechanisms by which pests affect the crop. These mechanisms are led by the com-
petition between the pest and the plant, between different pest species (e.g., for food
and space), and with other members of the ecological community (e.g., with preda-
tors or disease) and the abiotic environment. Pest damage can be caused directly
(i.e., the plant is eaten by a pest) or it can be caused indirectly, but always there is a
reduction in yield or quality due to competition for resources.
When the production of agricultural crops is declining in yield, the farmers often
expect a dramatic, magical treatment to make them green and healthy again, so that
the productivity increases. Over years, chemical pesticides had been the major con-
tributor against pests and diseases. The use of chemical pesticides was economi-
cally a viable strategy because they were cheap and effective. Such pesticides were
adopted in the 1940s with the use of dichloro-diphenyl-trichloroethane (DDT),
organophosphates, and carbamate pesticides (Nicholson 2007). Their long-term use
resulted in insecticide resistance. The contamination of soil and water and the harm-
ful residues of the agricultural products are the most serious problems of the use of
chemical pesticides causing tremendous damage to the environment, pest resis-
tance, and lethal effects on nontarget organisms (Abudulai et al. 2001). Chemical
fertilizers and pesticides are continuously accumulating in the environment, harm-
ing the ecosystem, causing pollution, and inflicting diseases at alarming levels
(Gerhardson 2002; Arora et al. 2010). The great use of pesticides has already caused
grave damage to health, ecosystems, and groundwater due to its survival in plants
for a long time as a residual. They also enter in the food chain and are found in meat
and dairy products and remain as residue in the soil and ecosystem for long periods
of time (Bisen et al. 2015; Keswani et al. 2013).
For the reasons given above, many farmers and growers over the world are trying to
reduce the amounts of conventional chemical pesticides used. Integrated pest man-
agement (IPM) is a program that combines different crop protection practices with
careful monitoring of pests and their natural enemies. Biopesticides are a particular
group used in IPM and offer the possibility to create a new generation of sustainable
agriculture products. In very general terms, according to the US EPA, biopesticides,
also known as biological pesticides, are derived from natural materials such as ani-
mals, plants, bacteria, and certain minerals (US Environmental Protection Agency
Pesticides 2014). Typically, biopesticides have unique modes of action and are
4 Regulatory Issues in Commercialization of Bacillus thuringiensis-Based… 71
considered reduced risk pesticides. Biopesticides are not used to eradicate pests but
to control pests with the advantage of a major selectivity and nontarget biological
safety. Biopesticides can be classified into three major classes: biochemical, plant-
incorporated protectants, and microbial pesticides, which are also known as BCAs.
Among the last group, there can be found microbial pesticides that function in mul-
tiple ways. The most widely used microbial pesticides are based on B. thuringiensis
(Bt), accounting for approximately 90 % of the biopesticide market in the USA
(Chattopadhyay et al. 2004).
The most widely used microbial biopesticide is the insect pathogenic bacterium
Bacillus thuringiensis (Bt), which produces an endotoxin protein called Cry during
sporulation that is able to lead to lysis of the gut cells when consumed by suscepti-
ble insects (Gill et al. 1992). The δ-endotoxin is very specific and can cause death
within 48 h (George and Crickmore 2012). It is safe to people, beneficial organisms,
and the environment. Bt biopesticides consist of a mix containing bacterial spores
and δ-endotoxin crystals mass-produced in fermentation tanks and formulated as a
sprayable product. Bt sprays are a great option for pest management on several
crops where resistance to synthetic chemical insecticides is a problem (Abdullah
2012). Bt sprays have also been used on broad-acre crops such as maize, soya bean,
and cotton. However, the use of Bt microbial biopesticide formulations has been
rather scarce due to the problems of narrow host range, low persistence on plants,
and inability of foliar application to reach the insects feeding inside the plants, not-
withstanding several biotechnological approaches for the development of improved
Bt biopesticides (Kaur 2007).
The problems of field application of Bt biopesticides have been overcome by Bt
transgenic crops. The total acreage of transgenic crops has been steadily increasing
with commercial cultivation of transgenic crops on 140 million hectares in 2010
(James 2010). The most widely grown Bt crop is cotton (Gossypium hirsutum L.),
accounting for 64% of global cotton area devoted to Bt crops, followed by corn (Zea
mays L.) accounting for 29% of global corn area. Bt cotton has been planted on an
increasingly large scale in India and China (Liu 2009; Chaudhary and Gaur 2010).
The use of Bt crops has resulted in increased yields and significant reductions of
insecticide application, thus generating economic and environmental improvements
(Shelton et al. 2008; Brookes and Barfoot 2008; Carpenter 2010). However, while
there are undoubted advantages of deployment of Bt transgenic crops for effective
insect pest control, certain concerns have been raised about the environmental safety
of Bt transgenic crops.
to humans and ecosystems and reduce concerns for development of pest resistance
to existing control tools.
The success of Bt-based biopesticide production depends on high-quality and
high-efficiency formulation processes, which must be safe and effective; the prod-
ucts must be easy to use and should have a long shelf life. The spore-crystal com-
plex is the active ingredient in commercial formulations, which is more effective to
use and cheaper to obtain than the crystals alone and must be helped by suitable
inert substance that can function to protect the spore-crystal complex or to increase
availability to insects. Bt sprays are used sporadically and typically over small areas
over cotton, fruit, and vegetable crops. However, the use of Bt spray as an insecti-
cide has several disadvantages: (1) Bt spray cannot be applied uniformly to all parts
of the plant, (2) it cannot be applied inside plant tissues, and (3) Bt is susceptible to
rapid degradation by UV light and removal by water runoff. Therefore, multiple
applications are required to provide extended pest protection. New Bt formulations
have consistently come to vegetable markets over the last number of years (Cerón
2001). The persistence of Bt spores in different environments has been reasonably
well studied (Petras and Casida 1985). Bt spores can survive for several years after
spray applications, although rapid declines in population and toxicity have been
noted (Addison 1993). The bioassays for Bt products have had a standardization
procedure. Before 1970, the standardization procedure was carried out through the
use of spore counts. However, there was no relationship between the number of
spores in a preparation and its insect killing power. Because of this actually stan-
dardization procedure is carried out using insect bioassay. Insect bioassay of Bt
products is expensive and time consuming and takes a relatively long time (4–7
days) to furnish information on the potency of the material. Due to these reasons,
insect bioassays have been replaced with chemical assays. There are several things
to take into consideration using chemical assays. The killing capacity of the product
toward target pest insects is the required information. The killing capacity of the
product is determined by both the quality and quantity of crystal toxin present, and
in some cases the crystal toxin requires the presence of spores to have toxicity
against pest insects. Chemical methods only measure the quantity of toxin present,
but not measure the quality of toxin present nor the presence, number, or viability
of Bt spores. Because of this, many laboratories still check their Bt preparations by
insect bioassay (Beegle and Yamamoto 1992).
The efficacy of Bt microbials applied to the surface of leaves is limited by the
fact that the formulation can be washed off by rain and the Cry proteins are inacti-
vated by sunlight within a few days of application (Federici and Siegel 2008). To
solve the problem of the damage of UV irradiation to B. thuringiensis, some chemi-
cal screens have been used. However, these chemical screens have some negative
impacts on the environment. In contrast, melanin is a natural pigment that is easily
biodegradable in the nature and can absorb radiation; therefore it is a perfect photo
protective agent, which has been used to protect Bt formulations from UV light
(Sansinenea and Ortiz 2015; Sansinenea et al. 2015).
With the development of biotechnology, it has been possible to introduce the
genes coding for Cry proteins into plants so that Cry proteins are expressed in the
4 Regulatory Issues in Commercialization of Bacillus thuringiensis-Based… 73
plant and are produced throughout the growing season to provide protection against
insect pests. The efficacy of Bt is dependent on its narrow spectrum of toxicity too.
Most of the Bt products in agriculture are targeted against lepidopteran insects.
Also, since it is only active when ingested by the specific target pests, topically
applied Bt is not effective against insects that normally bore into plant tissues such
as stem and fruit borers (i.e., Ostrinia nubilalis and Helicoverpa zea) and leaf min-
ers (i.e., Tuta absoluta and Phyllocnistis citrella). However, these limitations could
be overcome; O. nubilalis is effectively controlled by genetically modified corn that
expresses Bt toxin in its tissues (reviewed in (Sanahuja et al. 2011; Meissle et al.
2011)), while the addition of surfactant appears to increase the toxicity of topically
applied Bt against leaf miner P. citrella by increasing Bt penetration into the mines
(Shapiro et al. 1998).
5000
4500
4000
3500
3000
Biopesticide market
2500
size ($ million)
2000
1500
1000
500
0
2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023
North America Latin America E.U. Asia Australia Africa ROW
Source: Lux Research, Inc.
www.Luxresearchinc.com
Fig. 4.1 The biopesticide market is approximately $3 billion today and will rise above $4.5 billion
by 2023
fact that more than 53 % of the world biopesticide market is occupied by about 200
Bt-based products (CABI 2010), and almost 50 % of this is consumed by America
particularly in the USA and Canada (Guerra et al. 2001).
Today, biopesticides make up a small fraction of the total global crop protection
market at approximately $3 billion in value worldwide (Olson 2015) (See Fig. 4.1).
The growth would be regional, with Europe and Latin America projected to grow
most quickly in the coming 3 years, driven by tightening regulatory restrictions and
rapidly emerging insect resistance, respectively. Africa is poised for significant
growth, but in a more extended time frame. North America, which already accounts
for a large proportion of the market, will continue to grow at a slower rate than
Europe.
Since 2012, multiple acquisitions, licensing agreements, and partnerships with
values well into the hundreds of millions of dollars show the depth and breadth of
investment large companies are making in biopesticide development. The bigger
companies of the market such as Bayer, Syngenta, BASF, DuPont, Dow
AgroSciences, and Monsanto account for 70 % of the world pesticide sales
market.
pesticides are spread directly into nature, in contrast of industrial chemicals” (Blok
et al. 2006). Taking into consideration that biopesticides come from nature, these
objections do not apply in the same way, but this has not been fully recognized by
the regulatory system.
In the process of pesticide development, field testing is often necessary to evalu-
ate the efficacy of a pesticide. Briefly, when the size of the outdoor test acreage is
greater than a cumulative 10 acres of land or 1 surface acre of water, an experimen-
tal use permit (EUP) is required. Any food or feed crops involved in or affected by
the tests must be destroyed or consumed only by experimental animals unless a
tolerance has been established. These acreage limitations are applicable only for
outdoor terrestrial and aquatic uses. For those pesticides being tested on sites for
which acreage is not relevant (e.g., tree stumps, rodent control, structural treat-
ments, or bird repellents), the determination of the need for an EUP is made on a
case-by-case basis.
An EUP is of limited duration and requires that the test be carried out under
controlled conditions. For small-scale field tests of genetically modified microbial
pesticides or nonindigenous microbial pesticides that the USDA has not previously
acted upon, applicants must submit a notification to the EPA for the determination
of whether an experimental use permit is necessary, even if the testing is on less than
10 acres.
Registration of biopesticide is the main hurdle in the development, and most of
the time, registration is much more expensive than the production. Registration is
not only expensive but also time consuming (Ehlers 2006). The main problem is that
biopesticides contain active cells (live organisms), and these live forms are treated
like pathogens by the government agencies. Another issue is regarding the import
and export of biopesticides; again, it should be pointed that export and import of
chemical pesticide are much easier (as no one doubts on its integrity) in comparison
to the use of biopesticides.
The assessment of risks is important to approve or register new biological pesti-
cides and whether to renew the registration of old ones. Registration requires colla-
tion of data and preparation of dossier for submission to a national regulatory
authority. By these efforts, governments can speed up the process of approving safer
new pesticides and stopping the use of riskier ones.
Before a pesticide can be marketed and used in the USA, the Federal Insecticide,
Fungicide, and Rodenticide Act (FIFRA) requires that the EPA evaluates the pro-
posed pesticide to certificate that its use will not have unreasonable risks of harm to
human health and the environment. This involves an extensive review of health and
safety information. Pesticide registration is also the process through which the EPA
examines some points such as the ingredients of a pesticide; the site or crop on
which it is to be used; the amount, frequency, and timing of its use; and storage and
disposal instructions. A pesticide cannot legally be used, sold, or distributed if it has
not been registered with the EPA’s Office of Pesticide Programs.
As biopesticides are usually less toxic than chemical pesticides, biopesticide reg-
istrations may require a significantly reduced data set compared to conventional
78 E. Sansinenea
registrations. Additionally, there are reduced associated timelines and fees to help
expedite registration processes.
The manufacturer must submit data on a broad range of toxicological end points.
The Office of Pesticide Programs reviews toxicity and exposure information for
each active ingredient and dictates approved use conditions. The US EPA provides
a fast-track review and registration for biopesticides by combining lower data
requirements with high review priority.
In spite of the review process for biopesticides being more efficient in bringing
them to the market, the main focus of the EPA pesticide registration process is on
human health and safety. On the one hand, speed to market is an economically
important goal, but on the other hand, the environment and health data gaps must be
attended since otherwise it will lead to serious problems followed by backlash
against them.
4.5 Conclusions
The use and situation of biopesticides still remain in dilemma. Farmers find them-
selves confused and less confident in selecting biopesticides over the synthetics.
Despite the fact that presently biopesticides are being used everywhere in the world,
it is also known that developed countries seem to be ahead in their wider application
(Chandler et al. 2011).
Also countries like India are vastly dependent upon agriculture for not only feed-
ing their populations but also for the economy which depends majorly on this sector.
However, most of the challenges faced for the upliftment of biopesticides are funda-
mental and cosmopolitan. These include the efficacy of the microbial activity, sur-
vival of microorganisms, delivery systems, determining host range, avoiding injury
to nontarget organisms, consistency, performance in field conditions, economics,
government regulations, and confidence among the end users. Gelernter (2007) has
described the future of biocontrol in Asia, and according to him unreasonable
expectations for performance, inappropriate regulatory guidelines, lack of docu-
mentation on the uptake of microbial control strategies, difficulties in implementing
local production schemes, and inhibition of scientific exchange are the main hurdles
in establishment of biocontrol.
In spite of all these limitations, biopesticides are gradually becoming popular
around the world. While we cannot predict the growth of the biopesticide market to
the dollar, we are confident in our assessment that the industry will continue to
grow in the future. The applications of biopesticides have a bright future.
However, it is necessary to control the use of biopesticides, because some kinds
of products might result in environment pollution or be harmful to the natural
enemies.
4 Regulatory Issues in Commercialization of Bacillus thuringiensis-Based… 79
References
Abdullah MAF (2012) Use and efficacy of Bt compared to less environmentally safe alternatives.
In: Sansinenea E (ed) Bacillus thuringiensis biotechnology. Springer, Dordrecht, pp 19–39
Abudulai M, Shepard BM, Mitchell PL (2001) Parasitism and predation on eggs of Leptoglossus
phyllopus (L.) (Hemiptera: Coreidae) in Cowpea: impact of endosulfan sprays. J Agric Urban
Entomol 18:105–115
Addison JA (1993) Persistence and non-target effects of Bacillus thuringiensis in soil: a re-view.
Can J For Res 23:2329–2342
Ali S, Zafar Y, Ali MG, Nazir F (2008) Bacillus thuringiensis and its application in agriculture. Afr
J Biotechnol 9:2022–2031
Arora NK, Khare E, Maheshwari DK (2010) Plant growth promoting rhizobacteria: constraints in
bioformulation, commercialization, and future strategies. In: Maheshwari DK (ed) Plant
growth and health promoting bacteria. Springer, Berlin, pp 97–116
Beegle CC, Yamamoto T (1992) Invitation paper (C.P. Alexander Fund): history of Bacillus
thuringiensis Berliner research and development. Can Entomol 124:587–604
Bisen K, Keswani C, Mishra S, Saxena A, Rakshit A, Singh HB (2015) Unrealized potential of
seed biopriming for versatile agriculture. In: Rakshit A, Singh HB, Sen A (eds) Nutrient use
efficiency: from basics to advances. Springer, New Delhi, pp 193–206
Blok A, Jensen M, Kaltoft P (2006) Regulating pesticide risks in Denmark: expert and lay perspec-
tives. J Environ Policy Plan 8:309–330
Brar SK, Tyagi VRD, Valéro JR (2006) Recent advances in downstream processes and formula-
tions of Bacillus thuringiensis based biopesticide. Process Biochem 41:323–342
Brookes G, Barfoot P (2008) Global impact of biotech crops: socio-economic and environ-mental
effects 1996–2006. Ag Bio Forum 11:21–38
CABI (2010) The 2010 worldwide biopesticides: market summary. CPL Business Consultants,
London, p 40
Carpenter JE (2010) Peer-reviewed surveys indicate positive impact of commercialized GM crops.
Nat Biotech 28:319–321
Cerón JA (2001) Productos comerciales nativos y recombinantes a base de Bacillus thuringiensis.
In: Caballero P, Ferré J (eds) Bioinsecticidas: Fundamentos y aplicaciones de Bacillus thuringi-
ensis en el control integrado de plagas. Phytoma-España, pp 153–168
Chandler D, Bailey AS, Tatchell GM, Davidson G, Greaves GWP (2011) The development, regula-
tion and use of biopesticides for integrated pest management. Philos Trans R Soc Lond B Biol
Sci 1573:1987–1998
Chattopadhyay A, Bhatnagar NB, Bhatnagar R (2004) Bacterial insecticidal toxins. Crit Rev
Microbiol 30:33–54
Choudhary B, Gaur K (2010) Bt cotton in India: a country profile. ISAAA series of biotech crop
profiles. ISAAA, Ithaca. ISBN 978-1-892456-46-X
Ehlers RU (2006) Einsatz derBiotechnologieimbiologischenPflanzenschuz. Schnreihedtsch
Phytomed Ges 8:17–31
EPA (1998) EPA Registration Eligibility Decision (RED) Bacillus thuringiensis EPA 738-R-98- 004
Federici B, Siegel J (2008) Safety assessment of Bacillus thuringiensis and Bt crops used in insect
control. In: Hammond BG (ed) Food safety of proteins in agricultural biotechnology. CRC
Press, Boca Raton, pp 45–102
George Z, Crickmore N (2012) Bacillus thuringiensis applications in agriculture. In: Sansinenea E
(ed) Bacillus thuringiensis biotechnology. Springer, Dordrecht, pp 19–39
Gerhardson B (2002) Biological substitutes for pesticides. Trends Biotechnol 20:338–343
Gill SS, Cowles EA, Pietrantonio PV (1992) The mode of action of Bacillus thuringiensis endo-
toxins. Annu Rev Entomol 37:615–636
Guerra PT, Wong LJG, Roldán HM (2001) Bioinseticidas: Su empleo, produción y commercializa-
tion en México. Ciencia UANL 4:143–152
Huang DF, Zhang J, Song FP, Lang ZH (2007) Microbial control and biotechnology research on
Bacillus thuringiensis in China. J Invert Pathol 95:175–180
80 E. Sansinenea
James C (2010) Global view of commercialized transgenic crops: 2010. Brief no. 42. ISAAA
(International Service for Acquisition of Agri-biotech Applications), Ithaca, New York, USA.
http://www.isaaa.org/publications/briefs/Breif_.htm
Kabaluk JT, Svircev AM, GoettelMS WSG (eds) (2010) The use and regulation of microbial pes-
ticides in representative jurisdictions worldwide. IOBC Global, Canada, p 99
Kaur S (2007) Deployment of Bt transgenic crops: development of resistance and management
strategies in the Indian scenario. Biopest Int 3:23–42
Keswani C, Singh SP, Singh HB (2013) A superstar in biocontrol enterprise: Trichoderma spp.
Biotech Today 3:27–30
Kunimi Y (2007) Current status and prospects on microbial control in Japan. J Invertebr Pathol
95:181–186
Leahy J, Mendelsohn M, Kough J, Jones R, Berckes N (2014) Biopesticide oversight and registra-
tion at the U.S. environmental protection agency. In: Gross AD, Coats JR, Duke SO, Seiber JN
(eds) Biopesticides: state of the art and future opportunities, vol 1172, Symposium series.
Oxford University Press, Washington, DC, pp 3–18
Lewis W, van Lenteren C, Phatak S, Tumlinsen J (1997) A total system approach to sustainable
pest management. Proc Natl Acad Sci U S A 94:12243–12248
Liu W (2009) Effects of Bt transgenic crops on soil ecosystems: a review of a ten-year research in
China. Front Agric China 3:190–198
Marrone PG (2007) Barriers to adoption of biological control agents and biological pesticides,
CAB reviews: perspectives in agriculture, veterinary science, nutrition and natural resources
2(51). CAB International, Wallingford
Meissle M, Romeis J, Bigler F (2011) Bt maize and integrated pest management-a European per-
spective. Pest Manag Sci 67(9):1049–1058. doi:10.1002/ps.2221
Nicholson GM (2007) Fighting the global pest problem: preface to the special toxicon issue on
insecticidal toxins and their potential for insect pest control. Toxicon 49:413–422
Olson S (2015) An analysis of the biopesticide market now and where it is going. Outlooks on Pest
Manag 26:203–206
Petras SF, Casida LE Jr (1985) Survival of Bacillus thuringiensis spores in soil. Appl Environ
Microbiol 50:1496–1501
Sanahuja G, Banakar R, Twyman RM, Capell T, Christou P (2011) Bacillus thuringiensis: a cen-
tury of research, development and commercial applications. Plant Biotechnol J 9:283–300
Sansinenea E, Ortiz A (2015) Melanin: a photoprotection for Bacillus thuringiensis based biopes-
ticides. Biotechnol Lett 37:483–490
Sansinenea E, Salazar F, Ramirez M, Ortiz A (2015) An ultra-violet tolerant wild-type strain of
melanin-producing Bacillus thuringiensis. Jundishapur J Microbiol 8:e20910
Shapiro JP, Schroeder WJ, Stansly PA (1998) Bioassay and efficacy of Bacillus thuringiensis and
organosilicone surfactant against the citrus leaf miner (Lepidoptera: Phyllocnistidae). Florida
Entomol 81:201–210
Shelton AM, Romeis J, Kennedy GG (2008) IPM and insect protected transgenic plants: thoughts
for the future. In: Romeis J, Shelton AM, Kennedy GG (eds) Integration of insect-resistant,
genetically modified crops within IPM programs. Springer, Dordrecht, pp 419–429
U.S. Environmental Protection Agency, Office of Pesticide Programs, Web Page FIFRA. http://
www.epa.gov/pesticides/bluebook/FIFRA.pdf. Accessed 26 June 2014
WHO/IPCS (International Programme on Chemical Safety) (1999) Environmental health criteria
217: microbial pest control agent Bacillus thuringiensis. WHO, Geneva
Ziwen YH (2010) Bt research and development. Commercialization of Bacillus thuringiensis
insecticides in China. www.authorstream.com. Accessed 24 Sept 2010
Beauveria bassiana as Biocontrol Agent:
Formulation and Commercialization 5
for Pest Management
Abstract
Beauveria bassiana is the most widely used biocontrol agent against many major
arthropod pests. This ascomycetal fungus is able to produce infection structures
and synthesize a cocktail of proteins, enzymes, organic acids, and bioactive sec-
ondary metabolites, which are responsible for the entomopathogenic activity and
virulence. For commercial purposes, B. bassiana is usually formulated using
conidia with different stabilizing agents. Various types of formulation include
bait/solid, encapsulation, and emulsion. Commercialization and marketing strat-
egies, including alternative marketing channels, such as earthworm compost and
compost, along with the legal framework are addressed in this chapter.
Keywords
Beauveria bassiana • Biocontrol • Entomopathogen • Pest management
Although it has been suggested that hydrolytic enzymes represent the primary
infection mechanism that allows for penetration of fungal hyphae through the
arthropod cuticle (Ortiz-Urquiza and Keyhani 2013), adhesion to and interaction
with the epicuticular layer of the host must occur first. In B. bassiana, at least two
hydrophobins (Hyd1 and Hyd2) are in charge of fungal spore coat rodlet layer
assembly, thus contributing to cell surface hydrophobicity, adhesion to hydrophobic
surfaces, and virulence (Cho et al. 2007; Zhang et al. 2011). Assimilation of the
lipids, hydrocarbons, proteins, and other compounds included in the cuticular layer
requires the synthesis of different fungal enzymes, such as cytochrome P450, cata-
lases, esterases, long-chain alcohols, and aldehyde dehydrogenases (Pedrini et al.
2006, 2010, 2013; Ortiz-Urquiza and Keyhani, 2013). Other hydrolytic enzymes
related to virulence are known to be secreted by B. bassiana and include proteases,
5 Beauveria bassiana as Biocontrol Agent: Formulation and Commercialization… 83
Not only compounds from primary metabolism participate in the parasitization pro-
cess. Low molecular weight bioactive secondary metabolites produced in vitro and
in vivo by B. bassiana play an important role as (a) toxins that cause arthropod’s
death, (b) immunomodulators that aid the fungus to evade the host defense system,
(c) antimicrobials against competing microorganisms, and (d) defense molecules
against mycophagous organisms (Charnley 2003). B. bassiana has an enormous
potential to produce secondary metabolites, since 13 non-ribosomal peptide synthe-
tases (NRPS), 12 polyketide synthases (PKS), 7 NRPS-like, 1 PKS-like, 3 hybrid
NRPS–PKS, and 12 genes related to FAS/terpene/steroid biosynthesis are encoded
within its genome (Xiao et al. 2012). The known secondary metabolites produced
by this entomopathogenic fungus include cyclic peptides, such as beauvericin,
bassianolide, and beauverolides, and polyketide-derived pigments, such as
oosporein, tenellin, and bassianin, but only those genes involved in the biosynthesis
of beauvericin, bassianolide, tenellin, and oosporein have been functionally verified
(Roberts 1981; Strasser et al. 2000a, b; Vey et al. 2001; Molnar et al. 2010; Xu et al.
2008, 2009; Eley et al. 2007; Halo et al. 2008; Feng et al. 2015).
(Wu et al. 2002; Kouti et al. 2003) which have been suggested to trigger calcium-
sensitive cell apoptotic pathways (Jow et al. 2004; Wang and Xu 2012). Other
authors have reported that the apoptotic effect of beauvericin is mediated by Bc1-2
proteins, cytochrome c, and caspase 3 (Lin et al. 2005) and by the activation of the
JNK signaling pathway, inhibition of both TNFα-induced NF-kB activation, and
phosphorylation of ERK (p44/p42) (Wätjen et al. 2014).
Bassianolide is another cyclooligomer that might also be important during insect
pathogenesis (Xu et al. 2008, 2009), since this molecule, together with beauvericin,
has been isolated from extracts of Bombycis corpus inoculated by B. bassiana
(Kwon et al. 2000). This cyclic octodepsipeptide tetrameric ester of the dipeptidol
monomer d-hydroxyisovaleric acid–N-methyl-L-leucine is produced by B. bassi-
ana and Lecanicillium sp. (Verticillium lecanii) (Suzuki et al. 1977). This com-
pound exhibits antibacterial (against some M. tuberculosis), antimalarial, and
cytotoxic (against several tumor cell lines) activities (Kwon et al. 2000; Jirakkakul
et al. 2008). Bassianolide insecticidal properties are due to its ability to inhibit
acetylcholine-induced smooth muscle contraction (Nakajyo et al. 1983), thus induc-
ing atony and toxicity to different insect larvae (Suzuki et al. 1977; Champlin and
Grula 1979).
Other cyclic peptides include the beauverolides (beauveriolide or beauverilide)
and lipophilic and neutral cyclotetradepsipeptides that vary in amino acid composi-
tion and contain linear and branched β-hydroxy acid residues of variable length
(e.g., beauverolide M is made up of Val–Ala–Leu and contains 3-hydroxy-4-
methyloctanoic acid, whereas beauveriolide L is made up of Phe–Ala–Ile and con-
tains 3-hydroxy-4-methyldecanoic acid). These metabolites are produced by
entomopathogenic species of the genera Beauveria (including B. bassiana) and
Paecilomyces (Elsworth and Grove 1977; Jegorov et al. 1994). They seem not to
have bactericidal, fungicidal, or direct insecticidal effects, although they apparently
have an immunomodulatory role in insects (Jegorov et al. 1990; Mochizuki et al.
1993; Vilcinskas et al. 1999).
Tenellin and bassianin are yellow pigments with a 2-pyridone ring that have been
isolated from Beauveria species (Eley et al. 2007; McInnes et al. 1974). Bassianin
differs from tenellin by one chain extension in the ketide moiety. These two com-
pounds, in addition to oosporein, are able to inhibit erythrocyte membrane APTase
activity, which is likely a consequence of the ability of these pigments to promote
varying degrees of cell lysis by means of membrane disruption (Jeffs and
Khachatourians 1997). Although tenellin is not involved in the pathogenesis of B.
bassiana against honeycomb moth (Galleria mellonella), it can prevent iron-
generated reactive oxygen species toxicity in B. bassiana (Eley et al. 2007;
Jirakkakul et al. 2015).
5.3.2 Encapsulation
5.3.3 Emulsion
Some synthetic oils have been also evaluated as ingredients for emulsion
formulations, since they seemed to be more easily mixed and later applied to a water
surface, thereby improving the persistence of fungal spores after their application in
fields (Bukhari et al. 2011).
Usually, these emulsions are prepared in an oil-in-water formulation by adding a
surfactant (mainly Tween 20), mixing the oil phase with the aqueous phase contain-
ing the spore suspension. The aqueous phase with the conidial suspension is mixed
with sterilized oil at the effective concentration, and other optional ingredients such
as Triton X-100 (as nonionic surfactant), Na2CO3 (as stabilizer), and silicon (as
antifoaming agent) can be added. Finally, mixtures of these two phases are homog-
enized to get a stable formulation (Yacoub and Batta 2016).
The compatibility of most of these vegetable oils (and synthetic oils) has been
successfully evaluated on conidia from B. bassiana in terms of effectiveness, taking
into consideration parameters such as germination rate, vegetative growth, and
conidiogenesis (Sankar-Ummidi and Vadlamani 2014; Gomes et al. 2015).
Different oil emulsion formulations of B. bassiana have shown a variable reduc-
tion in spore germination, vegetative growth, and conidia production. Variation in
conidial germination due to different oils has been attributed to some qualitative
(and quantitative) composition of fatty acids, since different proportions of unsatu-
rated fatty acids contained in the oils, such as linoleic acid and oleic acids, have
antifungal properties. In this regard, the linseed oil emulsion formulation has shown
a maximum conidial germination rate, unlike other emulsion formulations contain-
ing even very low concentrations (1 %) of other oils (e.g., mustard and eucalyptus),
which have been reported as toxic for B. bassiana. In the case of eucalyptus oil, the
toxic effect has been attributed to its active ingredient citronellal (Sankar-Ummidi
and Vadlamani 2014).
Conidial germination in some oil emulsions (e.g., linseed) has been evaluated
under storage conditions (standard temperature of 30 ± 2 °C) for 12 months, show-
ing a significant decrease in conidial viability (deterioration in mycelium and unde-
tectable fungal conidia). Lower storage temperature is being evaluated to assure
further longevity of formulated conidia (Mishra et al. 2013).
In the case of insect pests, entomopathogenic fungi formulated in oil emulsions
show a clear increase in virulence, likely due to better ability of the oiled conidia to
adhere the lipid layer of insect cuticle through hydrophobic interactions, later facili-
tating germination and progression of the infection process (Ment et al. 2010).
Addition of some carriers, such as the clay bentonite, to oil-based liquid formula-
tions has been reported to improve the efficacy of infection of B. bassiana (Ritu
et al. 2012). The effectiveness of Beauveria emulsion formulations increases when
more complex pheromone trapping systems–oil emulsions are combined, since part
of the individuals are infected with a heavy load of spores directly by contact before
they leave the trap, thus providing an excellent and highly effective indirect infec-
tion way for other non-trapped individuals, mainly through their mating behavior
(Hajjar et al. 2015).
88 C. García-Estrada et al.
Emulsions are excellent spray carriers that increase the probability of direct con-
tact between fungal conidia and pests. Oils in the emulsion are reported to prevent
evaporation in field and increase in situ conidial retention. These properties repre-
sent further advantages of oiled emulsions of Beauveria, making this formulation an
excellent choice for the biocontrol of habitats difficult to penetrate (Mishra et al.
2013).
In an increasingly globalized world, the core facilities for fermentation and produc-
tion of B. bassiana are thousands of kilometers away from the market place. That is,
the first step in the distribution chain is the export–import process.
The Harmonized System 6-digit number (HS code) is given to each product capable
of passing through customs. It is an international system respected by the vast
majority of countries. The fundamental problem concerning international trade of
B. bassiana is the lack of a specific item in the HS for these products. This creates
difficulties in custom processes, as each country has a specific interpretation of the
code, thus requiring arbitrary documentation and inspections.
In general, the 3808 91code is recommended for this product (although it should
be contrasted with the local custom institution) because this tariff item includes
those products with insecticidal effect improperly described elsewhere. Also, the
3808 91code itself expressly refers to biopesticide products based on Bacillus
thuringiensis, a similar product in terms of effects and nature. The usual documen-
tation required in this process includes certificate of origin, supplier’s manufactur-
ing license, health certificate, letters of use, and destination, among others.
(a) Powder formulations: Four kilograms shall be mixed with 20 L of water. Stir
and wait until the carrier (usually talc) settles at the bottom of the container.
Then, take the liquid and mix with 500 L of water to apply it through the drip
irrigation system or through the foliar spray system.
(b) Liquid formulations: Directly mix the selected dosage (see below) with 500 L
of unchlorinated water.
5 Beauveria bassiana as Biocontrol Agent: Formulation and Commercialization… 89
5.4.3 Dosage
Commercial dosages greatly vary depending on the type of formulation, but in gen-
eral, assuming a CFU of 109 in liquid formulations and 108 in powder formulations,
3 L/Ha and 4 kg/Ha, respectively, should be applied to control pests. In the case of
severe infestation, apply every 2 weeks.
Like for every agricultural product, introduction of the biocontrol product in the
market is as important as the development of an innovative and effective formula-
tion which should follow effective strategies.
(a) Free trials: This is a well known but effective strategy, which must be conducted
by trained personnel and preferably in nonorganic crops. If the product works
well for this kind of crops, organic farmers will immediately assume that the
product will work also for their crops. However, when the tests are performed
in organic crops, conventional farmers believe that the product will not neces-
sarily work on their crop, because of the large amount of chemicals they apply.
(b) Creating a range of products (a system or a methodology): Farmers are much
more likely to buy a full range of products or a system than an isolated product
that is very different from the chemical products they are used to buy. In this
way, they will understand that we have to change how we understand agricul-
ture. It makes more sense for big and established chemical corporations to sim-
ply launch a new product (e.g., for the control of the tomato leaf miner), since
they already have an existing range of products. Organic companies must create
a new understanding of agriculture in order to be able to compete in the market
and survive in a sector that is mainly controlled by few chemical corporations.
90 C. García-Estrada et al.
B. bassiana is a fungus found in healthy soil, forming part of the immune system of
the plant. Along with this fungus, many other microorganisms conform microbial
communities that, together, create a biological balance capable of controlling many
pests and diseases.
Many studies describe the presence of B. bassiana in vermicompost and compost
(Anastasi et al. 2004). That is, there are other ways to ensure that B. bassiana is
present in crops and thus benefit from their effects. Applying vermicompost in the
planting substrate can achieve amazing results in controlling pests of great eco-
nomic impact, such as the red spider mite (Tetranychus urticae) and root-knot nem-
atodes (Meloidogyne spp.) (Arancon et al. 2002, 2007). This is particularly relevant
from the marketing point of view. Given the strict regulations required to bring B.
bassiana formulations to market, it is interesting for the business and consumer to
know that the use of a natural and ecological fertilizer as vermicompost also ensures
the presence of this fungus in the culture, which entails similar pest control
benefits.
The legal framework for the marketing of B. bassiana formulations greatly varies
depending on the country or region. However, in general, the greatest challenge is
that there is no specific regulation for entomopathogenic biopesticides. On the
contrary, these products are embedded in the existing regulations for plant
protection products. This fact is criticized by many companies, since powerful and
5 Beauveria bassiana as Biocontrol Agent: Formulation and Commercialization… 91
toxic chemical pesticides are considered in the same category as organic and
sustainable products.
Regarding the European Union, Regulation (EC) No. 1107/2009 of the European
Parliament and the Council (October 21, 2009) establishes the basis for regulating
the market of plant protection products. In short, this directive requires companies
to conduct a series of experiments including field trials, trials with animals, plants,
and insects. In practice, this process involves an average of 4–5-year evaluation
period by the authorities, which does not guarantee approval. During this evaluation
time, the sale of that product is not permitted. This is one of the major barriers for
the marketing of B. bassiana in Europe and is not very different from the existing
regulations in other regions of the world. This clearly benefits large corporations
with big economic capacities and is detrimental for small producers of organic
products.
5.6 Conclusion
References
Alurappa R, Bojegowda MR, Kumar V, Mallesh NK, Chowdappa S (2014) Characterisation and
bioactivity of oosporein produced by endophytic fungus Cochliobolus kusanoi isolated from
Nerium oleander L. Nat Prod Res 28:2217–2220
Anastasi A, Varese GC, Voyron S, Scannerini S (2004) Characterization of fungal biodiversity in
compost and vermicompost. Compost Sci Util 12:185
Arancon NQ, Edwards CA, Lee SS, Yardim F (2002) Management of plant parasitic nematodes by
use of vermicomposts. Proc Brighton Crop Protect Conf Pests Dis 2:705–710
Arancon NQ, Edwards CA, Yardim EN, Oliver TJ, Byrne RJ, Keeney G (2007) Suppression of
two-spotted spider mite (Tetranychus urticae), mealy bug (Pseudococcus sp) and aphid (Myzus
persicae) populations and damage by vermicomposts. Crop Protec 26:29–39
Bidochka MJ, Khachatourians GG (1991) The implication of metabolic acids produced by
Beauveria bassiana in pathogenesis of the migratory grasshopper, Melanoplus sanguinipes.
J Invertebr Pathol 58:106–117
Brewer D, Jen WC, Jones GA, Taylor A (1984) The antibacterial activity of some naturally occur-
ring 2, 5-dihydroxy-1, 4-benzoquinones. Can J Microbiol 30:1068–1072
Brown TP, Fletcher OJ, Osuna O, Wyatt RD (1987) Microscopic and ultrastructural renal pathol-
ogy of oosporein-induced toxicosis in broiler chicks. Avian Dis 31:868–877
Bukhari T, Takken W, Koenraadt CJ (2011) Development of Metarhizium anisopliae and Beauveria
bassiana formulations for control of malaria mosquito larvae. Parasit Vector 4:23
92 C. García-Estrada et al.
Calo L, Fornelli F, Ramires R, Nenna S, Tursi A, Caiaffa MF et al (2004) Cytotoxic effects of the
mycotoxin beauvericin to human cell lines of myeloid origin. Pharmacol Res 49:73–77
Champlin FR, Grula EA (1979) Non involvement of beauvericin in the entomopathogenicity of
Beauveria bassiana. Appl Environ Microbiol 37:1122–1126
Charnley AK (2003) Fungal pathogens of insects: cuticle-degrading enzymes and toxins. Adv Bot
Res 40:241–321
Cho EM, Kirkland BH, Holder DJ, Keyhani NO (2007) Phage display cDNA cloning and expres-
sion analysis of hydrophobins from the entomopathogenic fungus Beauveria (Cordyceps)
bassiana. Microbiology 153:3438–3447
Cole RJ, Kirksey JW, Cutler HG, Davis EE (1974) Toxic effects of oosporein from Chaetomium
trilaterale. J Agr Food Chem 22:517–520
Covarelli L, Beccari G, Prodi A, Generotti S, Etruschi F, Meca G et al (2015) Biosynthesis of
beauvericin and enniatins in vitro by wheat Fusarium species and natural grain contamination
in an area of central Italy. Food Microbiol 46:618–626
el-Basyouni SH, Vining LC (1966) Biosynthesis of oosporein in Beauveria bassiana (Bals.) Vuill.
Can J Biochem 44:557–565
Eley KL, Halo LM, Song Z, Powles H, Cox RJ, Bailey AM et al (2007) Biosynthesis of the
2-pyridone tenellin in the insect pathogenic fungus Beauveria bassiana. Chembiochem
8:289–297
Elsworth JF, Grove JF (1977) Cyclodepsipeptides from Beauveria bassiana Bals. Part 1.
Beauverolides H and I. J Chem Soc. Perkin 1(3):270–273
Fan YH, Fang WG, Guo SJ, Pei XQ, Zhang YJ, Xiao YH et al (2007) Increased insect virulence
in Beauveria bassiana strains overexpressing an engineered chitinase. Appl Environ Microbiol
73:295–302
Fang WG, Feng J, Fan YH, Zhang YJ, Bidochka MJ, Leger RJS et al (2009) Expressing a fusion
protein with protease and chitinase activities increases the virulence of the insect pathogen
Beauveria bassiana. J Invertebr Pathol 102:155–159
Faria MR, Wraight SP (2007) Mycoinsecticides and mycoacaricides: a comprehensive list with
worldwide coverage and international classification of formulation types. Biol Control
43:237–256
Feng P, Shang Y, Cen K, Wang C (2015) Fungal biosynthesis of the bibenzoquinone oosporein to
evade insect immunity. Proc Natl Acad Sci U S A 112:11365–11370
Fukuda T, Arai M, Yamaguchi Y, Masuma R (2004a) New beauvericins, potentiators of antifungal
miconazole activity, produced by Beauveria sp. FKI-1366. I. Taxonomy, fermentation, isola-
tion and biological properties. J Antibiot (Tokyo) 57:110–116
Fukuda K, Arai M, Yamaguchi Y, Masuma R, Tomoda H, Omura S (2004b) New beauvericins,
potentiators of antifungal miconazole activity produced by Beauveria sp FKI-1366 Structure
elucidation. J Antibiot (Tokio) 57:117–124
Goettel MS, Eilenberg J, Glare T (2010) Entomopathogenic fungi and their role in regulation of
insect populations. In: Gilbert LI, Gill SS (eds) Insect control: biological and synthetic agents.
Academic, Amsterdam, pp 387–432
Gomes SA, Paula A, Ribeiro A, Moraes C, Santos J, Silva CP et al (2015) Neem oil increases the
efficiency of the entomopathogenic fungus Metarhizium anisopliae for the control of Aedes
aegypti (Diptera: Culicidae) larvae. Parasit Vectors 8:669
Hajjar MJ, Ajlan AM, Al-ahmad MH (2015) New approach of Beauveria bassiana to control the
red palm weevil (Coleoptera: Curculionidae) by trapping technique. J Econ Entomol
108:425–432
Halo LM, Heneghan MN, Yakasai AA, Song Z, Williams K, Bailey AM et al (2008) Late stage
oxidations during the biosynthesis of the 2-pyridone tenellin in the entomopathogenic fungus
Beauveria bassiana. J Am Chem Soc 130:17988–17996
Hamill RL, Higgens GE, Boaz HE, Gorman M (1969) The structure of beauvericin, a new depsip-
eptide antibiotic toxic to Artemia salina. Tetrahedron Lett 49:4255–4258
He G, Yan J, Wu XY, Gou XJ, Li WC (2012) Oosporein from Tremella fuciformis. Acta Crystallogr
Sect E: Struct Rep Online 68:o1231
5 Beauveria bassiana as Biocontrol Agent: Formulation and Commercialization… 93
Huang B, Li CR, Li ZG, Fan MZ, Li ZZ (2002) Molecular Identification of the Teleomorph of
Beauveria bassiana. Mycotaxon 81:229–236
Jeffs LB, Khachatourians GG (1997) Toxic properties of Beauveria pigments on erythrocyte mem-
branes. Toxicon 35:1351–1356
Jegorov A et al. (1990) Are the depsipepeptides of Beauveria brongniartii involved in the entomo-
pathogenic process? In: Jegorov A, Matha V (eds) Proceeding of international conference on
biopesticides, theory and practice, pp 71–81
Jegorov A, Sedmera P, Matha V, Simek P, Zahradnícková H, Landa Z et al (1994) Beauverolides L
and La from Beauveria tenella and Paecilomyces fumosoroseus. Phytochemistry
37:1301–1303
Jirakkakul J, Punya J, Pongpattanakitshote S, Paungmoung P, Vorapreeda N, Tachaleat A et al
(2008) Identification of the nonribosomal peptide synthetase gene responsible for bassianolide
synthesis in wood-decaying fungus Xylaria sp. BCC1067. Microbiology 154:995–1006
Jirakkakul J, Cheevadhanarak S, Punya J, Chutrakul C, Senachak J, Buajarern T et al (2015)
Tenellin acts as an iron chelator to prevent iron-generated reactive oxygen species toxicity in
the entomopathogenic fungus Beauveria bassiana. FEMS Microbiol Lett 362:1–8
Jow G, Chou C, Chen B, Tsai J (2004) Beauvericin induces cytotoxic effects in human acute lym-
phoblastic leukemia cells through cytochrome c release, caspase 3 activation: the causative role
of calcium. Cancer Lett 216:165–173
Keswani C, Singh SP, Singh HB (2013) Beauveria bassiana: status, mode of action, applications
and safety issues. Biotech Today 3:16–20
Kirkland BH, Eisa A, Keyhani NO (2005) Oxalic acid as a fungal acaracidal virulence factor.
J Med Entomol 42:346–351
Kouti K, Lemmens M, Lemmens-Gruber R (2003) Beauvericin induced channels in ventricular
myocytes and liposomes. Biochim Biophys Acta 1609:203–210
Kwon HC, Bang EJ, Choi SU, Lee WC, Cho SY, Jung IY et al (2000) Cytotoxic cyclodepsipep-
tides of Bombycis corpus 101A. Yakhak Hoechi 44:115–118
Lewis MW, Robalino IV, Keyhani NO (2009) Uptake of the fluorescent probe FM4-64 by hyphae
and haemolymph-derived in vivo hyphal bodies of the entomopathogenic fungus Beauveria
bassiana. Microbiology 155:3110–3120
Li ZZ, Li CR, Huang B, Fan MZ (2001) Discovery and demonstration of the teleomorph of
Beauveria bassiana (Bals.) Vuill., an important entomogenous fungus. Chinese Sci Bull
46:751–753
Lin H, Lee Y, Chen B, Tsai M, Lu J, Chou C et al (2005) Involvement of Bc1-2 family, cytochrome
c and caspase 3 in induction of apoptosis by beauvericin in human non-small cell lung cancer
cells. Cancer Lett 230:248–259
Liu H, Zhao X, Guo M, Liu H, Zheng Z (2015) Growth and metabolism of Beauveria bassiana
spores and mycelia. BMC Microbiol 15:267
Manning RO, Wyatt RD (1984) Comparative toxicity of Chaetomium contaminated corn and vari-
ous chemical forms of oosporein in broiler chicks. Poultry Sci 63:251–259
Mao BZ, Huang C, Yang GM, Chen YZ, Chen SY (2010) Separation and determination of the
bioactivity of oosporein from Chaetomium cupreum. Afr J Biotechnol 9:5955–5961
McInnes AG, Smith DG, Wat CK, Vining LC, Wright JLC (1974) Tenellin and bassianin, metabo-
lites of Beauveria species. Structure elucidation with 15N- and doubly 13C-enriched com-
pounds using 13C nuclear magnetic resonance spectroscopy. J Chem Soc Chem Commun
1974:281–282
Ment D, Gindin G, Rot A, Soroker V, Glazer I, Barel S et al (2010) Novel technique for quantifying
adhesion of Metarhizium anisopliae conidia to the tick cuticle. Appl Environ Microbiol
76:3521–3528
Mishra S, Kumar P, Malik A (2013) Preparation, characterization, and insecticidal activity evalua-
tion of three different formulations of Beauveria bassiana against Musca domestica. Parasitol
Res 112:3485–3495
94 C. García-Estrada et al.
St Leger RJ, Cooper RM, Charnley AK (1986) Cuticle degrading enzymes of entomopathogenic
fungi: cuticle degradation in vitro by enzymes from entomopathogens. J Invertebr Pathol
47:167–177
St Leger RJ, Joshi L, Roberts DW (1997) Adaptation of proteases and carbohydrates of sapro-
phytic, phytopathogenic and entomopathogenic fungi to the requirements of their ecological
niches. Microbiology 143:1983–1992
Strasser H, Abendstein D (2000) Oosporein, a fungal secondary metabolite with antimicrobial
properties. IOBC/WPRS Bullet 23:113–115
Strasser H, Abendstein D, Stuppner H, Butt TM (2000a) Monitoring the distribution of secondary
metabolites produced by the entomogenous fungus Beauveria brongniartii with particular ref-
erence to oosporein. Mycol Res 104:1227–1233
Strasser H, Vey A, Butt TM (2000b) Are There Any Risks in Using Entomopathogenic Fungi for
Pest Control, with Particular Reference to the Bioactive Metabolites of Metarhizium,
Tolypocladium and Beauveria species? Biocontr Sci Technol 10:717–735
Sung JM, Lee JO, Humber RA, Sung GH, Shrestha B (2006) Cordyceps bassiana and Production
of Stromata in vitro Showing Beauveria Anamorph in Korea. Mycobiology 34:1–6
Suzuki A, Kanaoka M, IsogaiA MS, Ichinoe M, Tamura S (1977) Bassianolide, a new insecticidal
cyclodepsipeptide from Beauveria bassiana and Verticillium lecanii. Tetrahedron Lett
25:2167–2170
Toledo AV, de Remes Lenicov AMM, Lastra CCL (2010) Histopathology caused by the entomo-
pathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, in the adult planthopper,
Peregrinus maidis, a maize virus vector. J Insect Sci 10:35
Toshinori N, Kengo N, Kenji K, Tadao A (2004) Antifungal Activity of Oosporein from an
Antagonistic Fungus against Phytophthora infestans. Z Naturforsch 59:302–304
Vega FE, Posada F, Aime MC, Pava-Ripoli M, Infante F, Rehner SA (2008) Entomopathogenic
fungal endophytes. Biol Control 46:72–82
Vey A, Hoagland R, Butt TM (2001) Toxic metabolites of fungal biocontrol agents. In: Butt TM,
Jackson CW, Magan N (eds) Fungi as biocontrol agents: progress, problems and potential.
CAB International, Wallingford, pp 311–346
Vilcinskas A, Jegorov A, Landa Z, Götz P, Matha V (1999) Effects of beauverolide L and cyclo-
sporin A on humoral and cellular immune response of the greater wax moth, Galleria mel-
lonella. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 122:83–92
Wang Q, Xu L (2012) Beauvericin, a bioactive compound produced by fungi: a short review.
Molecules 17:2367–2377
Wätjen W, Debbab A, Hohlfeld A, Chovolou Y, Proksch P (2014) The mycotoxin beauvericin
induces apoptotic cell death in H4IIE hepatoma cells accompanied by an inhibition of NF-kB-
activity and modulation of MAP-kinases. Toxicol Lett 231:9–16
Wu SN, Chen H, Liu YC, Chiang HT (2002) Block of L-type Ca2+ current by beauvericin, a toxic
cyclopeptide, in the NG108-15 neuronal cell line. Chem Res Toxicol 15:854–860
Xiao G, Ying SH, Zheng P, Wang ZL, Zhang S, Xie XQ et al (2012) Genomic perspectives on the
evolution of fungal entomopathogenicity in Beauveria bassiana. Sci Rep 2:483
Xu Y, Orozco R, Wijeratne EM, Gunatilaka AA, Stock SP, Molnár I (2008) Biosynthesis of the
cyclooligomer depsipeptide beauvericin, a virulence factor of the entomopathogenic fungus
Beauveria bassiana. Chem Biol 15:898–907
Xu Y, Orozco R, Kithsiri Wijeratne EM, Espinosa-Artiles P, Leslie Gunatilaka AA, Patricia Stock
S et al (2009) Biosynthesis of the cyclooligomer depsipeptide bassianolide, an insecticidal
virulence factor of Beauveria bassiana. Fungal Genet Biol 46:353–364
Yacoub A, Batta YA (2016) Invert emulsion: Method of preparation and application as proper
formulation of entomopathogenic fungi. MethodsX 3:119–127
Zhang L, Yan K, Zhang Y, Huang R, Bian J, Zheng C et al (2007) High-throughput synergy screen-
ing identifies microbial metabolites as combination agents for the treatment of fungal infec-
tions. Proc Natl Acad Sci U S A 104:4606–4611
96 C. García-Estrada et al.
Zhang YJ, Feng MG, Fan YH, Luo ZB, Yang XY, Wu D et al (2008) A cuticle-degrading protease
(CDEP-1) of Beauveria bassiana enhances virulence. Biocontr Sci Technol 18:551–563
Zhang SZ, Xia YX, Kim B, Keyhani NO (2011) Two hydrophobins are involved in fungal spore
coat rodlet layer assembly and each play distinct roles in surface interactions, development and
pathogenesis in the entomopathogenic fungus, Beauveria bassiana. Mol Microbiol
80:811–826
Commercialization of Arbuscular
Mycorrhizal Technology in Agriculture 6
and Forestry
Abstract
The ecto- and endomycorrhizal fungi are commonly occurring mycorrhizas and
are very significant in relation to the growth of agricultural crops and forest trees.
Mycorrhizal technology can advantageously be applied in agricultural and horti-
cultural crops as well as forestry for better nutrient utilization offsetting ecologi-
cal and environmental concerns by reduced chemical input use, disease
management by reducing biotic stress by pathogenic fungi, and more effective
land use management. However, even though the inoculation of plants with
mycorrhiza is a familiar practice, the formulation of inocula with a dependable
and steady effect under field situation is still a bottleneck for their wider use. The
option of the technology for inocula production and of the carrier for the formu-
lation is key to their booming application. In this review, we focus on the status
of commercialization of mycorrhizal fungi as a gadget for enhancing plant
growth and productivity.
S. Pal
Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras
Hindu University, Varanasi 221005, India
Department of Biosciences, Integral University, Lucknow 226026, India
H.B. Singh
Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras
Hindu University, Varanasi 221005, Uttar Pradesh, India
A. Farooqui
Department of Biosciences, Integral University, Lucknow 226026, India
A. Rakshit (*)
Department of Soil Science and Agricultural Chemistry, Institute of Agricultural Sciences,
Banaras Hindu University, Varanasi 221005, India
e-mail: [email protected]
Keywords
Mycorrhizal technology • Biofertilizer • Transfer technology • Sustainable
agriculture
6.1 Introduction
Studies on the connections between plant, soil, and mycorrhizal fungi are shedding
light on their interrelationships, thus providing new possibilities to utilize them for
agriculture and forestry purposes. This symbiotic association results in distinct
enhancement in crop growth and development mediated by enlarged effectual
absorbing root surface (10X), nutrient absorption (N, P, Zn, Fe, B) and improved
water transport (2–3 10−5 mgs−1) in plants resulting endurance to avert water stress,
better outfitted to adverse effects of salt and also act to amplify intonation by
6 Commercialization of Arbuscular Mycorrhizal Technology in Agriculture and Forestry 99
symbiotic N2 fixing bacteria and provide resistance against plant disease (Hodge
2000: Rai et al. 2013). Arbuscular mycorrhizal fungi can account for 5–50 % of the
50 % of the microbial biomass in the soil. Efficient mycorrhizal symbiosis can sub-
stitute more than 225 kg P2O5 ha−1 (Rakshit et al. 2002). In field studies, the growth
and yield of crop species could be correlated to the mycorrhizal colonization rate.
Mycorrhizal expertise can also be successfully introduced with the on hand forestry
systems experienced to get better soil and crop productivity by allowing farmers to
sustainably reduce the use of fertilizers and/or by enhancing plant endurance. Fitting
mycorrhizal fungi can be included in nursery for raising mycorrhizal seedlings, and
transfer of seedlings to the field is a simple inoculation technique currently appro-
priate in plantation crops and trees (Hilderbrandt et al. 2002). These practical appli-
cations of mycorrhizal fungi become visible to have beneficial effects on soil
aggregation, thereby improving soil fertility, and may be important means of con-
trolling wearing away of topsoil. Cultural practices that augment the activity of
mycorrhizal fungi comprise of reduced tillage, crop rotations, cover crops, and
phosphorus management tool. Awareness in the application of these products is
going up due to the improvement in nutrient uptake efficiency and increasing soci-
etal anxiety for more green technologies in production and escalating expenses of
agrochemicals (Bisen et al. 2015, 2016). Furthermore, mycorrhizal biofertilizers
enjoy derived beneficial effects that would increase their convenience as
bioinoculants.
Although mycorrhizal fungi are found in 85 % of all plant families and occur in
many crop species for utmost growth, the extensive occurrence of these fungi in
virtually all soils confines the pressing needs for inoculation with these natural link-
ing fungi (Varma 1995, Newman 1988). Sophisticated scientific understanding on
mycorrhizal symbioses recently enhanced commercial prospective for the imple-
mentation of mycorrhizal technology in agriculture plant production horticulture,
forestry landscaping, phytoremediation in disturbed sites (Neill et al. 1991), and
other facets of the plant market.
Production of mycorrhizal inocula is a multifarious procedure as it requires
profit-making enterprises to develop the indispensable biotechnological proficiency
and capacity to counter to permissible, ethical, educational, and saleable require-
ments. There are a broad variety of formulation types which include both liquid and
solid and which can be tailored as per the requirement. Smart and high-class com-
mercial mycorrhizal inoculum is now available from an array of sources. Consortium
inocula containing mixtures of species of mycorrhizal fungi regularly give the best
response (Barea and Jeffries 1995). Mycorrhizal inoculum comes in granular, pow-
der, liquid, and tablet forms. Dry products comprise dusts, granules, and wettable
powders. Dusts have particle size ranging from 5 to 20 mm and contain <10 % of an
organism by weight. Granules are distinct masses 5–10 mm3 in size, pellets are
100 S. Pal et al.
>10 mm3, and briquettes are large blocks (5 g) up to several cubic centimeters.
These products include an inert carrier like charcoal, lignite, clay minerals (perlite,
vermiculite, and bentonite), starch polymers, dry fertilizers, and ground plant resi-
dues. Above all, the most significant aspect is to get the mycorrhizal propagules in
close proximity to the root systems of target plants. Presently a number of compa-
nies are producing mycorrhizal inocula for commercial purpose (Table 6.1). These
may include varying amounts of different species of fungi, different percentages of
viable spores, as well as additives such as fertilizers and hydrogels. Some inocu-
lants contain spores specific to particular species, while others contain a broad mix-
ture. When choosing commercially produced inoculants, it is important to look out
the specific plant requirement coupled with the existing soil conditions.
All through the last few years, there has been incredible explosion in market-
producing mycorrhizal fungi inocula and allied services for the retail and wholesale
segment. Clientele ranges from the familiar public and commercial growers to pub-
lic and private institutions (Tiwari et al. 2002). The growth in the mycorrhizal
industry is attributed to its growing body of scientific proof on the encouraging
6 Commercialization of Arbuscular Mycorrhizal Technology in Agriculture and Forestry 103
effects of mycorrhizal fungi on plant health, fitness, and productivity coupled with
mentoring by notable mycorrhizologist. In addition by tuning the appropriate inocu-
lants, economic feasibility of mycorrhizal technology is gaining more prominence.
Especially, there has been an appreciation in the market that mycorrhizal products
offer a sustainable strategy to plant production in the present format of climate-
resilient agricultural technology.
Even though during the last decade, mycorrhizal know-how reached a new height,
the bottlenecks of these applications should be vigilantly measured. There is a defi-
nite drawback to the obligate character of the AM symbiosis resulting in its inability
104 S. Pal et al.
to grow them in pure culture. According to current knowledge, they can only be
grown with plants in restricted conditions, with the addition in the inoculants imply-
ing that they cannot readily be multiplied in laboratory media. At present, in bulk of
the cases, mycorrhizal inoculum is produced as a non-sterile medium, either soil or
some other non-sterile substrate, which contains spores, hyphae, and colonized root
pieces. Because of this limitation, the present state of AM fungal inoculum produc-
tion technology makes direct application of the fungus inoculum in wide areas of
land burdensome and with poor cost-benefit ratio.
The rising sale of spurious bio-products and lack of vibrant quality control mech-
anism facilitate lower propagule number and their viability in many products. The
issue of spurious mycorrhizal products also has adverse impact on the health of
natural resource base. Further, the available commercial sources differ to a great
extent in the nature of carrier medium, the number of species claimed to be present,
and the number of active spores per unit weight or volume. The slower growth of
fungi in comparison to other microorganisms makes its hurdle in popularization in
large-scale agriculture.
6.6 Conclusion
Mycorrhizal fungi may be one option that can straightaway improve productivity in
natural and managed ecosystems without deterioration of natural resource base and
reduction of fertilizer costs and energy demands restoring economic efficiency and
environmental security. If better strains of mycorrhizal fungi were developed, they
could potentially advance growth of nearly all agronomic crops in a wide diversity
of soils throughout the globe. Both endomycorrhizae and ectomycorrhizae are in
commercial production on a small scale. Mycorrhizal inoculum production systems
in a gel-based carrier have been proposed and manufactured by some elite scientists
that are very concentrated and free from any microbial contamination. Such break-
throughs are necessary for mycorrhizal fungi to be practically used. The greatest
obstacle in the commercialization of mycorrhizal fungi appears to be the lack of
large-scale field tests under typical agroecology, adequate economics of the mycor-
rhizal fungi technology, and a growing unwillingness on the part of cultivators to
switch from an energy-intensive system to a new, but cheaper, energy-conservative
system using mycorrhizal fungi. Advocation and huge literacy drive of these geo-
graphically most widespread mutualistic associations could open up to the mycor-
rhizal industry an innovative approach for promoting a potential technology in
sustainable agricultural system ensuring their effective quality product availability
in the market. And with reference to commercialization strategy for mycorrhizal
technology to be victorious, it must be competent, efficient, and spotlight on out-
comes. Creating an entrepreneurial culture in the firm backed up by strong research
infrastructure, network, and funding is another prerequisite. In years to come, it is
highly likely that mycorrhizal biofertilizer can be a reliable partner with chemical
inputs bringing benefits in agronomic, economic, and social perspectives.
6 Commercialization of Arbuscular Mycorrhizal Technology in Agriculture and Forestry 105
References
Adholeya A (2003) Commercial production of AMF through industrial mode and its large scale
application. In: Proceedings of the 4th international conference on Mycorrhizae (ICOM4),
Montréal
Barea JM, Jeffries P (1995) Arbuscular mycorrhizas in sustainable soil plant systems. In: Varma A,
Hock B (eds) Mycorrhiza: structure, function, molecular biology and biotechnology. Springer,
Berlin/Heidelberg/New York, pp 521–559
Bisen K, Keswani C, Mishra S, Saxena A, Rakshit A, Singh HB (2015) Unrealized potential of
seed biopriming for versatile agriculture. In: Rakshit A, Singh HB, A Sen (eds) Nutrient use
efficiency: from basics to advances. Springer, New Delhi, pp 193–206
Bisen K, Keswani C, Patel JS, Sarma BK, Singh HB (2016) Trichoderma spp.: Efficient inducers
of systemic resistance in plants. In: Chaudhary DK, Verma A (eds) Microbial-mediated induced
systemic resistance in plants. Springer, Singapore, pp 185–195
Brundrett MC (1991) Mycorrhizas in natural ecosystem. Adv Ecol Res 21:171–173
Hilderbrandt U, Janetta K, Bothe H (2002) Towards growth of arbuscular mycorrhizal fungi inde-
pendent of a plant host. Appl Environ Microbiol 68:1919–1924
Hodge A (2000) Microbial ecology of arbuscular mycorrhiza. FEMS Microbiol Ecol 32:91–96
Neill EGO, Neill RVO, Norby RJ (1991) Hierarchy theory as a guide to mycorrhizal research on
large-scale problems. Environ Pol 73:271–284
Newman EI (1988) Mycorrhizal links between plants: their functioning and ecological signifi-
cance. Adv Ecol Res 18:243–270
Pal S, Singh HB, Rai A, Rakshit A (2013) Evaluation of different medium for producing on farm
arbuscular mycorrhizal inoculums. Int J Agric Environ Biotechnol 6:557–562
Pal S, Farooqui A, Rakshit A, Rai S, Rai A, Singh HB (2015) Mycorrhiza in a changing environ-
ment helps plants to deal stress. In: Sarma BK, Singh A (eds) Microbial empowerment in
agriculture – a key to sustainability and crop productivity. Biotech Books, New Delhi,
pp 109–128
Parewa HP, Rakshit A, Ali M, Lal B (2014) Arbuscular mycorrhizal fungi: a way to improve soil
quality. Popular Kheti 2:85–92
Rai A, Rai S, Rakshit A (2013) Mycorrhiza-mediated phosphorus use efficiency in plants. Environ
Exp Biol 11:107–117
Rakshit A (2015) Soil biodiversity: stars beneath our feet. Satsa Mukhapatra 9:43–48
Rakshit A, Bhadoria PBS, Das DK (2002) An overview of mycorrhizal symbioses. J Interacademicia
6:570–581
Tiwari P, Prakash A, Adholeya A (2002) Commercialization of arbuscular mycorrhizal fungi. In:
Arora (ed) Handbook of fungal biotechnology. Marcel Dekker, New York
Varma A (1995) Ecophysiology and application of arbuscular mycorrhizal fungi in arid soils. In:
Varma A, Hock B (eds) Mycorrhiza, structure, function, molecular biology and biotechnology.
Springer, Berlin/Heidelberg, pp 561–591
www.fao.org/docrep/article/wfc/xii/0961-b1.htm
Microbial Consortial Products
for Sustainable Agriculture: 7
Commercialization and Regulatory
Issues in India
Abstract
Rhizosphere microorganisms directly and indirectly influence the composition
and productivity of natural plant communities. Hence, belowground microbial
species richness has been proposed as a predictor of aboveground plant diversity
and productivity. Though research-based evidences clearly show the advantages
of microbial consortia-based products due to their multifunctionality, limited
attention is being given to develop quality standards for registration. This chapter
focuses on the uses, commercialization, and regulatory issues of various bacte-
rial consortia in sustainable agriculture.
Keywords
Consortia • Sustainable agriculture • Biofertilizers • Biopesticides • Rhizosphere
7.1 Introduction
Microbes are the most diverse communities on Earth that play a pivotal role in
Earth’s climatic, geological, geochemical, and biological process (Tringe et al.
2005; Xu 2006). The diverse genetic and functional groups of the soil microbial
population exert a critical impact on soil function (Barea et al. 2005; Avis et al.
2008), particularly in the root–soil microhabitat referred to as rhizosphere which is
considered as the hot spot for interaction between eukaryotes and prokaryotes
(Jones and Hinsinger 2008; Hinsinger et al. 2009; Raaijmakers et al. 2009).
Fig. 7.1 Schematic representation of the functions and interactions of the rhizomicrobiome
(Source: Mendes et al. 2013)
tolerance (Tringe et al. 2005; Zahir et al. 2009; Palaniyandi et al. 2013; Parihar et al.
2015; Shrivastava and Kumar 2015), production of phytoalexins/flavonoid-like
compounds, and enhancement of mineral uptake (Parmar and Dadarwal 1999). The
microbial community in the rhizosphere harbors members of few groups that
adversely affect plant growth and health, viz., pathogenic fungi, oomycetes, bacte-
ria, and nematodes (Raaijmakers et al. 2009; Damiani et al. 2012; Weller et al. 2012;
Sekar and Prabavathy 2014).
Rhizosphere-associated copious beneficial microbial groups with multi-
beneficial plant growth-promoting traits have been reported by many researchers
(Raupach and Kloepper 1998; Picard and Bosco 2008; Ryan et al. 2008; Hartmann
et al. 2009; Sekar and Prabavathy 2014; Viswanath et al. 2015; Krishnan et al. 2016;
Raju et al. 2016). Bacterial groups secrete signaling molecules that influence bacte-
rial gene expression and physiological behavior in a density-dependent manner
termed quorum sensing (QS) (Zhang and Pierson 2001; Schuhegger et al. 2006; Liu
et al. 2007; Viswanath et al. 2015); especially the rhizosphere regions were reported
to harbor high N-acyl homoserine lactone (AHL) population (Elasri et al. 2001;
DeAngelis et al. 2008; Viswanath et al. 2015). The QS-controlled phenotypes play
a vital role for successful inter-/intra-gene and host interactions, whether symbiotic
or pathogenic (Boyer and Wisniewski-Dyé 2009), and also influence interaction
110 J. Sekar et al.
with plants such as root colonization and induction of systemic resistance (Pang
et al. 2008; Hartmann et al. 2014).
During the past few decades, the interaction between rhizobacteria and plants
has been well explored and has resulted in the application of microbial products as
crop inoculants (biofertilizers/biopesticides), for increased crop biomass and dis-
ease suppression. Combined application of potential PGPR strains is termed as
microbial consortium (MC) which offers multi-beneficial plant growth-promoting
traits and provides solution to underpinning problems like drought, salinity, increas-
ing temperature, pest, and phytopathogenic infections in the agricultural system
leading to global food safety and security. Microbial consortia are inoculants in a
synergistic mixture which fulfill diverse functions in the rhizosphere and are the
most promising contenders for solving challenges linked to sustainable eco-friendly
agriculture (Jain et al. 2013).
Rhizobia and other PGPR share a common microhabitat, the root–soil interface,
where interaction between different microbial groups was reported during root col-
onization. Co-inoculation of rhizobia with other PGPR enhanced nodulation and
nitrogen fixation through the production of plant hormone, flavonoids, Nod factor,
or enzymes in pigeon pea and other legumes (Tilak et al. 2006; Dardanelli et al.
7 Microbial Consortial Products for Sustainable Agriculture: Commercialization… 111
2008; Remans et al. 2008; Medeot et al. 2010; Bansal and Srivastava 2012; Gupta
et al. 2015). Azospirillum, a free-living diazotroph, Azotobacter, Bacillus,
Pseudomonas, Serratia, and Enterobacter are a few genera that have been success-
fully used with rhizobium as co-inoculants (Gaind et al. 2007; Remans et al. 2008;
Cassán et al. 2009; Ahmad et al. 2011; Dashadi et al. 2011; Tajini et al. 2012;
Ahemad and Kibret 2014; Gopalakrishnan et al. 2014). Besides the indigenous rhi-
zobia community, inoculated diazotrophs like Azospirillum enhanced growth and
yield in leguminous crops upon inoculation and increased fixed nitrogen quantity
(Remans et al. 2008). Co-inoculation of A. lipoferum and R. leguminosarum bv.
trifolii improved nodulation in white clovers, pigeon pea, and chickpea (Deanand
et al. 2002). Most of the studies showed co-inoculation of Azospirillum, and
Rhizobium significantly increased both the upper and total nodule number, acety-
lene reduction activities, faster 15 N dilution, and the total macro- and micronutrient
mineral content as compared to other inoculants (Rodelas et al. 1996; German et al.
2000; Dardanelli et al. 2008; Askary et al. 2009; Cassán et al. 2009; Dashadi et al.
2011). Mehboob et al. (2013) extensively reviewed the effects of co-inoculation of
rhizobia with various rhizospheric bacteria. Azotobacter was found to be a potential
co-inoculant with rhizobium and enhanced the production of phytohormones and
vitamins (Chandra and Pareek 2002; Qureshi et al. 2009; Dashadi et al. 2011;
Akhtar et al. 2012). Co-inoculation of G. intraradices, Pseudomonas striata, and
Rhizobium showed significant increase in plant growth, number of pods, and chlo-
rophyll content in chickpea root rot (Akhtar and Siddiqui 2008).
Combination of Rhizobium with Bacillus strains was reported to improve root
structure and nodule formation in bean, pigeon pea, and soybean (Halverson and
Handelsman 1991; Srinivasan et al. 1997; Rajendran et al. 2008; Schwartz et al.
2013). Significant increase in root weight and seed yield of chickpea was reported
upon inoculation of Rhizobium with B. subtilis OSU-142 and B. megaterium M-3
(Elkoca et al. 2010). Interaction of Paenibacillus lentimorbus NRRL B-30488 and
Piriformospora indica DSM 11827 and their consortia with native rhizobia popula-
tion in the rhizosphere of Cicer arietinum enhanced nodulation, thereby increasing
plant growth (Nautiyal et al. 2010). When R. tropici CIAT899 was co-inoculated
with Chryseobacterium balustinum Aur9, it resulted in increased root hair forma-
tion and infection sites leading to early nodule development and increased nodule
formation (Estevez et al. 2009). A mixture of Bacillus atrophaeus and Burkholderia
cepacia significantly reduced vascular wilt and corm rot in gladiolus diseases and
enhanced plant growth by the elicitation of defense enzymes under field and green-
house condition (Shanmugam et al. 2011).
Combined application of IAA-producing Pseudomonas sp. and Mesorhizobium
sp. increased nodule formation and plant dry weight compared to Mesorhizobium
alone inoculated and uninoculated (Malik and Sindhu 2011) plants. Similar effects
were observed in chickpea upon co-inoculation with Mesorhizobium sp. and P.
aeruginosa (Verma et al. 2013; Verma et al. 2014). Comparable plant growth-
promoting effects along with antagonistic activities against F. oxysporum and R.
solani were observed in chickpea by co-inoculation of Mesorhizobium, Azotobacter
chroococcum, P. aeruginosa, and T. harzianum.
112 J. Sekar et al.
Synergistic interaction between PGPR and AMF has been reported to increase yield
and biomass in several plants under nursery and field conditions (Jia et al. 2004;
Singh et al. 2008; Adesemoye et al. 2009; Singh et al. 2009; Wang et al. 2011; Tajini
et al. 2012). Rhizosphere microorganisms either interfere or benefit mycorrhiza
establishment (Pivato et al. 2009; Bonfante and Genre 2010; Miransari 2011; Tajini
et al. 2012; Aroca et al. 2013). The beneficial effects exerted by the so-called mycor-
rhiza helper bacteria (MHB), a term referring to bacteria which enhance mycorrhiza
formation, were reported by Frey-Klett et al. (2007). AMF and PGPR mycorrhiza
helper bacteria interaction has beneficial implication in agriculture (Rabie et al.
2005; Aliasgharzad et al. 2006; Gamalero et al. 2008; Miransari 2011; Wang et al.
2011; Armada et al. 2015).
Co-inoculation of AMF with one or more PGPR has been reported to enhance
growth and productivity in different crops (Dutta and Podile 2010; Reddy and
Saravanan 2013). Several studies have reported the positive interactions between
AMF and a wide range of PGPR, including phosphate-solubilizing bacteria, nodule-
forming N2-fixing rhizobia, and free-living Azospirillum spp., Bacillus sp., and
Pseudomonas sp. (Gamalero et al. 2008; Singh et al. 2009). Co-inoculation of AMF
and PGPR was reported to have a synergistic effect on plant growth especially under
growth-limited conditions (Vivas et al. 2003a, b). Among the microbial groups,
PGPR and AMF promote activities which improve agricultural development (Barea
et al. 2005). The bioinoculants AMF and PGPR had a significant effect on grain
quality, for instance, the phosphorus content doubled in the bioinoculant-applied
rainfed wheat, both in greenhouse and field experiments (Roesti et al. 2006).
Co-inoculation of AM fungi and biocontrol agents resulted in the suppression of
soilborne pathogens such as Fusarium and Rhizoctonia. Enhanced bioprotection
results by the combination of mechanism exhibited by individual organisms, such
as competition, altered root exudates, morphological changes in the root system,
antibiosis, and activation of plant defense response (Saldajeno et al. 2008).
The AM symbiosis in legumes and its role in improving nodulation and nitrogen
fixation by legume–rhizobia association either at the colonization or symbiotic
functional stage have been reported (Lesueur et al. 2001; Lesueur and Sarr 2008;
Azcón and Barea 2010). Positive effects of the combination of mycorrhizal fungi
and/or PGPR on plant growth and plant health as biostimulators, biofertilizers, and
bioprotectants have been described by many authors (Barea et al. 2002; Azcón and
Barea 2010; Sharma et al. 2016). Arbuscular mycorrhizal fungi (AMF) and rhizobia
are the most important symbionts for the plant to acquire nutrients efficiently and to
promote growth. Tajini et al. (2012) used Glomus intraradices, a potential P mobi-
lizer, and R. tropici CIAT899, a nitrogen fixer, to increase the phosphorus-use effi-
ciency for symbiotic nitrogen fixation in common bean (Phaseolus vulgaris L.).
Co-inoculation of rhizobia and arbuscular mycorrhizal fungi (AMF) promoted
growth of soybean under low phosphorus and nitrogen conditions, indicated by
increased shoot dry weight (Wang et al. 2011).
114 J. Sekar et al.
Boby and Bagyaraj (2003) reported the effect of G. mosseae, P. fluorescens, and
T. viride consortium against soilborne root-rot wilt caused by Fusarium chlamydo-
sporum in Coleus forskohlii. Consortia of T. viride and G. mosseae decreased the
disease severity and enhanced maximum growth compared to other combinations.
Another study by Singh et al. (2009) reported the most effective suppression of
root-rot wilt in C. forskohlii by a consortium of AM fungus G. fasciculatum and P.
fluorescens. Though in both the reports consortium showed enhanced biocontrol
activity against root-rot wilt, the combination of efficient compatible strains in the
consortium contributes to more efficient control of the pathogen.
Consortium of Bradyrhizobium sp. BXYD3 and G. mosseae significantly
decreased the severity of Cylindrocladium parasiticum incidence in soybean by
altering the pathogen defense-related (PR) genes PR2, PR3, PR4, and PR10 expres-
sion level (Gao et al. 2012). A combined bio-inoculation of 2,4-diacetylphlorogluci
nol-producing PGPR strains and AMF synergistically improved the nutritional
quality of the grain in three Indian rainfed wheat without negatively affecting
mycorrhizal growth (Roesti et al. 2006), and in addition it stimulated both mycelial
development and spore germination in G. mosseae and enhanced root colonization
in tomato (Barea et al. 1998). Combined application of AM fungus F. mosseae with
Paenibacillus and Pantoea spp. enhanced all the biometric parameters in French
bean especially the total shoot dry biomass and fruit yield.
Rhizobium and AMF co-inoculation increased leaf area and biomass production
in broad bean (Vicia faba), AMF colonization increased the supply of P, and
Rhizobium facilitated N accumulation (Jia et al. 2004). The application of a consor-
tium of microbial inoculants such as mycorrhiza and Azospirillum brasilense effec-
tively increased plant growth and enhanced the ability of plants to alleviate drought
and nutrient stress (Azcón and Barea 2010). AM fungus G. intraradices enhanced
growth, photosynthetic efficiency, and antioxidative response in rice against drought
stress (Ruiz-Sanchez et al. 2010).
Kamal et al. (2016) evaluated the impact of Streptomyces labedae (SB-9),
Streptomyces flavofuscus (SA-11), Pseudomonas poae (KA-5), P. fluorescens (KB-
7), and G. intraradices consortium combination which showed pronounced increase
in the finger millet plant growth under drought condition.
Seed priming with consortia of T. harzianum and fluorescent pseudomonas
decreased the Fusarium wilt incidence, increased seed germination by 22–48 %,
and reduced the germination period (Srivastava et al. 2010). The enhanced perfor-
mance of microbial consortia compared to single inoculation is reported in several
crops including legumes (Antoun et al. 1998; Valdenegro et al. 2001; Ane et al.
2004; Bagyaraj and Kehri 2012; Bagyaraj 2014). Consortium product “Shu Dekang”
showed significant control of several phytopathogenic infestations like leaf speck
disease, banana wilt, and root-knot disease (Zheng et al. 2010). Thus, PGPR consor-
tia with multiple functions provide multiple growth-promoting and stress-tolerant
benefits in plants.
7 Microbial Consortial Products for Sustainable Agriculture: Commercialization… 115
The global climate is a great challenge for the agricultural sector, as predicted
increases in salinity, drought, and rising temperature cause abiotic stress in the plant
which reduce crop productivity (Grover et al. 2011; Larson 2013). About 60 % of
the global geographical area faces soil degradation either by waterlogging or salin-
ity or alkalinity, which threatens food security, the situation being worse in higher
rainfall areas where waterlogging follows shortly after the rains (Singh 2000).
Plant-associated microbial communities have received considerable attention for
their ability to confer many of the same benefits to crop productivity and stress
resistance as have been achieved through plant breeding programs (Mayak et al.
2004; Barrow et al. 2008; Marulanda et al. 2009; Mapelli et al. 2013). Microbial
symbionts are capable of conferring multiple stress tolerance against both abiotic
and biotic stress (Mayak et al. 2004; Rodriguez et al. 2008) benefits in both mono-
cot and dicot crop species (Timmusk and Wagner 1999; Redman et al. 2002; Zhang
et al. 2008).
Application of microbial inoculants specially consortia will be one of the solu-
tions to alleviate plant abiotic stress and enhance plant growth and productivity
under stress conditions (Yang et al. 2009; Jain et al. 2013). Multiple beneficial PGP
and abiotic stress-resistant strains, efficient 2,4-DNT-degrading consortia composed
of Burkholderia, Variovorax, Bacillus, Pseudomonas, and Ralstonia spp., have been
reported (Shirley et al. 2000; Snellinx et al. 2003) to enhance the root length of
Arabidopsis under 2,4-DNT stress, by doubling the root length within 9 days (Thijs
et al. 2014).
Co-inoculation of A. brasilense with R. tropici on bean relieved negative effects
of salt stress and nod gene transcription (Dardanelli et al. 2008). Microbial consor-
tium comprising of P. fluorescens (PHU094), Trichoderma (THU0816), and
Rhizobium (RL091) enhanced the expressions of defense systems like antioxidant
enzymes superoxide dismutase and peroxidase activities (Singh et al. 2013) under
stress. The response of rice plants to inoculation with an AMF and A. brasilense
consortia under drought stress conditions was due to enhanced ascorbate accumula-
tion. The effect of A. brasilense was pronounced only when mycorrhizal coloniza-
tion was established; thus, the bacterial and fungal consortia were responsible for
the protection of plant against plant pathogens (Ruiz-Sanchez et al. 2011). PGPR
consortium of endophytic bacterium P. pseudoalcaligenes in combination with B.
pumilus-treated plants showed increased concentrations of NPK and reduced con-
centrations of Na and Ca in paddy under saline conditions (Jha and Subramanian
2013). Co-inoculation of P. fluorescens Aur6 and Chryseobacterium balustinum
Aur9 in three field experiments induced systemic resistance in rice against rice blast
and increased rice productivity and grain quality under saline conditions (Lucas
et al. 2009).
Under drought stress cucumber seedlings treated with consortium product of Shu
Dekang containing B. cereus AR156, B. subtilis SM21, and Serratia sp. XY21
showed enhanced photosynthetic efficiency, less wilt symptoms, decreased leaf
monodehydroascorbate (MDA), increased leaf proline content, enhanced induced
116 J. Sekar et al.
Biofertilizer commercialization began with the rhizobia product in the year 1895 by
Nobbe and Hiltner under the brand of “Nitragin.” In India, N. V. Joshi first started
the commercialization of rhizobium for the growth promotion of leguminous plant
(Rivas et al. 2015). During its ninth five-year plan, the Ministry of Agriculture initi-
ated the popularization and promotion of biofertilizer production, developing stan-
dards for different biofertilizers, training, and utilization by launching National
Project on Development and Use of Biofertilizers (NPDB), and a National
Biofertilizer Development Centre was established, with six regional centers (Ghosh
2004). The government of India and state governments took several measures for
promoting the production of biofertilizers by providing grants and subsidies at dif-
ferent levels.
The Ministry of Agriculture passed a new decree on the control of biofertilizer
production and marketing standards with regard to different kinds of microorgan-
isms. The product should fulfill seven quality parameters like physical form, mini-
mum count of viable cells, contamination level, pH, particle size in the case of
carrier-based materials, maximum moisture percent by weight of carrier-based
products, and efficiency character. In bacterial bioproducts the minimum viable
cells to be maintained is 5 × 107 CFU g−1 for solid carrier or 1 × 108 CFU ml−1 for
liquid carrier. For products containing mycorrhizal fungi, at least 100 viable propa-
gules must be present per gram of product. Nitrogen-fixing efficiency of biofertil-
izer product should be capable of fixing at least 10 mg N g−1 of sucrose consumed
and for phosphate solubilization product a zone of solubilization at least 5 mm in a
media. AMF products should provide 80 infection points in roots g−1 of inoculum
(Ministry of Agriculture 2009).
Markets and Markets (2015) report shows that the biofertilizer market is pro-
jected to grow at a CAGR of 14.0 % from 2015 to 2020 and is expected to reach US
$1.88 billion by 2020. Leading players in the biofertilizer market include Gujarat
State Fertilizers & Chemicals Ltd. (India), Novozymes A/S (Denmark), Rizobacter
Argentina S.A. (Argentina), Camson Bio Technologies Limited (India), and
Lallemand, Inc. (Canada) (RNR Market Research 2014). Biofertilizer market in
118 J. Sekar et al.
Asia is strongly influenced by the government and its policies to promote sustain-
able and green agriculture. Around US $1.5 billion has been spent on the develop-
ment of biofertilizer and biopesticide products (Rivas et al. 2015). Currently there is
an increase in organic agriculture practice in the country with around 1,000,000 ha
under organic cultivation (Keshri 2016).
In India, around 100 public and private companies are involved in biofertilizer
production, and the list of a few companies and their consortial products are listed
in Table 7.1 (Rivas et al. 2015). Biofertilizer production and consumption have
gained importance in the recent times in India (Pindi and Satyanarayana 2012). The
average consumption in the country is about 45,000 t per annum, while its produc-
tion is less than half of the consumption. The maximum production capacity lies in
Agro Industries Corporation followed by state agriculture departments, National
Biofertilizers Development Centres, State Agricultural Universities, and private
sectors (Mazid and Khan 2014).
Worldwide the use and demand for biopesticides are rising due to the increased
awareness of pesticide residue-free crops. The global-level estimate for microbial
products in 2014 was US $ 2,183 million which is projected to double by US $ 4556
million in 2019 with a CAGR of 15.3 %. Of the several microbial types, the bacte-
rial segment accounted for the largest share (US $1.6 billion). Similar to biopesti-
cides, market for biofertilizers at global level is projected to reach US $1.88 billion
by 2020 at a CAGR of 14.0 % from 2015 to 2020 (Markets and Markets 2015).
Globally, more than 200 biopesticide active ingredients are registered, and 700
products are available in the market. In the case of India, 15 biopesticides were
registered as on 2008 under IA (1968), and its market share is only 4.2 % of the
overall pesticide market; however, it is predicted to increase at an annual growth
rate of 10 % (Suresh 2012). While its growth was multifold during the past years,
NAAS (2013) reported around 400 registered biopesticide active ingredients and
over 1250 actively registered biopesticide products in Indian markets. It shows the
awareness among farmers as well as policy support of the government to use the
ecologically safe products for pest management. However, there is no specific men-
tion about microbial consortium among 400 registered biopesticides individually.
At the international level, the regulatory frameworks differ widely among differ-
ent countries. In the USA, biopesticide production is institutionalized under a sepa-
rate division as “Biopesticides and Pollution” within the Environmental Protection
Agency (EPA). To maintain the quality, it specified good laboratory practices regu-
latory testing for microbial biopesticides in 1983 as EPA M guidelines. Following
the line, in 1996 the Japanese Ministry of Agriculture, Forestry and Fisheries
(JMAFF) harmonized its system with guidelines of EPA. Similarly in Europe,
biopesticides are evaluated through the European Pesticide Regulation EC No.
1107/2009 which promotes the production of less harmful substances, and it has
7 Microbial Consortial Products for Sustainable Agriculture: Commercialization… 119
been promoting the registration of low-risk products for pest control through
(2009/128/EC) simple and transparent registration protocols (Villaverde et al.
2014). Canada follows only the safety test and the rest of the countries need data of
both safety and efficacy tests. The EPA, JMAFF, and EC regulations toward biopes-
ticides are developed in such a way that it requires less data when compared to
chemical products and reduced the time to process the registration applications. In
this context, the International Organization for Biological Control of Noxious
Animals and Plants (2010) carried out a global-level review on the use of biopesti-
cides and regulatory measures. It stressed the need for streamlining the registration
process through harmonizing data requirements and protocols for risk assessments.
In India, any microorganisms used for pest and disease management require regis-
tration for both production and sale with the Central Insecticides Board (CIB) of the
Ministry of Agriculture as per the Insecticides Act (IA), 1968, of the Government of
India (GOI) and Insecticides Rules, 1971, which were recently replaced by the
Pesticides Management Bill 2008. The biopesticides are considered as generally
regarded as safe (GRAS) under this act, and to promote its production and use, it
provides the benefit of priority in processing of registration as well as provisional
registration. Thus, the producers can register the product either for regular registra-
tion under section 9 (3) or for provisional registration under section 9 (3B) of the
IA. While applying for registration, the data on product characterization, safety,
toxicology, efficacy, and labeling are necessary. In addition to the priority and pro-
visional registration for biopesticides in the Act, the registration protocols are made
easier and accept generic data for any new products containing strains which are
already registered. Such affirmative clauses are inbuilt in the Act which shows the
interest of the government in promoting the safe products for pest management
similar to other countries. In order to regulate the commercial production of these
products, the Government of India established four different bodies to regulate the
biopesticide production. The Central Insecticides Board (CIB) is involved in devel-
oping appropriate policies, and the Registration Committee (RC) is responsible to
register the products for production. Whereas the Central Insecticides Laboratory
(CIL) is in charge to monitor the quality of the products available in the market,
finally the State Department for Agriculture (SDA) issues the manufacturing license
and performs quality check. However, coordination among the four bodies plays a
vital part in ensuring the registration and availability of quality products in the mar-
ket. Recently, efforts were taken to harmonize the IA of 1968 with the Organization
for Economic Cooperation and Development (OECD) during 2000s on the methods
and approaches to assess biological pesticides. On this basis, CIB has rationalized
the guidelines and data requirements for registration and infrastructure necessary
for production of the biopesticides (NAAS 2013). However, research studies on
how the harmonization eased the process of registration are yet unavailable. On the
other side, as per the notification dated March 26, 1999, of the Central Insecticides
Board, Ministry of Agriculture, biopesticide was put under the Insecticide Schedule
Act 1968, and hence, the generation of toxicological data became a prerequisite for
the registration of biopesticide. In spite of the relatively abundant number of patents
for microbial pesticides, the number of commercial applications has not been as
7 Microbial Consortial Products for Sustainable Agriculture: Commercialization… 121
dramatic as expected due to the high cost involved in toxicologic analysis, biosafety,
and environmental concerns (Montesinos 2003).
7.11 Conclusion
Though the performance of the PGPR and its consortia has been proved to promote
plant growth and enhance productivity in the field conditions by several strains in
different crops, the use of these products has not been popular among farmers due
to several reasons such as (1) lack of awareness among farmers and (2) availability
and supply of quality bioproducts. A survey conducted by Srinivas and Bhalekar
(2013) reported the communication gap that exists between farmers and manufac-
tures, miscommunication about the quality of the product, and sustainability of bio-
fertilizer as the major hurdle. In natural conditions and in disease-suppressive soil,
the existence of mixture of microbial antagonists (Lemanceau and Alabouvette
1991) has been reported. Hence, augmentation of compatible strains of PGPR to
infection court will mimic the natural environment and could broaden the spectrum
of biocontrol against different plant pathogens. Efficiency of biocontrol agents
could be increased by the development of compatible strain mixtures of different
biocontrol organisms by considering the following norms (Raupach and Kloepper
1998). While developing a consortial formulation, the following needs to be
addressed: (1) compatible strain combination that differs in the pattern of plant col-
onization, (2) compatible strain combination with broad spectrum of action against
different plant pathogens, (3) compatible strain combination with different modes
122 J. Sekar et al.
References
Adesemoye AO, Torbert HA, Kloepper JW (2009) Plant growth-promoting rhizobacteria allow
reduced application rates of chemical fertilizers. Microb Ecol 58(4):921–929
Ahemad M, Kibret M (2014) Mechanisms and applications of plant growth promoting rhizobacte-
ria: Current perspective. J King Saud Uni Sci 26:1–20
Ahmad M, Zahir ZA, Asghar HN, Asghar M (2011) Inducing salt tolerance in mung bean through
coinoculation with rhizobia and plant-growth-promoting rhizobacteria containing 1-aminocyc
lopropane-1-carboxylate deaminase. Can J Microbiol 57:578–589
Akhtar MS, Siddiqui ZA (2008) Biocontrol of a root-rot disease complex of chickpea by Glomus
intraradices, Rhizobium sp. and Pseudomonas straita. Crop Protect 27(3):410–417
Akhtar N, Qureshi M, Iqbal A, Ahmad M, Khan K (2012) Influence of Azotobacter and IAA on
symbiotic performance of Rhizobium and yield parameters of lentil. J Agric Res 50:361–372
Akila R, Rajendran L, Harish S, Saveetha K, Raguchander T, Samiyappan R (2011) Combined
application of botanical formulations and biocontrol agents for the management of Fusarium
oxysporum f. sp. cubense (Foc) causing Fusarium wilt in banana. Biol Control 57:175–183
Aliasgharzad N, Reza M, Neyshabouri Salimi G (2006) Effects of arbuscular mycorrhizal fungi
and Bradyrhizobium japonicum on drought stress of soybean. Biologia 19:324–328
Ane JM, Kiss GB, Riely BK, Penmetsa RV, Oldroyd GE, Ayax C, Levy J, Debelle F, Baek JM,
Kalo P, Rosenberg C, Roe BA, Long SR, Denarie J, Cook DR (2004) Medicago truncatula
DMI1 required for bacterial and fungal symbioses in legumes. Science 303:1364–1367
Antoun H, Beauchamp C, Goussard N, Chabot R, Lalande R (1998) Potential of Rhizobium and
Bradyrhizobium species as plant growth promoting rhizobacteria on non-legumes: effect on
radishes (Raphanus sativus L.). Plant Soil 204:57–67
Armada E, Azcon R, Lopez-Castillo OM, Calvo-Polanco M, Ruiz-Lozano JM (2015)
Autochthonous arbuscular mycorrhizal fungi and Bacillus thuringiensis from a degraded
Mediterranean area can be used to improve physiological traits and performance of a plant of
agronomic interest under drought conditions. Plant Physiol Biochem 90:64–74
Aroca R, Porcel R, Ruiz-Lozano JM (2007) How does arbuscular mycorrhizal symbiosis regulate
root hydraulic properties and plasma membrane aquaporins in Phaseolus vulgaris under
drought, cold or salinity stresses? New Phytol 173:808–816
7 Microbial Consortial Products for Sustainable Agriculture: Commercialization… 123
Aroca R, Ruiz-Lozano JM, Zamarreno AM, Paz JA, Garcia-Mina JM, Pozo MJ, Lopez-Raez JA
(2013) Arbuscular mycorrhizal symbiosis influences strigolactone production under salinity
and alleviates salt stress in lettuce plants. J Plant Physiol 170:47–55
Askary M, Mostajeran A, Amooaghaei R, Mostajeran M (2009) Influence of the co-inoculation
Azospirillum brasilense and Rhizobium meliloti plus 2,4-D on grain yield and N, P, K content
of Triticum aestivum (cv. Baccros and Mahdavi). Am Eurasian J Agric Environ Sci 5:296–307
Avis TJ, Gravel V, Antoun H, Tweddell RJ (2008) Multifaceted beneficial effects of rhizosphere
microorganisms on plant health and productivity. Soil Biol Biochem 40:1733–1740
Azcón R, Barea J-M (2010) Mycorrhizosphere interactions for legume improvement. In: Khan
MS, Zaidi A, Musarrat J (eds) Microbes for legume improvement. Springer, New York,
pp 237–271
Bagyaraj D, Kehri H (2012) AM fungi: importance, nursery inoculation and performance after
outplanting. In: Bagyaraj D, Tilak K, Kehri H (eds) Microbial diversity and functions. New
India Publishing Agency, New Delhi, pp 641–668
Bagyaraj JD (2014) Ecology of arbuscular mycorrhizal fungi. In: Kharwar RN, Upadhyay RS,
Dubey NK, Raghuwanshi R (eds) Microbial diversity and biotechnology in food security.
Springer, New Delhi, pp 133–146
Bano A, Fatima M (2009) Salt tolerance in Zea mays (L). following inoculation with Rhizobium
and Pseudomonas. Biol Fertil Soils 45:405–413
Bansal R, Srivastava JP (2012) Antioxidative defense system in pigeon pea roots under water log-
ging stress. Acta Physiol Plant 34:515–522
Barea J, Pozo M, Azcon R, Azcon-Aguilar C (2005) Microbial co-operation in the rhizosphere.
J Exp Bot 56:1761–1778
Barea JM, Andrade G, Bianciotto VV, Dowling D, Lohrke S, Bonfante P, O’Gara F, Azcon-Aguilar
C (1998) Impact on arbuscular mycorrhiza formation of Pseudomonas strains used as inocu-
lants for biocontrol of soil-borne fungal plant pathogens. Appl Environ Microbiol
64:2304–2307
Barea JM, Azcon R, Azcon-Aguilar C (2002) Mycorrhizosphere interactions to improve plant fit-
ness and soil quality. Antonie Van Leeuwenhoek 81:343–351
Barrow JR, Lucero ME, Reyes-Vera I, Havstad KM (2008) Do symbiotic microbes have a role in
plant evolution, performance and response to stress? Commun Integr Biol 1:69–73
Bashan Y, de-Bashan LE (2005) Bacteria. In: Hillel D (ed) Encyclopedia of soils in the environ-
ment, vol 1. Elsevier, Oxford, pp 103–115
Berg G, Smalla K (2009) Plant species and soil type cooperatively shape the structure and function
of microbial communities in the rhizosphere. FEMS Microbiol Ecol 68(1):1–13
Bisen K, Keswani C, Mishra S, Saxena A, Rakshit A, Singh HB (2015) Unrealized potential of
seed biopriming for versatile agriculture. In: Rakshit A, Singh HB, Sen A (eds) Nutrient use
efficiency: from basics to advances. Springer, New Delhi, pp 193–206
Bisen K, Keswani C, Patel JS, Sarma BK, Singh HB (2016) Trichoderma spp.: Efficient Inducers
of Systemic Resistance in Plants. In: Chaudhary DK, Verma A (eds) Microbial-mediated
Induced Systemic Resistance in Plants. Springer, Singapore, pp 185–195
Boby V, Bagyaraj D (2003) Biological control of root-rot of Coleus forskohlii Briq. using micro-
bial inoculants. World J Microbiol Biotechnol 19(2):175–180
Bonfante P, Genre A (2010) Mechanisms underlying beneficial plant-fungus interactions in mycor-
rhizal symbiosis. Nat Commun 27:1–48
Boyer M, Wisniewski-Dyé F (2009) Cell–cell signalling in bacteria: not simply a matter of quo-
rum. FEMS Microbiol Ecol 70:1–19
Brimecombe MJ, De Leij FA, Lynch JM (2007) Rhizodeposition and Microbial Populations. In: R
Pinton, Z Varanini, P Nannipieri (eds) The rhizosphere: biochemistry and organic substances at
the soil-plant interface. CRC Press/Taylor & Francis Group, Boca Raton/London/New York,
pp 73–109
Buée M, de Boer W, Martin F, van Overbeek LS, Jurkevitch E (2009) The rhizosphere zoo: An
overview of plant-associated communities of microorganisms, including phages, bacteria,
archaea, and fungi, and some of their structuring factors. Plant Soil 321:189–212
124 J. Sekar et al.
Bulgarelli D, Schlaeppi K, Spaepen S, Ver Loren van Themaat E, Schulze-Lefert P (2013) Structure
and functions of the bacterial microbiota of plants. Annu Rev Plant Biol 64:807–838
Cai A, Xu H, Shao X, Zhu P, Zhang W, Xu M, Murphy DV (2016) Carbon and Nitrogen mineral-
ization in relation to soil particle-size fractions after 32 years of chemical and manure applica-
tion in a continuous maize cropping system. PLoS One 11:e0152521
Cassán F, Perrig D, Sgroy V, Masciarelli O, Penna C, Luna V (2009) Azospirillum brasilense Az39
and Bradyrhizobium japonicum E109, inoculated singly or in combination, promote seed ger-
mination and early seedling growth in corn (Zea mays L.) and soybean (Glycine max L.). Eur
J Soil Biol 45:28–35
Cavaglieri L, Orlando J, Etcheverry M (2009) Rhizosphere microbial community structure at dif-
ferent maize plant growth stages and root locations. Microbiol Res 164:391–399
Chandra R, Pareek R (2002) Effect of rhizobacteria in urdbean and lentil. Indian J Pulses Res
15(2):152–155
Chaparro JM, Badri DV, Vivanco JM (2014) Rhizosphere microbiome assemblage is affected by
plant development. ISME J 8:790–803
Chaparro JM, Sheflin AM, Manter DK, Vivanco JM (2012) Manipulating the soil microbiome to
increase soil health and plant fertility. Biol Fertil Soils 48:489–499
Couillerot O, Combes-Meynet E, Pothier JF, Bellvert F, Challita E, Poirier MA, Rohr R, Comte G,
Moenne-Loccoz Y, Prigent-Combaret C (2011) The role of the antimicrobial compound
2,4-diacetylphloroglucinol in the impact of biocontrol Pseudomonas fluorescens F113 on
Azospirillum brasilense phytostimulators. Microbiology 157:1694–1705
Damiani I, Baldacci-Cresp F, Hopkins J, Andrio E, Balzergue S, Lecomte P, Puppo A, Abad P,
Favery B, Herouart D (2012) Plant genes involved in harbouring symbiotic rhizobia or patho-
genic nematodes. New Phytol 194:511–522
Dardanelli MS, Fernández de Córdoba FJ, Espuny MR, Rodríguez Carvajal MA, Soria Díaz ME,
Gil Serrano AM, Okon Y, Megías M (2008) Effect of Azospirillum brasilense coinoculated with
Rhizobium on Phaseolus vulgaris flavonoids and Nod factor production under salt stress. Soil
Biol Biochem 40:2713–2721
Dashadi M, Khosravi H, Moezzi A, Nadian H, Heidari M, Radjabi R (2011) Co-inoculation of
Rhizobium and Azotobacter on growth indices of faba bean under water stress in the green
house condition. Adv Stud Biol 3:373–385
de Boer M, Bom P, Kindt F, Keurentjes JJ, van der Sluis I, van Loon LC, Bakker PA (2003) Control
of Fusarium wilt of radish by combining Pseudomonas putida strains that have different
disease-suppressive mechanisms. Phytopathology 93:626–632
De Deyn G, Raaijmakers C, Van der Putten W (2004) Plant community development is affected by
nutrients and soil biota. J Ecol 92:824–834
de Jensen CE, Percich J, Graham P (2002) Integrated management strategies of bean root rot with
Bacillus subtilis and Rhizobium in Minnesota. Field Crop Res 74:107–115
Deanand B, Patil A, Kulkaarni J, Algawadi A (2002) Effect of plant growth promoting rhizobacte-
ria on growth and yield of pigeon pea (Cajanus cajan L.) by application of plant growth pro-
moting rhizobacteria. Microbiol Res 159:371–394
DeAngelis KM, Lindow SE, Firestone MK (2008) Bacterial quorum sensing and nitrogen cycling
in rhizosphere soil. FEMS Microbiol Ecol 66:197–207
Dennis P, Miller A, Hirsch P (2010) Are root exudates more important than other sources of rhi-
zodeposits in structuring rhizosphere bacterial communities? FEMS Microbiol Ecol
72:313–327
Dunne C, Moënne‐Loccoz Y, McCarthy J, Higgins P, Powell J, Dowling D, O’Gara F (1998)
Combining proteolytic and phloroglucinol-producing bacteria for improved biocontrol of
Pythium-mediated damping-off of sugar beet. Plant Pathol 47:299–307
Dutta S, Podile AR (2010) Plant growth promoting rhizobacteria (PGPR): the bugs to debug the
root zone. Crit Rev Microbiol 36:232–244
Elasri M, Delorme S, Lemanceau P, Stewart G, Laue B, Glickmann E, Oger PM, Dessaux Y (2001)
Acyl-homoserine lactone production is more common among plant-associated Pseudomonas
spp. than among soilborne Pseudomonas spp. Appl Environ Microbiol 67:1198–1209
7 Microbial Consortial Products for Sustainable Agriculture: Commercialization… 125
Elkoca E, Turan M, Donmez MF (2010) Effects of single, dual and triple inoculations with Bacillus
subtilis, Bacillus megaterium and Rhizobium leguminosarum bv. Phaseoli on nodulation, nutri-
ent uptake, yield and yield parameters of common bean (Phaseolus vulgaris l. cv.‘elkoca-05’).
J Plant Nutr 33:2104–2119
Estevez J, Dardanelli M, Megias M, Rodríguez-Navarro D (2009) Symbiotic performance of com-
mon bean and soybean co-inoculated with rhizobia and Chryseobacterium balustinum Aur9
under moderate saline conditions. Symbiosis 49:29–36
Farajzadeh D, Yakhchali B, Aliasgharzad N, Sokhandan-Bashir N, Farajzadeh M (2012) Plant
growth promoting characterization of indigenous Azotobacteria isolated from soils in Iran.
Curr Microbiol 64:397–403
Figueiredo MV, Burity HA, Martínez CR, Chanway CP (2008) Alleviation of drought stress in the
common bean (Phaseolus vulgaris L.) by co-inoculation with Paenibacillus polymyxa and
Rhizobium tropici. Appl Soil Ecol 40:182–188
Fox SL, O’Hara GW, Bräu L (2011) Enhanced nodulation and symbiotic effectiveness of Medicago
truncatula when co-inoculated with Pseudomonas fluorescens WSM3457 and Ensifer
(Sinorhizobium) medicae WSM419. Plant Soil 348:245–254
Franzini VI, Azcon R, Mendes FL, Aroca R (2010) Interactions between Glomus species and
Rhizobium strains affect the nutritional physiology of drought-stressed legume hosts. J Plant
Physiol 167:614–619
Frey-Klett P, Garbaye J, Tarkka M (2007) The mycorrhiza helper bacteria revisited. New Phytol
176:22–36
Gaind S, Rathi MS, Kaushik BD, Nain L, Verma OP (2007) Survival of bio-inoculants on
fungicides-treated seeds of wheat, pea and chickpea and subsequent effect on chickpea yield.
J Environ Sci Health B 42:663–668
Gamalero E, Berta G, Massa N, Glick BR, Lingua G (2008) Synergistic interactions between the
ACC deaminase-producing bacterium Pseudomonas putida UW4 and the AM fungus
Gigaspora rosea positively affect cucumber plant growth. FEMS Microbiol Ecol 64:459–467
Gao X, Lu X, Wu M, Zhang H, Pan R, Tian J, Li S, Liao H (2012) Co-inoculation with rhizobia
and AMF inhibited soybean red crown rot: from field study to plant defense-related gene
expression analysis. PLoS One 7:e33977
German MA, Burdman S, Okon Y, Kigel J (2000) Effects of Azospirillum brasilense on root mor-
phology of common bean (Phaseolus vulgaris L.) under different water regimes. Biol Fertil
Soils 32:259–264
Ghosh N (2004) Promoting Biofertilisers in Indian Agriculture. Econ Polit Wkly 39:5617–5625
Gopalakrishnan S, Sathya A, Vijayabharathi R, Varshney RK, Gowda CLL, Krishnamurthy L
(2014) Plant growth promoting rhizobia: challenges and opportunities. 3 Biotech 5:355–377
Gray E, Smith D (2005) Intracellular and extracellular PGPR: commonalities and distinctions in
the plant–bacterium signaling processes. Soil Biol Biochem 37:395–412
Grover M, Ali SZ, Sandhya V, Rasul A, Venkateswarlu B (2011) Role of microorganisms in adap-
tation of agriculture crops to abiotic stresses. World J Microbiol Biotechnol 27:1231–1240
Gupta A, Saxena A, Gopal M, Tilak K (1998) Effect of plant growth promoting rhizobacteria on
competitive ability of introduced Bradyrhizobium sp. (Vigna) for nodulation. Microbiol Res
153:113–117
Gupta R, Bisaria VS, Sharma S (2015) Effect of agricultural amendments on Cajanus cajan
(pigeon pea) and Its rhizospheric microbial communities – a comparison between chemical
fertilizers and bioinoculants. PLoS One 10:e0132770
Halverson LJ, Handelsman J (1991) Enhancement of soybean nodulation by Bacillus cereus
UW85 in the field and in a growth chamber. Appl Environ Microbiol 57:2767–2770
Hartmann A, Rothballer M, Hense BA, Schroder P (2014) Bacterial quorum sensing compounds
are important modulators of microbe-plant interactions. Front Plant Sci 5:131
Hartmann A, Schmid M, van Tuinen D, Berg G (2009) Plant-driven selection of microbes. Plant
Soil 321:235–257
Hinsinger P, Bengough AG, Vetterlein D, Young IM (2009) Rhizosphere: biophysics, biogeochem-
istry and ecological relevance. Plant Soil 321:117–152
126 J. Sekar et al.
Hooper DU, Chapin FS, Ewel JJ, Hector A, Inchausti P, Lavorel S, Lawton JH, Lodge DM, Loreau
M, Naeem S, Schmid B, Setälä H, Symstad AJ, Vandermeer J, Da W (2005) Effects of biodi-
versity on ecosystem functioning: a consensus of current knowledge. Ecol Monogr 75:3–35
Jain A, Singh A, Singh BN, Singh S, Upadhyay RS, Sarma BK, Singh HB (2013) Biotic stress
management in agricultural crops using microbial consortium. In: Maheshwari KD (ed)
Bacteria in agrobiology: disease management. Springer, Berlin/Heidelberg, pp 427–448
Jain A, Singh A, Singh S, Singh HB (2015) Biological management of Sclerotinia sclerotiorum in
pea using plant growth promoting microbial consortium. J Basic Microbiol 55(8):961–972
Jain A, Singh S, Kumar Sarma B, Bahadur Singh H (2012) Microbial consortium-mediated repro-
gramming of defence network in pea to enhance tolerance against Sclerotinia sclerotiorum.
J Appl Microbiol 112(3):537–550
Jha Y, Subramanian R (2013) Paddy plants inoculated with PGPR show better growth physiology
and nutrient content under saline condition. Chilean J Agricult Res 73:213–219
Jia Y, Gray VM, Straker CJ (2004) The influence of Rhizobium and arbuscular mycorrhizal fungi
on nitrogen and phosphorus accumulation by Vicia faba. Ann Bot 94:251–258
Jones DL, Hinsinger P (2008) The rhizosphere: complex by design. Plant Soil 312:1–6
Jones DL, Nguyen C, Finlay RD (2009) Carbon flow in the rhizosphere: carbon trading at the
soil–root interface. Plant Soil 321:5–33
Kamal R, Gusain YS, Sharma IP, Sharma S, Sharma A (2016) Impact of arbuscular mycorrhizal
fungus, Glomus intraradices, Streptomyces and Pseudomonas spp. strain on finger millet
(Eleusine coracana L.) cv Korchara under water deficit condition. Afr J Biotechnol
14:3219–3227
Kathiravan R, Jegan S, Ganga V, Prabavathy VR, Tushar L, Sasikala C, Ramana CV (2013)
Ciceribacter lividus gen. nov., sp. nov., isolated from rhizosphere soil of chick pea (Cicer ari-
etinum L.). Int J Syst Evol Microbiol 63:4484–4488
Keshri S (2016) Organic food – harvesting a mirage. http://mediaindiaeu/sector/organic-food/
Keswani C, Singh SP, Singh HB (2013) A superstar in biocontrol enterprise: Trichoderma spp.
Biotech Today 3:27–30
Keswani C, Mishra S, Sarma BK, Singh SP, Singh HB (2014) Unraveling the efficient application
of secondary metabolites of various Trichoderma. Appl Microbiol Biotechnol 98:533–544
Kloepper JW (1993) Plant growth-promoting rhizobacteria as biological control agents. In:
Metting FB (ed) Soil microbial ecology: applications in agricultural and environmental man-
agement. Marcel Dekker Inc, New York
Kloepper JW, Lifshitz R, Zablotowicz RM (1989) Free-living bacterial inocula for enhancing crop
productivity. Trends Biotechnol 7:39–44
Kohler J, Caravaca F (2010) An AM fungus and a PGPR intensify the adverse effects of salinity on
the stability of rhizosphere soil aggregates of Lactuca sativa Roldan. Soil Biol Biochem
42:429–434
Kohler J, Hernández JA, Caravaca F, Roldán A (2008) Plant-growth-promoting rhizobacteria and
arbuscular mycorrhizal fungi modify alleviation biochemical mechanisms in water-stressed
plants. Funct Plant Biol 35:141–151
Krishnan R, Menon RR, Tanaka N, Busse HJ, Krishnamurthi S, Rameshkumar N (2016)
Arthrobacter pokkalii sp nov, a novel plant associated Actinobacterium with plant beneficial
properties, isolated from saline tolerant Pokkali rice, Kerala, India. PLoS One 11:e0150322
Kumar H, Bajpai VK, Dubey RC, Maheshwari DK, Kang SC (2010) Wilt disease management and
enhancement of growth and yield of Cajanus cajan (L) var. Manak by bacterial combinations
amended with chemical fertilizer. Crop Prot 29:591–598
Larson C (2013) Climate change. Losing arable land, China faces stark choice: adapt or go hungry.
Science 339:644–645
Lau JA, Lennon JT (2011) Evolutionary ecology of plant-microbe interactions: soil microbial
structure alters selection on plant traits. New Phytol 192:215–224
Lemanceau P, Alabouvette C (1991) Biological control of fusarium diseases by fluorescent
Pseudomonas and non-pathogenic Fusarium. Crop Prot 10:279–286
7 Microbial Consortial Products for Sustainable Agriculture: Commercialization… 127
Lesueur D, Ingleby K, Odee D, Chamberlain J, Wilson J, Tiki Manga T, Sarrailh JM, Pottinger A
(2001) Improvement of forage production in Calliandra calothyrsus: methodology for the
identification of an effective inoculum containing Rhizobium strains and arbuscular mycor-
rhizal isolates. J Biotechnol 91:269–282
Lesueur D, Sarr A (2008) Effects of single and dual inoculation with selected microsymbionts
(rhizobia and arbuscular mycorrhizal fungi) on field growth and nitrogen fixation of Calliandra
calothyrsus Meissn. Agrofor Syst 73:37–45
Liu X, Bimerew M, Ma Y, Muller H, Ovadis M, Eberl L, Berg G, Chernin L (2007) Quorum-
sensing signaling is required for production of the antibiotic pyrrolnitrin in a rhizospheric bio-
control strain of Serratia plymuthica. FEMS Microbiol Lett 270:299–305
Lucas JA, Ramos Solano B, Montes F, Ojeda J, Megias M, Gutierrez Mañero FJ (2009) Use of two
PGPR strains in the integrated management of blast disease in rice (Oryza sativa) in Southern
Spain. Field Crop Res 114:404–410
Lynch JM (1990) Introduction: some consequences of microbial rhizosphere competence for plant
and soil. In: Lynch JM (ed) The rhizosphere. Wiley, New York, pp 1–10
Malik DK, Sindhu SS (2011) Production of indole acetic acid by Pseudomonas sp.: effect of coin-
oculation with Mesorhizobium sp. Cicer on nodulation and plant growth of chickpea (Cicer
arietinum). Physiol Mol Biol Plants 17:25–32
Malusa E, Vassilev N (2014) A contribution to set a legal framework for biofertilisers. Appl
Microbiol Biotechnol 98:6599–6607
Mapelli F, Marasco R, Rolli E, Barbato M, Cherif H, Guesmi A, Ouzari I, Daffonchio D, Borin S
(2013) Potential for plant growth promotion of rhizobacteria associated with Salicornia grow-
ing in Tunisian hypersaline soils. Biomed Res Int 2013:248078
Markets and Markets (2015) Biofertilizers market by type (Nitrogen-fixing, phosphate-solubilizing
& potash- mobilizing), microorganisms (Rhizobium, Azotobacter, Azospirillum, Cyanobacteria
& phosphate-solubilizing bacteria), application, crop type & by region – Global Forecast to
2020
Marulanda A, Barea JM, Azcon R (2009) Stimulation of plant growth and drought tolerance by
native microorganisms (AM Fungi and Bacteria) from dry environments: mechanisms related
to bacterial effectiveness. J Plant Growth Regul 28:115–124
Mayak S, Tirosh T, Glick BR (2004) Plant growth-promoting bacteria that confer resistance to
water stress in tomatoes and peppers. Plant Sci 166:525–530
Mazid M, Khan TA (2014) Future of bio-fertilizers in Indian agriculture: an overview. Int J Agri
Food Res 3:10–23
Medeot D, Paulucci N, Albornoz A, Fumero M, Bueno M, Garcia M, Woelke M, Okon Y, Dardanelli
M (2010) Plant growth promoting rhizobacteria improving the legume–rhizobia symbiosis.
Springer, New York
Mehboob I, Naveed M, Zahir ZA, Sessitsch A (2013) Potential of rhizosphere bacteria for improv-
ing Rhizobium-legume symbiosis. In: Arora NK (ed) Plant microbe symbiosis: fundamentals
and advances. Springer, New Delhi, pp 305–349
Mendes R, Garbeva P, Raaijmakers JM (2013) The rhizosphere microbiome: significance of plant
beneficial plant pathogenic and human pathogenic microorganisms. FEMS Microbiol Rev
37:634–663
Ministry of Agriculture (2009) Biofertilizers and organic fertilizers covered in fertilizer (Control)
Order, 1985 (as amended, March 2006 and November 2009). Government of India
Miransari M (2011) Interactions between arbuscular mycorrhizal fungi and soil bacteria. Appl
Microbiol Biotechnol 89:917–930
Montesinos E (2003) Development, registration and commercialization of microbial pesticides for
plant protection. Int Microbiol 6:245–252
NAAS (2013) Biopesticides – Quality Assurance. Policy Paper No. 62. New Delhi
Nadeem SM, Naveed M, Zahir ZA, Asghar HN (2013) Plant–microbe interactions for sustainable
agriculture: Fundamentals and recent advances. In: Arora KN (ed) Plant Microbe Symbiosis:
Fundamentals and Advances. Springer India, New Delhi, pp 51–103
128 J. Sekar et al.
Nautiyal CS, Chauhan PS, DasGupta SM, Seem K, Varma A, Staddon WJ (2010) Tripartite inter-
actions among Paenibacillus lentimorbus NRRL B-30488, Piriformospora indica DSM 11827,
and Cicer arietinum L. World J Microbiol Biotechnol 26:1393–1399
Nunes da Rocha U, Plugge CM, George I, van Elsas JD, van Overbeek LS (2013) The rhizosphere
selects for particular groups of Acidobacteria and Verrucomicrobia. PLoS One 8:e82443
Ordookhani K, Khavazi K, Moezzi A, Rejali F (2010) Influence of PGPR and AMF on antioxidant
activity, lycopene and potassium contents in tomato. Afr J Agric Res 5:1108–1116
Palaniyandi SA, Yang SH, Zhang L, Suh JW (2013) Effects of actinobacteria on plant disease sup-
pression and growth promotion. Appl Microbiol Biotechnol 97:9621–9636
Pandey P, Maheshwari DK (2007) Bioformulation of Burkholderia sp. MSSP with a multispecies
consortium for growth promotion of Cajanus cajan. Can J Microbiol 53(2):213–222
Pang Y, Liu X, Ma Y, Chernin L, Berg G, Gao K (2008) Induction of systemic resistance, root colo-
nisation and biocontrol activities of the rhizospheric strain of Serratia plymuthica are depen-
dent on N-acyl homoserine lactones. Eur J Plant Pathol 124:261–268
Parihar P, Singh S, Singh R, Singh VP, Prasad SM (2015) Effect of salinity stress on plants and its
tolerance strategies: a review. Environ Sci Pollut Res Int 22:4056–4075
Park J, Bolan N, Megharaj M, Naidu R (2010) Isolation of Phosphate-Solubilizing Bacteria and
characterization of their Effects on Lead Immobilization. Pedologist 53:67–75
Parmar N, Dadarwal KR (1999) Stimulation of nitrogen fixation and induction of flavonoid-like
compounds by rhizobacteria. J Appl Microbiol 86:36–44
Picard C, Bosco M (2008) Genotypic and phenotypic diversity in populations of plant-probiotic
Pseudomonas spp. colonizing roots. Naturwissenschaften 95:1–16
Picard C, Di Cello F, Ventura M, Fani R, Guckert A (2000) Frequency and biodiversity of
2,4-diacetylphloroglucinol-producing bacteria isolated from the maize rhizosphere at different
stages of plant growth. Appl Environ Microbiol 66:948–955
Pindi PK, Satyanarayana SDV (2012) Liquid microbial consortium- a potential tool for sustainable
soil health. J Biofertil Biopest 3:124
Pivato B, Offre P, Marchelli S, Barbonaglia B, Mougel C, Lemanceau P, Berta G (2009) Bacterial
effects on arbuscular mycorrhizal fungi and mycorrhiza development as influenced by the bac-
teria, fungi, and host plant. Mycorrhiza 19:81–90
Prasanna R, Bidyarani N, Babu S, Hossain F, Shivay YS, Nain L (2015) Cyanobacterial inocula-
tion elicits plant defense response and enhanced Zn mobilization in maize hybrids. Cogent
Food Agricult 1:998507
Qureshi M, Ahmad M, Naveed M, Iqbal A, Akhtar N, Niazi K (2009) Co-inoculation with
Mesorhizobium ciceri and Azotobacter chroococcum for improving growth, nodulation and
yield of chickpea (Cicer arietinum L.). Soil and Environment (Pakistan)
Raaijmakers J, Paulitz T, Steinberg C, Alabouvette C, Moënne-Loccoz Y (2009) The rhizosphere:
a playground and battlefield for soilborne pathogens and beneficial microorganisms. Plant Soil
321:341–361
Rabie GH, Aboul-Nasr MB, Al-Humiany A (2005) Increased salinity tolerance of cowpea plants
by dual inoculation of an arbuscular mycorrhizal fungus Glomus clarum and a nitrogen-fixer
Azospirillum brasilense. Mycobiology 33:51–60
Rajendran G, Sing F, Desai AJ, Archana G (2008) Enhanced growth and nodulation of pigeon pea
by co-inoculation of Bacillus strains with Rhizobium spp. Bioresour Technol 99:4544–4550
Raju K, Sekar J, Vaiyapuri Ramalingam P (2016) Salinicola rhizosphaerae sp. nov., isolated from
the rhizosphere of the mangrove Avicennia marina L. Int J Syst Evol Microbiol
66:1074–1079
Ramos-Solano B, Algar E, Garcia-Villaraco A, Garcia-Cristobal J, Lucas Garcia JA, Gutierrez-
Manero FJ (2010) Biotic elicitation of isoflavone metabolism with plant growth promoting
rhizobacteria in early stages of development in Glycine max var. Osumi J Agric Food Chem
58:1484–1492
Raupach GS, Kloepper JW (1998) Mixtures of plant growth-promoting rhizobacteria enhance bio-
logical control of multiple cucumber pathogens. Phytopathology 88:1158–1164
7 Microbial Consortial Products for Sustainable Agriculture: Commercialization… 129
Schlaeppi K, Bulgarelli D (2015) The plant microbiome at work. Mol Plant Microbe Interact
28:212–217
Schnitzer SA, Klironomos JN, Hillerislambers J, Kinkel LL, Reich PB, Xiao K, Rillig MC, Sikes
BA, Callaway RM, Mangan SA, van Nes EH, Scheffer M (2011) Soil microbes drive the clas-
sic plant diversity-productivity pattern. Ecology 92:296–303
Schuhegger R, Ihring A, Gantner S, Bahnweg G, Knappe C, Vogg G, Hutzler P, Schmid M, Van
Breusegem F, Eberl L, Hartmann A, Langebartels C (2006) Induction of systemic resistance in
tomato by N-acyl-L-homoserine lactone-producing rhizosphere bacteria. Plant Cell Environ
29:909–918
Schwartz A, Ortiz I, Maymon M, Herbold C, Fujishige N, Vijanderan J, Villella W, Hanamoto K,
Diener A, Sanders E, DeMason D, Hirsch A (2013) Bacillus simplex—a little known PGPB
with anti-fungal activity—alters pea legume root architecture and nodule morphology when
coinoculated with Rhizobium leguminosarum bv. viciae. Agronomy 3:595
Sekar J, Prabavathy VR (2014) Novel Phl-producing genotypes of finger millet rhizosphere associ-
ated pseudomonads and assessment of their functional and genetic diversity. FEMS Microbiol
Ecol 89:32–46
Shanmugam V, Kanoujia N, Singh M, Singh S, Prasad R (2011) Biocontrol of vascular wilt and
corm rot of gladiolus caused by Fusarium oxysporum f. sp. gladioli using plant growth promot-
ing rhizobacterial mixture. Crop Prot 30:807–813
Sharma N, Yadav K, Aggarwal A (2016) Growth response of two Phaseolus mungo L. cultivars
induced by arbuscular mycorrhizal fungi and Trichoderma viride. Intl J Agron 2016:1–6
Shirley FN, Zhongqi H, Jim CS (2000) Strategies for aerobic degradation of nitroaromatic com-
pounds by bacteria. In: Spain JC, Hughes JB, Knackmuss H-J (eds) Biodegradation of nitroaro-
matic compounds and explosives. CRC Press, Boca Raton, pp 7–62
Shrivastava P, Kumar R (2015) Soil salinity: A serious environmental issue and plant growth pro-
moting bacteria as one of the tools for its alleviation. Saudi J Biol Sci 22:123–131
Singh A, Sarma BK, Upadhyay RS, Singh HB (2013) Compatible rhizosphere microbes mediated
alleviation of biotic stress in chickpea through enhanced antioxidant and phenylpropanoid
activities. Microbiol Res 168:33–40
Singh JS, Pandey VC, Singh DP (2011) Efficient soil microorganisms: A new dimension for sus-
tainable agriculture and environmental development. Agric Ecosyst Environ 140:339–353
Singh N, Pandey P, Dubey R, Maheshwari D (2008) Biological control of root rot fungus
Macrophomina phaseolina and growth enhancement of Pinus roxburghii (Sarg.) by rhizo-
sphere competent Bacillus subtilis BN1. World J Microbiol Biotechnol 24:1669–1679
Singh R, Parameswaran TN, Prakasa Rao EVS, Puttanna K, Kalra A, Srinivas KVNS, Bagyaraj
DJ, Divya S (2009) Effect of arbuscular mycorrhizal fungi and Pseudomonas fluorescens on
root-rot and wilt, growth and yield of Coleus forskohlii. Biocontrol Sci Technol 19:835–841
Singh RB (2000) Environmental consequences of agricultural development: a case study from the
Green Revolution state of Haryana, India. Agric Ecosyst Environ 82:97–103
Smith DL, Praslickova D, Ilangumaran G (2015) Inter-organismal signaling and management of
the phytomicrobiome. Front Plant Sci 6:722
Snellinx Z, Taghavi S, Vangronsveld J, van der Lelie D (2003) Microbial consortia that degrade 2,
4-DNT by interspecies metabolism: isolation and characterisation. Biodegradation 14:19–29
Srinivas A, Bhalekar DN (2013) Constraints faced by farmers in adoption of biofertilizers. Int J Sci
Res 3:2277–8179
Srinivasan M, Holl FB, Petersen DJ (1997) Nodulation of Phaseolus vulgaris by Rhizobium etli is
enhanced by the presence of Bacillus. Can J Microbiol 43:1–8
Srivastava R, Khalid A, Singh US, Sharma AK (2010) Evaluation of arbuscular mycorrhizal fun-
gus, fluorescent Pseudomonas and Trichoderma harzianum formulation against Fusarium oxy-
sporum f. sp. lycopersici for the management of tomato wilt. Biol Control 53:24–31
Suresh K (2012) Biopesticides: A need for food and environmental safety. J Biofertil Biopestici
3:e107
7 Microbial Consortial Products for Sustainable Agriculture: Commercialization… 131
Tajini F, Trabelsi M, Drevon JJ (2012) Combined inoculation with Glomus intraradices and
Rhizobium tropici CIAT899 increases phosphorus use efficiency for symbiotic nitrogen fixa-
tion in common bean (Phaseolus vulgaris L.). Saudi J Biol Sci 19:157–163
Thijs S, Weyens N, Sillen W, Gkorezis P, Carleer R, Vangronsveld J (2014) Potential for plant
growth promotion by a consortium of stress-tolerant 2,4-dinitrotoluene-degrading bacteria:
isolation and characterization of a military soil. Microb Biotechnol 7:294–306
Tilak KVBR, Ranganayaki N, Manoharachari C (2006) Synergistic effects of plant-growth pro-
moting rhizobacteria and Rhizobium on nodulation and nitrogen fixation by pigeonpea
(Cajanus cajan). Eur J Soil Sci 57:67–71
Tilman D, Fargione J, Wolff B, D’Antonio C, Dobson A, Howarth R, Schindler D, Schlesinger
WH, Simberloff D, Swackhamer D (2001) Forecasting agriculturally driven global environ-
mental change. Science 292:281–284
Timmusk S, Wagner EG (1999) The plant-growth-promoting rhizobacterium Paenibacillus poly-
myxa induces changes in Arabidopsis thaliana gene expression: a possible connection between
biotic and abiotic stress responses. Mol Plant Microbe Interact 12:951–959
Tokala RK, Strap JL, Jung CM, Crawford DL, Salove MH, Deobald LA, Bailey JF, Morra MJ
(2002) Novel plant-microbe rhizosphere interaction involving Streptomyces lydicus WYEC108
and the pea plant (Pisum sativum). Appl Environ Microbiol 68:2161–2171
Tringe SG, von Mering C, Kobayashi A, Salamov AA, Chen K, Chang HW, Podar M, Short JM,
Mathur EJ, Detter JC, Bork P, Hugenholtz P, Rubin EM (2005) Comparative metagenomics of
microbial communities. Science 308:554–557
Unno Y, Shinano T (2013) Metagenomic analysis of the rhizosphere soil microbiome with respect
to phytic acid utilization. Microbes Environ 28:120–127
Upadhyay SK, Singh JS, Saxena AK, Singh DP (2012) Impact of PGPR inoculation on growth and
antioxidant status of wheat under saline conditions. Plant Biol (Stuttg) 14:605–611
Vacheron J, Desbrosses G, Bouffaud ML, Touraine B, Moenne-Loccoz Y, Muller D, Legendre L,
Wisniewski-Dye F, Prigent-Combaret C (2013) Plant growth-promoting rhizobacteria and root
system functioning. Front Plant Sci 4:356
Valdenegro M, Barea JM, Azcón R (2001) Influence of arbuscular-mycorrhizal fungi, Rhizobium
meliloti strains and PGPR inoculation on the growth of Medicago arborea used as model
legume for re-vegetation and biological reactivation in a semi-arid mediterranean area. Plant
Growth Regul 34:233–240
Van der Heijden MG, Klironomos JN, Ursic M, Moutoglis P, Streitwolf-Engel R, Boller T,
Wiemken A, Sanders IR (1998) Mycorrhizal fungal diversity determines plant biodiversity,
ecosystem variability and productivity. Nature 396:69–72
van der Heijden MGA, Bardgett RD, Van Straalen NM (2008) The unseen majority: soil microbes
as drivers of plant diversity and productivity in terrestrial ecosystems. Ecol Lett 11:296–310
Verma JP, Yadav J, Tiwari KN, Jaiswal DK (2014) Evaluation of plant growth promoting activities
of microbial strains and their effect on growth and yield of chickpea (Cicer arietinum L.) in
India. Soil Biol Biochem 70:33–37
Verma JP, Yadav J, Tiwari KN, Kumar A (2013) Effect of indigenous Mesorhizobium spp. and
plant growth promoting rhizobacteria on yields and nutrients uptake of chickpea (Cicer arieti-
num L.) under sustainable agriculture. Ecol Eng 51:282–286
Villaverde JJ, Sevilla-Moran B, Sandin-Espana P, Lopez-Goti C, Alonso-Prados JL (2014)
Biopesticides in the framework of the European Pesticide Regulation (EC) No. 1107/2009.
Pest Manag Sci 70:2–5
Viswanath G, Jegan S, Baskaran V, Kathiravan R, Prabavathy VR (2015) Diversity and N-acyl-
homoserine lactone production by Gammaproteobacteria associated with Avicennia marina
rhizosphere of South Indian mangroves. Syst Appl Microbiol 38:340–345
Vivas A, Azcón R, Biró B, Barea J, Ruiz-Lozano J (2003a) Influence of bacterial strains isolated
from lead-polluted soil and their interactions with arbuscular mycorrhizae on the growth of
Trifolium pratense L. under lead toxicity. Can J Microbiol 49:577–588
132 J. Sekar et al.
Vivas A, Voros I, Biro B, Campos E, Barea JM, Azcon R (2003b) Symbiotic efficiency of autoch-
thonous arbuscular mycorrhizal fungus (G. mosseae) and Brevibacillus sp. isolated from cad-
mium polluted soil under increasing cadmium levels. Environ Pollut 126:179–189
Vyas P, Gulati A (2009) Organic acid production in vitro and plant growth promotion in maize
under controlled environment by phosphate-solubilizing fluorescent Pseudomonas. BMC
Microbiol 9:174
Wagg C, Jansa J, Schmid B, van der Heijden MG (2011) Belowground biodiversity effects of plant
symbionts support aboveground productivity. Ecol Lett 14:1001–1009
Wang CJ, Yang W, Wang C, Gu C, Niu DD, Liu HX, Wang YP, Guo JH (2012) Induction of drought
tolerance in cucumber plants by a consortium of three plant growth-promoting rhizobacterium
strains. PLoS One 7:e52565
Wang X, Pan Q, Chen F, Yan X, Liao H (2011) Effects of co-inoculation with arbuscular mycor-
rhizal fungi and rhizobia on soybean growth as related to root architecture and availability of N
and P. Mycorrhiza 21(3):173–181
Weller DM, Mavrodi DV, van Pelt JA, Pieterse CM, van Loon LC, Bakker PA (2012) Induced
systemic resistance in Arabidopsis thaliana against Pseudomonas syringae pv. tomato by
2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens. Phytopathology
102:403–412
Xu J (2006) Microbial ecology in the age of genomics and metagenomics: concepts, tools, and
recent advances. Mol Ecol 15:1713–1731
Yang JW, Kloepper JW, Ryu CM (2009) Rhizosphere bacteria help plants tolerate abiotic stress.
Trends Plant Sci 14:1–4
Yin D, Wang N, Xia F, Li Q, Wang W (2013) Impact of biocontrol agents Pseudomonas fluores-
cens 2P24 and CPF10 on the bacterial community in the cucumber rhizosphere. Eur J Soil Biol
59:36–42
Yokoyama S, Adachi Y, Asakura S, Kohyama E (2013) Characterization of Alcaligenes faecalis
strain AD15 indicating biocontrol activity against plant pathogens. J Gen Appl Microbiol
59:89–95
Zahir ZA, Ghani U, Naveed M, Nadeem SM, Asghar HN (2009) Comparative effectiveness of
Pseudomonas and Serratia sp. containing ACC-deaminase for improving growth and yield of
wheat (Triticum aestivum L.) under salt-stressed conditions. Arch Microbiol 191:415–424
Zahran HH (1999) Rhizobium-legume symbiosis and nitrogen fixation under severe conditions
and in an arid climate. Microbiol Mol Biol Rev 63:968–989
Zhang H, Kim MS, Sun Y, Dowd SE, Shi H, Pare PW (2008) Soil bacteria confer plant salt toler-
ance by tissue-specific regulation of the sodium transporter HKT1. Mol Plant Microbe Interact
21:737–744
Zhang Z, Pierson LS (2001) A second quorum-sensing system regulates cell surface properties but
not phenazine antibiotic production in Pseudomonas aureofaciens. Appl Environ Microbiol
67:4305–4315
Zheng Y, Liu H, Guo J (2010) Effects of biopesticide against root-knot nematodes in bitter melon.
China: plant bacteria disease and biocontrol, 365 p
Agriculturally Important
Microorganisms as Biofertilizers: 8
Commercialization and Regulatory
Requirements in Asia
Abstract
Biofertilizer refers to the different formulations of living preparations of agricul-
turally important beneficial microorganisms (bacteria/fungi) with certain desir-
able physiological and behavioral characters which are utilized for crop nutrition
management programs. The major attributes of quality of different biofertilizers
is well established and efforts are being made to regularize the production and
marketing of these biofertilizer products. During the last two decades, biofertil-
izer production and marketing exhibited phenomenal growth in most of the Asian
countries. Now time has come to form some set of rules assuring the quality of
biofertilizers available in the market. In any microbial biofertilizer formulation,
viable cell count is the crucial parameter, while the ability of organisms to fix
nitrogen or solubilize phosphorus, potassium, zinc, etc. is important efficiency
characters being considered in formulating the quality standards for biofertiliz-
ers. In the present chapter, the situation of biofertilizer quality control and regu-
latory mechanism in Asia are discussed.
Keywords
Biofertilizers • Quality regulations • Production standards • Agriculturally impor-
tant microorganisms
8.1 Introduction
While Asia is one of the biggest producers and consumers of biofertilizers, there are
still few untied threads which pose a major biosafety concern for environmentalist.
Firstly there is a used difference in the guidelines for mass production and commer-
cialization in the developed, developing, and underdeveloped countries. These
unparallel regulatory measures pose a huge setback for intercontinental trade.
Moreover, the expanding demand for organically grown food supplied provides an
excellent opportunity for developing agro-based economies specifically in Asia.
The growing demand increases the responsibility of the governmental regulatory
bodies, industries, and academia to employ strict regulatory guidelines for produc-
tion of high-quality biofertilizers, which have the desired effects as advertised.
Easy regulatory and registration framework based on advanced R&D in coun-
tries like China, India, Korea, Japan, and Taiwan has tremendously helped the
acceptance of biofertilizers. However, successful adoption of biofertilizers largely
depends upon the quality of the biofertilizer product and also sensitization and
training to the farmers (Banayo et al. 2012).
In Japan, the Tokachi Federation of Agricultural Cooperative (TFAC) in
Hokkaido produces rhizobium biofertilizers under the trade names Mamezo for
soybean and Azuki for bean (Sheng 2005; Sheng and He 2006). Central and local
government agencies in Taiwan support popularization of biofertilizers including
rhizobium, P-solubilizing bacteria, and mycorrhizal inoculants for horticultural
crops. From 1987 to 2006, enough inoculants were produced to inoculate approxi-
mately 65,091 ha of farmland. Farmer’s annual income also increased significantly
from using biofertilizers (Liu et al. 2012; Njira 2013).
In Vietnam, farmers in the Mekong River Delta have been using BioGro in rice,
and this resulted in disease reduction and higher yields (Jones and Darrah 1994).
8 Agriculturally Important Microorganisms as Biofertilizers: Commercialization… 135
Regular inspection
for quality control Density of Strains guaranteed
by authority (Colony forming unit)
under acts
India has a strong legal framework related to biofertilizer production. The Ministry
of Agriculture and Farmers Welfare, Government of India, issued an order in 2006
that brought biofertilizers under the Essential Commodities Act of 1955 and within
the order for the control of fertilizers of 1985. This was later amended in 2009,
2010, and 2015 which brought arbuscular mycorrhiza, Acetobacter, potash mobi-
lize, zinc solubilizer liquid, and carrier-based formulations under its preview of
FCO (1985). The term biofertilizer is legally defined as “the product containing
carrier based (solid or liquid) living microorganisms which are agriculturally useful
in terms of nitrogen fixation, phosphorus solubilization or nutrient mobilization, to
increase the productivity of the soil and/or crop.” The term is also covered under the
broad definition of fertilizers, which “means any substance used or intended to be
used as a fertilizer of the soil and/or crop.” The registration requirements, sampling
procedures, scale of sampling, and testing procedures along with other procedures
and requirements are clearly defined as:
• As per clause 13, subclause (1) (b) and (c), no person shall manufacture any
biofertilizer/organic fertilizer, unless it conforms to the standards set out in the
Part A of Schedule III and Schedule IV, respectively.
• Certificate of manufacture has to be obtained from registering authority under
clause 14, subclause (3) with requisite fee under clause 36.
• As per clause 21, subclause (ii) (a), every container of Biofertilizer/Organic fer-
tilizer has to be labeled as biofertilizer/organic fertilizers.
8.6.2.1 Packing
Biofertilizer shall be packed in suitable plastic bags/packs, thickness of which shall
not be less than 75–100 μm or in suitable plastic bottles.
8.6.2.2 Marking
Each polyethylene pack shall be marked legibly and indelibly with the following
information:
138 V. Pandey and K. Chandra
(a) In drawing, preparing, and handling the samples, the following precautions and
directions should be observed.
(b) Sampling shall be carried out by a trained and experienced person as it is essen-
tial that the samples should be representative of the lot to be examined.
(c) Samples in their original unopened packets should be drawn and sent to the
laboratory to prevent possible contamination of the samples during handling
and help in revealing the true condition of the material.
(d) Intact packets shall be drawn from a protected place not exposed to dampness,
air, light, dust, or soot.
(a) Lot – All units (containers in a single consignment of type of material belonging
to the same batch of manufacture) shall constitute a lot. If a consignment con-
sists of different batches, the container of the same batch shall be separated and
shall constitute a separate lot.
(b) Batch – Inoculants prepared from a batch fermentor or a group of flask (con-
tainer) constitutes a batch.
8 Agriculturally Important Microorganisms as Biofertilizers: Commercialization… 139
(c) For ascertaining conformity of the material to the requirements of the specifica-
tion, samples shall be tested from each lot separately.
(d) The number of packets to be selected from a lot shall depend on the size of the
lot and these packets shall be selected at random and in order to ensure the ran-
domness of selection procedure.
(a) The inspector shall take three packets as samples from the same batch. Each
sample constitutes a test sample.
(b) The samples should be sealed with the inspector’s seal after putting inside
FORM P. Identifiable details such as sample number’s code number or any
other details which enable its identification shall be marked on the cloth bags.
(c) Out of the three samples collected, one sample so sealed shall be sent to in-
charge laboratory notified by the state government under clause 29 or to NCOF/
RCOFs. Another sample shall be given to the manufacturer, or importer, or
dealer as the case may be. The third sample shall be sent by the inspector to his
next higher authority for keeping in safe custody. Any of the latter two samples
shall be sent for referee analysis under subclause (2) of clause 29B of FCO.
(d) The number of samples to be drawn from the lot is as under:
As per clause 2a, subclause (1) (1A) and (1B), samples of biofertilizers/organic
fertilizers are to be tested in National Centre of Organic Farming (NCOF) and
Regional Centre of Organic Farming (RCOF), Bengaluru, Bhubaneswar, Imphal,
Jabalpur, Nagpur, and Panchkula or other notified state laboratories.
(a) Organic fertilizer/biofertilizer inspectors are persons notified under FCO, who
is delegated the powers of drawl of samples of organic fertilizers and biofertil-
izers, as per the procedure laid down in the FCO.
(b) Inspectors of biofertilizer and organic fertilizer are appointed under clause 27B
and should possessing the following qualifications:
140 V. Pandey and K. Chandra
(iv) pH 6.0–7.5
(v) Total viable propagules/gram of 100/g of finished product
product, minimum
(vi) Infectivity potential 80 infection points in test roots/gram of
mycorrhizal inoculum used
(vii) Tolerance limit The viable propagules shall not be less than 80
(i) Individual viable count in liquid- CFU minimum in a mixture of any two or
based formulation more of following microorganisms:
CFU minimum Rhizobium or Azotobacter
or Azospirillum: 1×108 ml−1
CFU minimum PSB: 1×108 ml−1
CFU minimum KSB: 1×108 ml−1
(ii) Total viable count of all the CFU minimum 5×108 cells ml−1 of liquid-
biofertilizer organisms in the product based formulation
(iii) Contamination level No contamination at any dilution
(iv) pH 5.0–7.0
(v) Efficiency character
Rhizobium Nodulation test positive
Azotobacter The strain should be capable of fixing at least
10 mg N fixation/gm of carbon source
Azospirillum The strain should be capable of fixing at least
10 mg N fixation/gm of malate applied
PSB Minimum 5 mm zone of solubilization zone
on PSB media having at least 3 mm thickness
KSB Minimum 5 mm zone of solubilization zone
on KSB media having at least 3 mm
thickness
144 V. Pandey and K. Chandra
8.8 Conclusion
References
Banayo NPM, Cruz PCS, Aguilar EA, Badayos RB, Haefele SM (2012) Evaluation of biofertiliz-
ers in irrigated rice: effects on grain yield at different fertilizer rates. Agriculture 2:73–86
Bhattacharyya P, Kumar D, Pandey V, Paliwal MK (2002) Sustainable cotton production with
biofertilizers. Agrolook 3:22–24
Bisen K, Keswani C, Mishra S, Saxena A, Rakshit A, Singh HB (2015) Unrealized potential of
seed biopriming for versatile agriculture. In: Rakshit A, Singh HB, Sen A (eds) Nutrient use
efficiency: from basics to advances. Springer, India, pp 193–206
Herridge DF, Peoples MB, Boddey RM (2008) Global inputs of biological nitrogen fixation into
agricultural systems. Plant Soil 311:1–18
Jones DL, Darrah PR (1994) Role of root derived organic acids in the mobilization of nutrients
from the rhizosphere. Plant Soil 166:247–257
Keswani C, Singh SP, Singh HB (2013) A superstar in biocontrol enterprise: Trichoderma spp.
Biotech Today 3:27–30
Liu D, Lian B, Dong H (2012) Isolation of Paenibacillus sp. and assessment of its potential for
enhancing mineral weathering. Geomicrobiol J 29:413–421
Naveed M, Mehboob I, Shaker MA, Hussain MB, Farooq M (2015) Biofertilizers in Pakistan:
initiatives and limitations. Int J Agric Biol 17:411–420
Njira KOW (2013) Microbial contributions in alleviating decline in soil fertility. Br Microbiol Res
J 3:724–742
Pandey V, Kumar D, Paliwal MK (2001) Biofertilizers for spices crop production. Agrolook
2:23–25
Ram RM, Keswani C, Mishra S, Bisen K, Tripathi R, Ray S, Singh V, Singh HB (2016)
Comprehensive approaches in plant disease management. In: Sarma BK, Singh A (eds)
Microbial empowerment in agriculture: a Key to sustainability and crop productivity. Biotech
Books, New Delhi, pp 417–443
8 Agriculturally Important Microorganisms as Biofertilizers: Commercialization… 145
Sheng XF (2005) Growth promotion and increased potassium uptake of cotton and rape by a potas-
sium releasing strain of Bacillus edaphicus. Soil Biol Biochem 37:1918–1922
Sheng XF, He LY (2006) Solubilization of potassium bearing minerals by a wild type strain of
Bacillus edaphicus and its mutants and increased potassium uptake by wheat. Can J Microbiol
52:66–72
Suh JS, Jiarong P, Toan PV (2006) Quality control of biofertilizers. Biofertilizers Manual. Forum
for Nuclear Cooperation in Asia, Japan, pp 112–115
The Fertilizer (Control) Order 1985. (2015) The fertiliser association of India, 10 Shaheed Jit
Singh Marg, New Delhi, p 278
Yadav AK, Chandra K (2014) Mass production and quality control of microbial inoculants. Indian
Natn Sci Acad 80(2):483–489
Part III
Biopesticide and Biofertilizer Regulatory
Requirements in South and Southeast Asia
Research, Development
and Commercialisation of Agriculturally 9
Important Microorganisms in Malaysia
Abstract
Agriculture is considered as one of the most important economic sector in
Malaysia. Intensive agriculture in Malaysia is highly based on usage of agricul-
tural inputs such as fertilisers and pesticides. Both the agriculture and plantation
sectors are operated by using imported fertilisers and pesticides. An injudicious
usage of these two inputs in agricultural sectors has created many environmental
and health issues. A possible solution to avoid this fertiliser and pesticide crisis
may rest with groups of microorganisms that have the capacity to provide the
nutrients needed and protection against pests for crops. These beneficial
microorganism-based products are known as biofertilisers and biopesticide. The
current chapter discusses the research, development and commercialisation of
agriculturally important microorganisms in Malaysia.
Keywords
Biofertilisers • Biopesticides • Pesticides • Agriculturally important
microorganisms
G. Krishnen (*)
Crop and Soil Science Research Centre,
MARDI Head Quarters, 43400 Serdang, Selangor, Malaysia
e-mail: [email protected]
M.R.M. Noor • A. Jack • S. Haron
Director General Office, MARDI Head Quarters, 43400 Serdang, Selangor, Malaysia
to make optimal use of indigenous input sources. Biofertilisers and biopesticides are
two of indigenous inputs which were successfully used for partial or full replace-
ment of synthetic fertiliser and pesticides in various crops globally. Based on our
own experiences, low dose of chemical fertiliser (1/3rd recommended rate) in com-
bination with biofertiliser had produced same amount of rice yield as recommended
dose of chemical fertiliser, suggesting that partial replacement of chemical fertiliser
is possible. Biopesticides are natural pest and disease control strategy used in
Integrated Pest Management (IPM) and successfully reduced the amount of pesti-
cide application on the field (Allahyari et al. 2016). Although both biofertiliser and
biopesticide were used for a long time in crops production, the performance of these
inputs are inconsistent. Therefore production of excellent quality biofertilisers and
biopesticide products is a prerequisite for product performance and reliability.
Research and development on biofertiliser and biopesticides are crucial for innova-
tion of excellent quality biofertiliser and biopesticide products with reliable perfor-
mance that can be commercialised. In Malaysia, the Government is encouraging the
R&D activities through National Policy on Science, Technology and Innovation
(NPSTI).
There are various funding options available for R&D and commercialisation for
innovation generation in Malaysia. The funds offered by various agencies are shown
in Table 9.1. Among the fund providing agencies, the Ministry of Science,
Technology and Innovation (MOSTI) and Ministry of Education (MOE) are the
main research funders. Basic research funds (FRGS, TRGS and LRGS) are open for
implementation of activities such as theory development, concept and new ideas
152 G. Krishnen et al.
Biopesticides are pest control tool derived from biological resources including sev-
eral types of pest management strategies through predatory, parasitic and antagonis-
tic activities (Soleder and Lacey 2013; Glare et al. 2012). Biopesticide are naturally
occurring or derived substance or an organism which controls pests by non-toxic
means. There are wide spectrums of potential products that can be classified as
biopesticide:
In this chapter, only the microbial biopesticide have been discussed due to their
growing significance in plant disease management in Malaysia.
Although biopesticide has been used for a long time in Malaysian agriculture,
their performance was less satisfactory; thus, farmers have negative perceptions.
The main concern for farmers is that they desire very quick effects of biopesticide
equivalent to chemical pesticides. In the past decade, at least 96 research projects on
biopesticides were executed by universities and PRIs (Table 9.3). The main micro-
organism types used for biopesticide researches in Malaysia were fungi
(Metarhizium, Beauveria, Trichoderma and Paecilomyces), bacteria (Bacillus and
Pseudomonas) and viruses (Nucleopolyhedrovirus and bacteriophage). The main
research focus was bioprospection of microbial biopesticide, biopesticide
9 Research, Development and Commercialisation of Agriculturally Important… 155
Technology transfer
Problem identification
The technology is evaluated for its commercial potential by TCO based on the
criteria such as proof of concept, novelty of innovation, technology competitiveness
and potential market. Once the technology is transferred to the end user, TCO evalu-
ates the impact of technology after certain period of time. Feedbacks on the results
and problems of the technology are evaluated and the corrective action is taken
immediately. Generally, other agencies in Malaysia also implement the same pro-
cess with minor changes or modifications.
(i) Licencing
(ii) Consultation
(iii) Contract manufacturing (OEM)
(iv) Outright sale
(v) Joint venture
(vi) Spin-off companies
(vii) Leasing
(viii) Profit sharing
Among them, (i–iv) are the most common methods used for technology commer-
cialisation in MARDI. At least, three biofertilisers and biopesticide technologies
were commercialised using licencing method.
160 G. Krishnen et al.
(i) Permit to import sample – for sample importation for related authority analysis
(small consignment up to 2 kg or 2 l)
(ii) Import permit – for bulk consignment importation for marketing
For this application, applicants have to enclose a verification letter from organ-
iser or related party. Application can be submitted to Crop Protection and Quarantine
Section, Department of Agriculture of Malaysia (JPK) by filling the off-module
import permit application form (EP-4A Form) and enclose a banker’s draft, postal
order or money order valued RM 15.00 and paid to the Director General of
Agriculture. Incomplete EP-4A Form will be rejected by the authority. Applicants
are requested to apply for the permit 30 days before the importation of microbial
product samples.
The information requested in the EP-4A form is as listed below:
For the materials that are imported in for the first time, the amount of organic
fertiliser, microorganisms and materials containing microorganism allowed is 2 l,
2 kg or 5 units per samples (test tubes/ampoules) for analytical purposes. For
importing sample, the entry point will only be the Kuala Lumpur International
Airport (KLIA). Applicant has to pay RM 340.00 by using the method mentioned
above as analysis fee for the samples. Five sets of the biofertilisers and biopesticide
samples, each weighing 250 g (powder/solid) or 250 ml (liquid), need to be
164 G. Krishnen et al.
Any import and export of materials into Malaysia requires an import/export permit.
The permit needs to be applied electronically (ePermit) through an appointed ven-
dor Dagang Net Technologies Sdn. Bhd. (DNT). Importer, exporter, forwarding
agent and individual including foreigner have to be registered with Dagang Net for
ePermit access. Registration form can be downloaded (www.dagangnet.com) or
obtained from DNT branches in the country. A registration fee of RM 500 applied
for corporate company and RM 200 for Small and Medium Enterprises (SME).
A fee of RM 200 is applied for annual renewal of ePermit.
ePermit application for any import of organic fertilisers, biofertilisers, biopesti-
cides, microorganism and material containing microorganisms is required to import
sample as prerequisite for permit application from JPK, Department of Agriculture
of Malaysia. Once the ePermit application is registered with DNT, the applicant
needs to inform JPK to initiate the online application. The applicant is requested to
open a deposit account to enable import/export permit online application using
ePermit Deposit Form EP-1. The minimal amount needed for a deposit account
opening is RM 150.00.
Before the bulk consignment of biofertilisers and biopesticides arrives, the JPK
personnel will visit the warehouse where the products will be stored. If the person-
nel is satisfied with the warehouse condition for storage, the import permit will be
granted. The JPK personnel will also collect the samples from three consecutive
bulk consignments of the product arrived and will be analysed again, and the results
will be presented to the MOBO committee. If the analysis data shown there has any
discrepancies, the import permit will be revoked.
9 Research, Development and Commercialisation of Agriculturally Important… 165
If compared with 10 years ago, relatively more Malaysian farmers are willing to try
biofertilisers and biopesticides for their crop production. The inconsistency in qual-
ity and efficacy are fading the farmers’ acceptance and confidence on these prod-
ucts. The uncertainty in product efficacy is mainly caused by the lack of stringent
quality control during production, transportation and storing of these products. Lack
of regulatory control and standard further diminish the quality of these products;
thus, the farmers do not have good perception on the products. Therefore, standards
and regulation for production of biofertilisers and biopesticides with high-quality
products are urgently needed to increase the acceptance of these products.
Malaysia does not have any standards for biofertilisers and biopesticide yet.
Currently the Fertiliser Technical Committee under the Department of Standards
Malaysia had just set-up a working group to draft a Malaysian standard for biofer-
tilisers. Hopefully, in another 2–3 years, Malaysia may have its own standard to
regulate the quality of biofertilisers sold in Malaysia.
Biofertilisers and biopesticide were two of the focal areas of agricultural biotech-
nology of Malaysian National Biotechnology Policy. Therefore research, develop-
ment and commercialisation of these two products have become the priority of the
government. Various incentives such as BioNexus status were given to the related
companies to commercialise their products. BioNexus is a special status awarded to
qualified international and Malaysian biotechnology companies which enjoy fiscal
incentives, grants and other guarantees to assist growth. BioNexus status is awarded
to qualified companies undertaking value-added biotechnology and/or life sciences
activities. To date, there were 11 biofertiliser and biopesticide companies which
have been given BioNexus status as recognising the importance of these technolo-
gies. The government efforts in encouraging sustainable production system and
green technologies also contribute to the expansion of biofertiliser and biopesticide
usage among farmers.
166 G. Krishnen et al.
Biofertilisers and biopesticide can play an important role in sustainable crop pro-
duction. They have a huge potential to partially or fully replace the chemical fertil-
iser and pesticides. The inconsistency in the performance of these both inputs needs
to be addressed. The biofertiliser and biopesticide products need to be more com-
petitive, versatile and effective. New types of biofertilisers which can be used for a
wide range of crops are urgently needed, while for biopesticide, products active
against wide range of pest are needed. Collaboration with private companies is cru-
cial for efficient transfer of technology and commercialisation. More incentives to
biofertiliser and biopesticide companies in support of ‘green technology’ for food
safety and societal well-being will encourage private commitment in production
and commercialisation of these products.
References
Allahyari MS, Damalas CA, Ebadattalab M (2016) Determinants of integrated pest management
adoption for olive fruit fly (Bactrocera oleae) in Roudbar, Iran. Crop Prot 84:113–120
Bisen K, Keswani C, Mishra S, Saxena A, Rakshit A, Singh HB (2015) Unrealized potential of
seed biopriming for versatile agriculture. In: Rakshit A, Singh HB, Sen A (eds) Nutrient use
efficiency: from basics to advances. Springer, New Delhi, pp 193–206
Chandler D, Davidson G, Grant WP, Greaves J, Tatchell GM (2008) Microbial biopesticides for
integrated crop management: an assessment of environmental and regulatory sustainability.
Trend Food Sci Technol 19:275–283
Dardak RA, Adham KA (2014) Transferring agricultural technology from government research
institution to private firms in Malaysia. Proc Social Behav Sci 115:346–360
DOA (2015) Booklet Statistik Tanaman 2015, Department of Agriculture Malaysia, p 115
DOA (2016) Fertilizer imports in Malaysia (quantity in tonnes) 2005–2013, p 3
FAO (2016) Agricultural resources – pesticide use for Malaysia
Glare T, Caradus J, Gelernter W, Jackson T, Keyhani N, Kohl J, Marrone P, Morin L, Stewart A
(2012) Have biopesticides come of age? Trend Biotechnol 30:250–258
Keswani C, Singh SP, Singh HB (2013) A superstar in biocontrol enterprise: Trichoderma spp.
Biotech Today 3:27–30
Mishra S, Singh A, Keswani C, Saxena A, Sarma BK, Singh HB (2015) Harnessing plant-microbe
interactions for enhanced protection against phytopathogens. In: Arora NK (ed) Plant microbe
symbiosis– applied facets. Springer, New Delhi, pp 111–125
OECD (2014) “Malaysia”, in OECD Science, Technology and Industry Outlook 2014, OECD
Publishing. http://dx.doi.org/10.1787/sti_outlook-2014-64-en
Pemandu (2011) www.etp.pemandu.gov.my
Sporleder M, Lacey LA (2013) Biopesticides. In: Alyokhin A, Vincent C, Giodanengo P (eds)
Insect pests of potato: global perspective on biology and management 1st edition, Academic
Press. Oxford, UK, pp 463–497
Vessey JK (2003) Plant growth promoting rhizobacteria as bio-fertilizers. Plant Soil 255:571–586
Development and Application
of Agriculturally Important 10
Microorganisms in India
Abstract
Significant expansion in agricultural yield brought by green revolution has not
been converted into a replica for prolonged agricultural growth. Disproportionate
reliance on chemical fertilizers mainly nitrogen and phosphorus and non-
judicious use of pesticides have led to water pollution and untenable burden on
the agricultural ecosystems. This alarming situation has paved way for augmen-
tation in biofertilizer and biopesticide uses, as commercially available alternative
for improving soil quality, and utilization of different resources more effectively
for escalating crop production. The coverage of biofertilizer and biopesticide
usage in various states of India and its influence on agricultural production has
been studied intensively in the current work. The uses of biofertilizers and
biopesticides have been promoted actively by both state and central government
in India. This chapter comprehensively discusses the current scenario and vari-
ous regulatory and commercialization challenges associated with the use of
biopesticides and biofertilizers. In this chapter few general recommendations
have been suggested to enhance the promotion and acceptance of green agricul-
ture in India.
Keywords
Biofertilizer • Biopesticides • Regulation • Pollution
10.1 Introduction
Table 10.1 List of representative biopesticides registered in India under section 9(3) of the
Insecticides Act, 1968
Biopesticides Taxus Formulations Targets Trade name
Bacillus thuringiensis Bacterium 5.0 % WP, Lepidopteran Tacibio
subsp. israelensis 5.0 % AS pests
B. thuringiensis subsp. 5.0 % WP, Lepidopteran Bio-Dart
kurstaki 7.5 % WP pests
Pseudomonas 0.5 %, 1.0 % Soilborne diseases Biomonas
fluorescens WP
Trichoderma viride Fungus 1.0 % WP Soilborne Bioderma
pathogens
Beauveria bassiana 2.15 % WP, Coffee berry Myco-Jaal
10 % SC or borer,
1.0 %, 1.15 % diamondback
moth,
grasshoppers,
whiteflies, aphids
Ampelomyces quisqualis 2.0 % WP Powdery mildew Bio-Dewcon
Trichoderma harzianum 0.5 %, 1.0 %, Soilborne Biozim
2.0 % WP pathogens
Metarhizium anisopliae 1.0 %, 1.5 % Coleoptera, Biomet
WP Lepidoptera,
termites,
mosquitoes,
leafhoppers,
beetles, grubs
Paecilomyces lilacinus 1.0 % Whitefly Yorker
Verticillium lecanii 1.15 % Whitefly, coffee Verisoft
green bug,
homopteran pests
Verticillium 1.0 % WP Nematodes
chlamydosporium
Nuclear polyhedrosis Virus 0.43 %, 0.5 %, Helicoverpa Helicide
virus of Helicoverpa 0.64 %, 2.0 % armigera
armigera
Nuclear polyhedrosis 0.5 %, 2.0 % Spodoptera litura Spodocide
virus of Spodoptera
litura
Note: WP wettable powder, AS aqueous solution or aqueous suspension, SC suspension
concentrate
behind (Bailey et al. 2010). The positive impacts of biopesticides have failed to dif-
fuse into the Indian biopesticide market and consequently it is lagging behind,
growing at a slow pace. According to a study, India’s share of the global biopesti-
cide market was approximately 2.89 % during 2005, which gradually increased to
4.5 % by 2010.
In India, until October 2013, only 14 types of biopesticides with their formula-
tions were registered under the Insecticide Act, 1968 (Table 10.1). Poor technology
170 H.B. Singh et al.
In India, biopesticide is included in the Insecticide Act of 1968, which frames gen-
eral guidelines with respect to pesticide’s bioefficacy and safety to human beings
and ecosystem. The Central Insecticides Board & Registration Committee (CIBRC)
is the main regulatory body under this Act. CIB as the apex advisory body maintains
a strong network of eminent scientists from all disciplines concerned. The RC is
involved in granting registrations and licenses to the amateur biopesticide manufac-
turers. A standard procedure is followed during this entire process. The novel
biopesticide formulation is scrutinized by various quality check protocols, and its
potential risk associated with human health and ecosystem is properly analyzed.
Inclusion of biopesticides in the integrated pest management (IPM) program initi-
ated by the government was the first stepping stone toward the rise of biopesticide
products in India. The main government agencies behind the promotion and cre-
ation of awareness among farmer’s community about biopesticides in India are the
Ministry of Agriculture and Farmers Welfare and the Department of Biotechnology
(DBT). The Central IPM Centre (Faridabad), the National Centre for IPM (NCIPM)
under ICAR, and the Directorate of Biological Control are also among the key play-
ers promoting diffusion of biopesticides in agricultural fields (Alam 1994). The
DBT funds research in development of biopesticides and also helps in generation of
toxicological data for registration purposes. There are also agencies like the National
Agricultural Research System (NARS) and the National Board of Accreditation
(NBA) whose primary responsibility lies in conducting various standard quality
control tests of biopesticides. Besides they are also actively involved in training the
officers of the state departments of agriculture in quality control protocols. The state
government’s role in implementing IPM programs is indispensable. Their IPM pro-
grams include purchase and distribution of biopesticides to farmers at affordable
10 Development and Application of Agriculturally Important Microorganisms in India 171
Table 10.2 CIB guidelines/data requirements for registration of biopesticides for minimum infra-
structure facilities to be created by the manufacturers of biopesticides
Sl.
no. Particulars
1 Guidelines/data requirements for registration of baculoviruses – nuclear polyhedrosis
viruses (NPV) and granulosis viruses (GV) u/s 9(3B) and 9(3) of the Insecticide Act,
1968
1.1 Indian standards – baculoviruses, nuclear polyhedrosis viruses (NPV) and granulosis
viruses (GV) specifications
2 Guidelines/data requirements for registration of antagonistic fungi u/s 9(3B) and 9(3) of
the Insecticide Act, 1968
2.1 Indian standards – antagonistic fungi specifications
3 Guidelines/data requirements for registration of entomogenous fungi u/s 9(3B) and 9(3)
of the Insecticide Act, 1968
3.1 Indian standards – entomopathogenic fungi – specifications
4 Guidelines/data requirements for registration of antagonistic bacteria u/s 9(3B) and 9(3)
of the Insecticide Act, 1968
4.1 Indian standards – antagonistic bacteria – specifications
5 Guidelines/data requirements for registration of entomotoxic bacteria technical and
formulation u/s 9(3B) and 9(3) of the Insecticide Act, 1968
5.1 Indian standards – entomotoxic bacteria – specifications
6 Guidelines for minimum infrastructural facilities to be created by the manufacturers of
microbial biopesticides (antagonistic fungi, entomopathogenic fungi, antagonistic
bacteria, and entomotoxic bacteria)
7 Guidelines for minimum infrastructural facilities to be created by the manufacturers for
baculoviruses (NPV, GV)
8 Guidelines for minimum infrastructural facilities to be created by the manufacturers of
botanical biopesticides (Pyrethrum, azadirachtin, Cymbopogon, etc.)
Source: http://www.cibrc.nic.in/guidelines.htm
price. This in turn has created a market for and also encouraged the private com-
mercial production of microbial pesticides (Rabindra and Grzywacz 2010).
(a) There is still no ordered mechanism or procedure for the reverification of the
issued registration certificates for biopesticides.
(b) Manufacturing licenses for biopesticides are issued on paper, but their existence
in actual business market remains in ambiguity, and till date there is an absence
of proper mechanism to track them.
(c) Various case studies on market scenario confirm the quality concerns, espe-
cially from amateur producers who find it difficult to align their products as per
the standards set by regulatory bodies.
10 Development and Application of Agriculturally Important Microorganisms in India 173
business, and companies will only develop these products if there is a long-term
profit in doing so.
Fig. 10.1 Pie chart showing percentage distribution of biofertilizers in different regions of India
176 H.B. Singh et al.
Indian legal framework related to biofertilizers is quite robust. The Indian Ministry
of Agriculture issued an order in 2006 which included biofertilizers under the
Essential Commodities Act of 1955.
Agro Industries Foundation (BAIF) and Kumar Krishi Mitra Bio Products Pvt. Ltd.
(KKM), carried out an extensive field exercise. The media used are pamphlets, bro-
chures, audiovisual material, advertisements, demonstrations and setting up of trial/
demo farms, dealer workshops, and discussion forums. They carried out interviews
with farmers, dealers, extension workers, and officers. The empirical findings and
observations from these exercises from multiple stakeholders’ perspectives are
listed as follows.
10.4 Conclusion
products with improved shelf and field life, emphasis should be laid on developing
novel formulations and carrier materials. We need to develop safety indices for
biopesticide and biofertilizer formulations including acceptable levels of inorganic
contaminants. There should be a strict and transparent monitoring of quality of
biopesticides and biofertilizers at various stages of production, marketing, procure-
ment, and applications to avoid diffusion of spurious products in the market. For
achieving this, proper effort should be taken in developing more testing laboratories
with adequate infrastructure and manpower. For bioefficacy testing, referral labs
with good infrastructural facilities may be notified by the CIB. Besides, the CIB
should form a technical expert committee comprising of scientists to oversee the
infrastructural facilities of biopesticide and biofertilizer production units before
granting registration under section 9(3B) or 9(3). The existing production units
should be subjected to accreditation. Also the development of farmer-friendly tech-
nology should be the prime focus of all the research.
To reduce farmers’ risk and increase acceptance of biopesticides and biofertiliz-
ers by them, some insurance or buyback of product scheme should be initiated. The
cost of such schemes can be jointly shared by the government and the manufactur-
ing firm. Public-private collaboration in research, production, and commercializa-
tion should be encouraged. The industry must promote standards for biopesticides
and biofertilizers to translate their value in agriculture, forestry, and other target
markets. A strong academic-industry alliance is necessary for scaling up the com-
mercialization of biopesticides and biofertilizers. A road map may be developed for
putting this agenda into implementation.
Indigenous knowledge of bio-inputs should be validated and documented and its
integration with scientific knowledge should be undertaken. There should be devel-
opment of data banks freely accessible for reference and use. Therefore, it is imper-
ative to bring out manuals for supporting the development of high-quality
biopesticides and biofertilizers. Harmonization of international regulations is
required. The fact that the use of biopesticides and biofertilizers is a knowledge-
intensive input is highlighted. The issues of data protection and IPR must be
addressed. Registration requirement for biopesticides and biofertilizers could be
relaxed and rationalized. Since some of these agents have the ability to increase
plant growth, they should be considered as plant growth-promoting agents for relax-
ing registration requirements like toxicological data. There is an urgent need to
develop bar coding of microbes used in commercial production of biopesticides and
biofertilizers. Microbial consortia can be developed for better results. Accordingly,
the CIB may amend the existing rules for biopesticide and biofertilizer registration.
Selection of proper strain/species of biocontrol agents is the key factor for overall
success. More focus may be given to develop low-cost technologies for mass pro-
duction of biopesticides and biofertilizers. Attention should be given to develop
postharvest disease management practices. A concerted effort of research institutes,
universities, nongovernment organizations (NGO), and government organizations is
required to elevate the stature of biopesticides and biofertilizers which in turn will
facilitate their diffusion in the Indian market.
180 H.B. Singh et al.
References
Alam G (1994) Biotechnology and sustainable agriculture: lessons from India, Technical paper no.
103. OECD Development Centre, Paris
Alam G (2000) A study of biopesticides and biofertilisers in Haryana, India. Gatekeeper series no.
93 IIED, London. http://pubs.iied.org/pdfs/6348IIED.pdf
Arora NK, Khare E, Maheshwari DK (2010) Plant growth promoting rhizobacteria: constraints in
bioformulation, commercialization, and future strategies. In: Maheshwari DK (ed) Plant
growth and health promoting bacteria. Springer, Berlin, pp 97–116
Bailey KL, Boyetchko SM, Längle T (2010) Social and economic drivers shaping the future of
biological control: a Canadian perspective on the factors affecting the development and use of
microbial biopesticides. Biol Control 52:221–229
Bashan Y (1998) Inoculants of plant growth promoting bacteria use in agriculture. Biotech Adv
6:729–770
Bisen K, Keswani C, Mishra S, Saxena A, Rakshit A, Singh HB (2015) Unrealized potential of
seed biopriming for versatile agriculture. In: Rakshit A, Singh HB (eds) Nutrient use effi-
ciency: from basics to advances. Springer, New Delhi, pp 193–206
Bisen K, Keswani C, Patel JS, Sarma BK, Singh HB (2016) Trichoderma spp.: efficient inducers
of systemic resistance in plants. In: Chaudhary DK, Verma A (eds) Microbial-mediated induced
systemic resistance in plants. Springer, Singapore, pp 185–195
Darbro JM, Thomas MB (2009) Spore persistence and likelihood of aeroallergenicity of entomo-
pathogenic fungi used for mosquito control. Am J Trop Med Hyg 80:992–997
Desai S, Kumar GP, Amalraj ELD, Talluri VR, Peter AJ (2016) Challenges in regulation and reg-
istration of biopesticides: an overview. In: Singh DP, Singh HB, Prabha R (eds) Microbial
inoculants in sustainable agricultural productivity. Springer, New Delhi, pp 301–308
Doekes G, Larsen P, Sigsgaard T, Baelum J (2004) IgE sensitization to bacterial and fungal biopes-
ticides in a cohort of Danish greenhouse workers: the BIOGART study. Am J Ind Med
46:404–407
Evans J, Wallace C, Dobrowolski N (1993) Interaction of soil type and temperature on the survival
of Rhizobium leguminosarum bv Viciae. Soil Biol Biochem 25:1153–1160
Iida A, Sanekata M, Fujita T, Tanaka H, Enoki A, Fuse G, Kanai M, Rudewicz PJ, Tachikawa E
(1994) Fungal metabolites XVI structures of new peptaibols, trichokindins I–VII, from the
fungus Trichoderma harzianum. Chem Pharm Bull 42:1070–1075
Keswani C, Singh SP, Singh HB (2013) A superstar in biocontrol enterprise: Trichoderma spp.
Biotech Today 3:27–30
Keswani C, Mishra S, Sarma BK, Singh SP, Singh HB (2014) Unraveling the efficient application
of secondary metabolites of various Trichoderma. Appl Microbiol Biotechnol 98:533–544
Kulshrestha S (2004) The status of regulatory norms for biopesticides in India. In: Kaushik E (ed)
Biopesticides for sustainable agriculture: prospects and constraints. Energy Research Institute,
New Delhi
Kumar S, Singh A (2015) Biopesticides: present status and the future prospects. J FertilPestic
6:e129
Market and Market (2013) Report code: CH 1266 Global biopesticides market – trends and fore-
casts (2012–2017), India
Marrone PG (2007) Barriers to adoption of biological control agents and biological pesticides,
CAB reviews: perspectives in agriculture, veterinary science, nutrition and natural resources
2(51). CAB International, Wallingford
Mazid M, Khan TA, Mohammad F (2011) Potential of NO and H2O2 as signaling molecules in
tolerance to abiotic stress in plants. J Indus Res Technol 1:56–68
Mishra S, Singh A, Keswani C, Saxena A, Sarma BK, Singh HB (2015) Harnessing plant-microbe
interactions for enhanced protection against phytopathogens. In: Arora NK (ed) Plant microbe
symbiosis– applied facets. Springer, New Delhi, pp 111–125
10 Development and Application of Agriculturally Important Microorganisms in India 181
Rabindra RJ (2005) Current status of production and use of microbial pesticides in India and the
way forward. In: Rabindra RJ, Hussaini SS, Ramanujam B (eds) Microbial biopesticide formu-
lations and applications: Technical document no. 55. Project Directorate of Biological Control,
Bangalore, pp 1–12
Rabindra RJ, Grzywacz D (2010) India. In: Kabaluk JT, Svircev AM, Goettel MS, Woo SG (eds)
The use and regulation of microbial pesticides in representative jurisdictions worldwide. IOBC
Global, p 99
Singh HB, Singh BN, Singh SP, Sarma BK (2012) Exploring different avenues of Trichoderma as
a potent bio-fungicidal and plant growth promoting candidate-an overview. Rev Plant Pathol
5:315–426
Singhal V (2004) Biopesticides in India. In: Kaushik N (ed) Biopesticides for sustainable agricul-
ture, prospects and constraints. TERI, Delhi, pp 31–39
Tittabutr P, Teamthisong K, Buranabanyat B, Teaumroong N, Boonkerd N (2012) Gamma irradia-
tion and autoclave sterilization peat and compost as the carrier for rhizobial inoculant produc-
tion. J Agri Sci 4:59–64
Regulatory Requirements
and Registration of Biopesticides 11
in the Philippines
Abstract
Traditional agriculture, in a perpetual effort to maximize productivity, have
always relied on synthetic pesticides to control pest infestations. However, usage
of these synthetic chemicals has an inadvertent adverse effect on the environ-
ment. Thus, they are not sustainable and there is a need to slowly decrease its
usage in favor of pesticidal products that are more environment-friendly in
nature. An alternative to traditional pesticides is biological pesticides or biopes-
ticides. Biopesticides are biocontrol chemicals derived from natural resources
such as plants, animals, minerals, or microorganism such that the usage of which
is without the threat of environmental contamination and pollution. Moreover,
most biopesticides have been proven to be at par if not better relative to the domi-
nant synthetic pesticides in the market. Thus, biopesticides have been constantly
promoted as an alternative to traditional and inorganic pesticides. Despite this,
biopesticide usage in the Philippines and the world in general remains relatively
diminutive. Inefficiencies in registration of new biopesticide products are in part
responsible. Regulation of pesticides and biopesticides in general is governed by
the Fertilizer and Pesticide Authority (FPA) agency in the Philippines. The lack
of general interest in biopesticides locally has been attributed to insufficient
trainings and extensions to farmers, insufficient manufacturing capacity to sati-
ate even the meager demand, lack of biopesticide inoculant resources reposi-
tory, limited to absence of linkages between local government units and farmers,
and cultural tendency – the 50-year-old habit of the massive application of fertil-
izers and pesticides brought about by the practices in green revolution.
Recommendations to promote local biopesticides utilization include partner-
ships with the private sector to facilitate mass production and commercialization
if government or state-owned biopesticides formulation plant is not possible,
Keywords
Biopesticides • Formulation • Fertilizers • Pesticides
11.1 Introduction
Pest-induced stress and infestations on crops has had a tremendous impact on the
productivity of crops throughout the history of agriculture. Various outbreaks have
led to widespread destruction of food crops ultimately leading to famine and the
death of millions of people. This is especially true for underprivileged countries
where the economy heavily relies on agriculture. Thus, it is of utmost importance to
minimize and manage the damage brought about by such infestation. Pest infesta-
tions have been generally managed through the use of traditional inorganic pesti-
cides. The first generation of synthetic pesticides is in the form of highly toxic
compounds, such as arsenic and cyanide-based chemicals. Later on, it was deemed
highly unsafe leading to the dawn of synthetic organic compounds. The application
of these organochlorine compounds with a broad-spectrum effect, however, has a
huge negative impact on non-target beneficial organisms. Moreover, these com-
pounds are highly stable and tend to persist in nature leading to the accumulation
and magnification in biological systems. The Philippine pesticide industry emerged
in the 1950’s, coinciding with the popularity in usage of the said compounds such
as DDT, 2,4-D, endrin, and malathion (Elazequi 1989).
Although traditional methods of pest control have made a huge impact in the con-
trol of pest infestations, the negative ecological impacts cannot be ignored. An eco-
friendly alternative to broad-spectrum traditional chemical pesticides is biopesticides.
According to FPA (2016), they can be in the form of biochemical pest control agents
including pheromones, kairomones, allomones, and hormones; natural plant regula-
tors such as auxins and enzymes; and microbial pest control agents such as bacteria,
fungi, protozoa, and virus-based products. It has the advantage of having no chemical
residues and thus has no known adverse effect on the environment (Gupta and Dikshit
2010; Keswani et al. 2014; Bisen et al. 2015). In addition, most biopesticides have a
narrow spectrum of effect and are thus not lethal to non-target organisms.
The Philippines is largely an agricultural country where agriculture accounts for
11.3 % of the current GDP according to World Bank (2014). In addition, 24 % of
the total export and 46 % of the total employment are from agriculture (Bureau of
Agricultural Statistics, 2003). Important agricultural commodities of the Philippines
include rice, corn, mangoes, pineapples, and coconut largely because they either are
a staple food crop or they comprise a large proportion of the agricultural export
(Bureau of Agricultural Statistics, 2004). Pesticide application has been a major
factor in the increase of productivity for these crops. It is estimated that in 1990,
pesticide industry in the Philippines is valued at around 100 million USD - 40 % of
11 Regulatory Requirements and Registration of Biopesticides in the Philippines 185
which was used on rice whereas 20 % was allocated for vegetables (Gaston 1994).
Small-scale farmers which comprise the bulk of the agricultural industry in the
Philippines are heavily reliant on old and broad-spectrum effect pesticides. This is
understandable because these synthetic formulations are inexpensive, easily applied
and as well as effective. Biopesticides remain largely overlooked despite consider-
able effort put into research and extension by various local institutions. In spite of
its lukewarm success, the Philippine biopesticide industry has a wide array of prod-
ucts commercially and locally available.
Biofertilizers are not only limited in function as plant growth enhancers but also act
as a preventative measure against pathogen infection. Some of these products are
Vesicular Arbuscular Mycorrhizal Root Inoculant (VAMRI) (Fig. 11.1). It is com-
posed of chopped dried corn roots infected with Glomus mosseae or Glomus fascicu-
latum, an arbuscular mycorrhizal fungus. It can serve both as a biocontrol agent and
a biofertilizer. The biological agent allows for a better absorption of nutrients espe-
cially non-mobile elements such as phosphorus and zinc. VAMRI greatly reduces
disease incidence caused by Fusarium oxysporum, Pythium spp., Phytophthora
infestans, and Ralstonia solanacearum. It also induces resistance against burrowing
nematodes such as Radopholus similis in banana seedling plants. However, the
degree of effectiveness of VAMRI varies depending on the plant host, variety, culti-
var, VAM inoculant, density of application, and soil fertility. It has been found to be
effective inoculants for various crop species such as Capsicum spp. (pepper),
Solanum melongena (eggplant), Lycopersicon esculentum (tomato), Carica spp.
(papaya), Musa spp. (banana), Ananas comosus (pineapple), Citrullus lanatus
(watermelon), Allium cepa (onion), Zea mays (corn), Saccharum officinarum
(sugarcane), Arachis hypogaea (peanut) and some fruit crops and ornamental plants.
Application of VAMRI can potentially replace 50–100 % of the fertilizer require-
ment depending on the aforementioned factors. Currently, VAMRI is widely used
by onion farmers in the northern part of the Philippines. Trainings and extensions
have been conducted regarding its use across the Philippines, although it is still
insufficient owing to financial constraints (Brown et al. 2013, 2006, 2002, 1998).
Another BIOTECH-UPLB product primarily used as a plant growth enhancer is the
Brown Magic (Fig. 11.2). The product is also a mycorrhizal-based inoculant used for
orchids. It acts as a biofertilizer as well as biocontrol agent against soil-borne diseases of
orchids. The fungal inoculant is composed of sclerotium or fruiting bodies of fungi and
mycelia collected and isolated from orchid roots. Application of Brown Magic increases
orchids’ tolerance and resistance against pathogens and diseases. As a biofertilizer,
it induces early flowering in orchids, production of more suckers and longer spikes.
Mykovam on the other hand is a soil-based biofertilizer that can also act as a
biocontrol agent. It contains spores, infected root propagules, and VAM fungi. The
mode of action in growth promotion is similar to VAMRI – it increases the effec-
tiveness of nutrient absorption. A positive side effect of inoculation is the increased
tolerance to pathogens, environmental stresses such as water deficit and heavy metal
exposure. The product is economical and relatively easy to use. It only needs to be
applied once, and it has the capacity to replace 60–85 % of the commercial fertil-
izers used to plant growth. The product can be applied to a wide array of plants such
as vegetables, fruit crops, trees, and ornamentals with the exception of orchids.
Biogroe, on the other hand, consists of plant growth promoting rhizobacteria
(PGPR) that positively influence root growth as well as solubilize nutrients. The
presence of PGPR in the roots also influences the plants’ capacity to defend itself
against pathogens through out competition and production of metabolites inhibitory
to the growth of pathogens. It can be used on crops such as rice, sugarcane, vegeta-
bles, and some ornamentals.
11 Regulatory Requirements and Registration of Biopesticides in the Philippines 187
11.2.2 Biopesticides
increasing range and effectiveness, directly proportional to the severity of the infes-
tation (Lavinaa et al. 2001; Adams and McClintock 1991).
The utilization of bacterial toxins has also been explored for the biocontrol of
Lepidopteran insect pests in the form of Bt toxin. The mechanism is similar to NPV,
such that the toxin is only activated at alkaline pH. The toxin incapacitates the target
by affecting the digestive system leading to deprivation and ultimately death.
Bactrolep is a Bt-based formulation that is especially effective against Asian corn
borer (Ostrinia furnacalis) and diamond back moth (Fig. 11.3) (Padua et al. 1990,
1984, 1980). Asian corn borer infestation accounts for 4–30 % of yield loss during
dry season which is magnified during wet seasons (Gonzales 2000).
Currently, the development and usage of genetically modified plants are under
stringent regulations in the Philippines.
Another plant-derived pesticide that was developed in the Institute of Plant
Breeding in UPLB is the transgenic papaya resistant to papaya ringspot virus
(PRSV). The virus causes papaya mosaic disease which affects not just papayas
(Carica spp.) but cucurbits in general. Infected plants develop mosaic and chlorosis
in the leaves. Advanced infection exhibits young leaf distortion leading to a shoe-
string appearance, which could be mistaken as mite infestation. Infection at an early
stage of the plant shows symptoms of stunted growth. Fruits from the infected
plants, on the other hand, manifest ringspots. The development of the genetically
modified papaya resistant to PRSV was through the use of coat protein (cp) gene
technology conducted through particle bombardment. The technology made use of
a fragment of CP gene derived from Philippine PRSV strain (Magdalita et al. 2004).
Section IX of the Presidential Decree 1144 promulgated in May 1977 marks the
establishment of the Fertilizer and Pesticide Authority (FPA) responsible for the
regulation of the quality of pesticides/biopesticides, such that the commercially
released pesticide has minimal health and environmental hazard and ensures that
its price is set reasonably. Consequently, all pesticides and biopesticides as well
as their handlers have to be registered under FPA, prior to product release and
commercialization. Under FPA, the Pesticide Technical Advisory Committee is
responsible for governing the registration, setting of the requirements, issuing
labeling guidelines, approving or disapproving of license application, handing
out certification on approved pest control operators, regulating imports, and lim-
iting the availability of toxic agricultural products. The detailed registration pro-
cess under FPA is shown in Fig. 11.4 (FPA 2016). The registrant is required to
submit a technical grade sample of the formulated product along with an applica-
tion, data package, and samples of the technical material and analytical standards
to FPA. The data package includes prior information on the identity of the regis-
trant, product itself, manufacturing process, product composition, physical and
chemical characteristics, bioefficacy data, toxicological data which includes
information on long- and short-term toxicity studies, human safety data, environ-
mental hazard, and transport and residues in food. The application is then
reviewed by experts from different institutions and universities where the results
and recommendations are submitted to the Technical Advisory Committee. The
committee then forwards the recommendations to FPA which then approves or
disapproves the registration. If the application is approved, further instructions
on the labeling pictorially depicting hazards will be required in compliance to
World Health Organization’s recommendation. Each registration is subject for
renewal every 3 years.
Unfortunately, regulation of pesticidal products under the FPA has not been rig-
idly enforced. As a result, banned products still lurk the market, and their residues
are still detected on biological systems. This is in part due to the financial con-
straints leading to the inability to enforce its mandate. An added effect of this con-
straint would be product analyses inefficiencies (Lu et al. 2010).
Biopesticides are biocontrol chemicals, the active ingredient of which has been
derived from natural resources such as plants, animals, or microorganisms. Owing
to the nature of its source, the mode of action of biopesticides is considerably differ-
ent than that of traditional inorganic and synthetic chemicals. Data requirements for
the registration of such needs to be different as well. Some types of biopesticides
that is generally required to be registered are botanical pesticides, microbial pesti-
cides, and biochemical and macrobials such as biocontrol insects. Southeast Asian
nations in general have a few biopesticides that are registered in the form of neem
and rotenone.
11 Regulatory Requirements and Registration of Biopesticides in the Philippines 191
Data
FPA Complete?
NO
Consultants YES
Review/Assess
Data
Data
NO
YES
FPA integrates
Consultant’s
Report
YES
Accept?
NO
For CAT II or
Questionable
Data
PPTAC
Recommends
YES Full
Recommend
Accept?
NO Registration Conditional
Fig. 11.4 Schematic diagram of the pesticide registration process under the FPA (FPA 2016)
The root cause of the meager consumer demand and usage of biopesticides in the
Philippines lies in part with the regulatory policies regulating the registration of
pesticide and biopesticide products, administrative policies subsequent to the devel-
opment of technologies, and absence of well-defined linkages between the local
government units and farmers.
The regulatory policies associated with the regulation of pesticides and biopesti-
cides in the Philippines present stringency in biopesticide registration even though
it is a well-known fact that it, costs less, is eco-friendly and sustainable in nature.
This is because biopesticide registration process is almost similar to the registration
process of traditional pesticides. Consequently, the registration of new biopesticides
would cost a sizable amount of money. It is therefore recommended that the cost of
biopesticide registration be atleast lower especially if the registrant is a government
research institution or university. This is reflected on a very limited number of
biopesticides that have been registered under FPA as compared to the vast array of
synthetic pesticides registered though it is due to a very limited number of research-
ers developing and testing new biopesticides. This presents the area that require
urgent attention. Moreover, there is limited access to researchers on biological
resources for biopesticides development. Thus, it is recommended that a regional
bank for starter inoculants concerning biopesticides be established. It would func-
tion as a repository of biological materials found having the potential for use in
biopesticide development.
In the Philippines, the support of the development of a product or technology
ends with the product development. At this point it is up to the researcher to seek
grants in order to conduct further research, extensions, and seminars to increase the
public awareness regarding the availability, advantages, and disadvantages in the
usage of the product. Failure to do so means that limited investments on the product
will be made. Bridging this gap between the researchers and farmers, most of the
time, is the burden of the researchers. The tendency therefore is that most products
tend to be manufactured in the research institution itself, which in most cases lack
the capacity for mass production. Due to this reason, biopesticides in general are not
readily available in the market unlike synthetic pesticides. Moreover, added to the
burden of manufacturing is the cost of informing, conducting seminars, and adver-
tising which serves to increase the awareness of the public on the product. An alter-
native to commercialization could be through the establishment of linkages with the
local government units and nongovernment offices. The linkage will aggressively
extend the technology to farmers through integrated pest management programs in
the Philippines.
Biopesticides with its narrow spectrum of effect tend to make the application
procedures more complex, such that the farmers without the proper trainings will
naturally be daunted and intimidated by the task of using and applying biopesti-
cides. This is in stark contrast to traditional pesticides wherein application tends to
be the same way – spray as much as you can in an area as wide as you can. In
11 Regulatory Requirements and Registration of Biopesticides in the Philippines 193
addition, most farmers have the unfounded notion that biopesticides are less effec-
tive than traditional pesticides (Rola 1990). More importantly, farmers are used to
the practice of application of traditional pesticides. The green revolution half a cen-
tury ago has led to the wide usage of high-yielding varieties exceedingly dependent
on the massive usage of inorganic fertilizers and pesticides to maintain high yields.
Such is the case in Masagana 99 during the 1970s wherein high-yielding varieties
of rice have been widely promoted nationwide despite limited information on its
pest susceptibility. Moreover, cultural tendencies of the locals are inclined toward
religiosity and superstition. The dominant religion therefore has a high degree of
influence and the power to impose its stand on various issues, such as its opposition
to GM crops. Biopesticides in the form of GM crops therefore have a very limited
acceptance in the Philippines. Recent court ruling on Bt eggplant has prohibited
further field testing trials in the country citing the reason as lack of scientific con-
sensus on the risk involved. GM crops have been therefore frequently presented as
a technology that poses a high degree of risk. Consequently, the locals in general
naturally have an unfounded aversion and reluctance to use the technology.
Commercialization, after product development, is another facet that needs to be
critically addressed. Of the multitude of biopesticides and biofertilizers developed
in the Philippines, only a few have gained private investment for manufacturing and
commercialization. The reality is more grim for biopesticides as compared to that
for biofertilizers as it faces a far more competitive market with a myriad of fast-
acting and broad-ranged pesticides. Traditional synthetic pesticides in the
Philippines are manufactured en masse by multinational companies with stream-
lined production and commercialization process. Thus, their products are inexpen-
sive, although not necessarily affordable to most local farmers, widely available,
and widely known. Investments should be made by the government and partner-
ships with the private sector must be promoted. In this approach, private-owned
manufacturing would have the prerogative to set prices with the intent of competi-
tively maximizing profit. Thus, it is recommended that a research institution be
granted the benefit to create an establishment which would serve as the factory or
mixing plants for the biopesticides. This arrangement will allow for the maximum
benefit for the research institution and the farmers.
Trainings and extensions must be conducted in various parts of the country so
that pesticide users are well informed of the advantage of using biopesticide and
biofertilizers. Consequently, relevant and comprehensive training must be con-
ducted for the trainers to ensure the quality of the trainings. This endeavor is to
guarantee that farmers who will attend the training program will have the capacity
to plan and decide their strategy for pest and disease management. The farmers must
be aware of the proper application and amount of pesticides to use whether using
biopesticides or traditional synthetic pesticides.
Although research on biopesticides started three decades ago, it was only in 2011
that its promotion and wide adoption were implemented through the Republic Act
10068 or Organic Agriculture Act. The formation of the committee to develop
guidelines and protocols for the certification and registration is still ongoing in the
form of Bureau of Agriculture and Food Standards.
194 M.B. Brown et al.
11.5 Conclusion
References
Adams JR, Mc Clintock JT (1991) Nuclear polyhedrosis viruses of insects. In: Adams JR, Bonami
JR (eds) Atlas of invertebrate viruses. CRC Press, Boca Raton, pp 89–226
Bisen K, Keswani C, Mishra S, Saxena A, Rakshit A, Singh HB (2015) Unrealized potential of
seed biopriming for versatile agriculture. In: Rakshit A, Singh HB, Sen S (eds) Nutrient use
efficiency: from basics to advances. Springer, New Delhi, pp 193–206
Brown MB (1998) On-farm application of vesicular-arbuscular mycorrhizae on priority agricul-
tural crops. UPLB, College Laguna, Philippines
Brown MB (2002) Biotechnology for health program: endomycorrhizae as biological control
agents for soil-and root-borne plant diseases. #1248. UPLB, College, Laguna, Philippines
Brown MB (2006) Management of soil-borne disease on onions in rice-vegetable system using
specific biological control agents (vesicular-arbuscular mycorrhizae, VAM). UPLB, College,
Laguna, Philippines
Brown MB (2013) Philippine country project on biofertilizer technologies for sustainable agricul-
ture. TR #1344. UPLB, College, Laguna, Philippines
Bureau of Agricultural Statistics (2000–2004) Census of agriculture. Department of agriculture,
Quezon City, Philippines
Cuevas VC, Sinohin AM, Orajay JI (2005) Performance of selected Philippine species of
Trichoderma as biocontrol agents of damping off pathogens and as growth enhancer of vegeta-
bles in farmers’ field. Philipp Agric Sci 88:63–71
De Jesus LRA, Gabo RR (2000) Life history and host range of the mango Pulp Weevil, sternoche-
tus frigidus (Fabr.) in Palawan, Philippines. Philipp Agric 83:145–150
ELAZAQUI (1989) Policy issues in pesticides. UPLB, College, Philippines
Fertilizer and Pesticide Authority (2016) Pesticide regulatory policies in the Philippines. http://
www.dbm.gov.ph/wp-content/OPCCB/OPIF2012/DA/.pdf. Accessed Mar 2016
Food and Agriculture Organization (2012) Guidance for harmonizing pesticide regulatory man-
agement in southeast Asia. RAP Publication 2012/13
Gaston CP (1994) Pesticide regulatory policies of selected countries in Asia, Technical report no.
2. Regional Agribusiness Project
Gonzales L (2000) Breaking new ground – the prospects of enhancing the corn sector’s global
competitiveness through biotechnology. STRIVE Foundation, Philippines
11 Regulatory Requirements and Registration of Biopesticides in the Philippines 195
Gupta S, Dikshit AK (2010) Biopesticides: an eco-friendly approach for pest control. J Biopesticides
3(1):186–188
Javier PA, Brown MB (2007) Bio-fertilizers and bio-pesticides research and development at
UPLB. Crop Protection Cluster, University of the Philippines Los Baños (UPLB) College,
Laguna 4031
Javier PA, Morallo-Rejesus B (1986) Insecticidal activity of black pepper (Piper nigrum L.)
extracts. Phillipp Entomol 6:517–525
Keswani C, Mishra S, Sarma BK, Singh SP, Singh HB (2014) Unraveling the efficient application
of secondary metabolites of various Trichoderma. Appl Microbiol Biotechnol 98:533–544
Lavinaa BA, Padua LE, Wua FQ, Shirataa N, Ikedaa M, Kobayashiaa M (2001) Biological charac-
terization of a nucleopolyhedrovirus of Spodoptera litura (Lepidoptera: Noctuidae) isolated
from the Philippines. Biol Control 20(1):39–47
Leonardo RP (1983) Field evaluation of the insecticidal activity of makabuhai against three major
insect pests of rice. B.S. Thesis, UPLB, College, Laguna p 51
Lu JL, Cosca KZ, Del Mundo J (2010) Trends of pesticide exposure and related cases in the
Philippines. J Rural Med 5(2):153–164
Magdalita PM, Valencia LD, Ocampo Atid, Tabay RT, Villegas VN (2004) Towards development
of PRSV resistant papaya by genetic engineering. Institute of Plant Breeding, College of
Agriculture, University of the Philippines
Morallo-Rejesus B (1984) Status and prospects of botanical pesticides in the Philippines. Second
SEARCA Professorial Chair Lecture. 29 August 1984. UPLB, College, Laguna
Padua LE, Federici BA (1990) Development of mutants of the mosquitocidal bacterium Bacillus
thuringiensis subspecies morrisoni (PG-14) toxic to lepidopterous or dipterous insects. FEMS
Microbiol Lett 66(1–3):257–262. doi:10.1111/j.1574-6968.1990.tb04007.x
Padua LE, Ohba M, Aizawa K (1980) The isolates of Bacillus thuringiensis serotype 10 with a
highly preferential toxicity to mosquito larvae. J Invertebr Pathol 36:180–186.
doi:10.1016/0022-2011(80)90022-1
Padua LE, Ohba M, Aizawa K (1984) Isolation of a Bacillus thuringiensis strain (serotype 8a:8b)
highly and selectively toxic against mosquito larvae. J Invertebr Pathol 44(1):12–17.
doi:10.1016/0022-2011(84)90040-5
Rola AC (1990) Fertilizer and pesticide policies: growth, equity and environmental sustainability.
Seminar paper presented during the 4th FSSRI-CPDS Seminar Series on Policy Support to
Farming Systems Development, UPLB, College, Laguna, Philippines
World Bank (2014) http://data.worldbank.org/indicator/NV.AGR.TOTL.ZS. Accessed Mar 2016
Wu F (2007) Bt corn and impact on mycotoxins CAB reviews: perspectives in agriculture, veteri-
nary science. Nutr Nat Resour 2(60)
Yorobe JM, Quicoy CB (2006) Economic impact of Bt corn in the Philippines. Philippine Agric
Scientist 89(3):258–267
Biofertilizer Research, Development,
and Application in Vietnam 12
Pham Van Toan
Abstract
Biofertilizers are products containing living microorganisms having the ability to
convert nutritionally important elements from unavailable to available form and
enhance the plant growth through biological processes. The book chapter sum-
marized the research, development, application, and regulatory requirements of
biofertilizer in Vietnam and contains the results of isolation of nitrogen-fixing
(Rhizobium, Azotobacter, Azospirillum), phosphate-solubilizing (Bacillus), and
pathogen-antagonistic (Bacillus, Pseudomonas) microorganism, the evaluation
of their biological activities, the effect on the growth, the yield of crops, and the
economical effects to the crop production. Briefly informations about the regula-
tory requirement of biofertilizer in Vietnam are also included in the paper.
Keywords
Nitrogen fixation • Phosphate solubilization • Pathogen antagonistic • Biofertilizer
• Multifunctional biofertilizer
12.1 Introduction
Fertilizers play an important role in agriculture and are accountable for about 40 %
increase in crop yield. Vietnam is predominantly an agricultural country with more
than 10 million ha of agricultural land, so the demand for fertilizers is high. For the
past 10 years, the mineral fertilizer supply has been exponentially increasing.
However, the fertilizer use efficiency (FUE) is still low which is estimated at
40–45 % for nitrogen, 25–30 % for phosphorus, and 60 % for potassium (Nguyen
Van Bo 2014).
Biofertilizers contain beneficial microorganisms, which are applied to seed,
plant surfaces, or soil, colonize the rhizosphere (region around the surface of the
roots) or the interior of the plant, and promote growth by increasing the supply or
availability of primary nutrients to the host plant. Subsequent application of biofer-
tilizer reduces dependence on chemical fertilizers, thereby allowing the farmers to
cut the cost of agrochemical input.
Research on N-fixing and P-solubilizing microorganisms in Vietnam started two
decades ago. The results of the study showed that N-fixing inoculant can reduce
nitrogen application by 30–60 kg N/ha/year and increase the crop yield by 5–25 %
(Tables 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, and 12.9). P-solubilizing inoc-
ulants and rock phosphate can replace 30–50 % mineral phosphorus fertilizer with-
out significant change in crop yield. This chapter will discuss the environment
biofertilizer research, development, application, and regulatory requirements of
biofertilizer in Vietnam based on the research projects (Nguyen Kim Vu 1995;
Pham Van Toan 2000, 2005; Luong Huu Thanh and Pham Van Toan 2008).
From governmental budgets and international budgets in the last two decades, many
research projects were carried out in Vietnam with more than ten research institu-
tions and universities and more than 100 scientists working on the biofertilizer
12 Biofertilizer Research, Development, and Application in Vietnam 199
Rhizobial Plant nitrogen Ration of 15N-enriched and total plant N-fixing activity
strains (mg/pot) nitrogen content (%) (%) (mg/pot)
Control 51.67 0.285 – 18.2
MAR 337 86.17 0.225 21.1 21.7
TAL 1000 87.94 0.215 24.6 24.2
NC-92 94.68 0.212 25.6 24.6
98 90.97 0.208 27.0 24.7
THA 201 89.01 0.206 27.7 24.8
Isolates having high biological activities were tested for the growth, yield, and
nutrition uptake of crops. Data in Tables 12.7 and 12.8 shows the N-fixing ability of
rhizobia to form more nodules in groundnut as well as effects on growth and yield
of tested crops in comparison to untreated control. Rhizobial-inoculated peanut fer-
tilized with 30 % mineral N reduction has the same N content, green biomass, and
pod yield as the control fertilized with 100 % required mineral N fertilizer.
P-solubilizing microbes inoculated to rice and vegetable have positive effects on
growth, P uptake, and crop yield. Table 12.9 illustrates the effect of P-solubilizing
Bacillus on the growth of potato. The results showed that the combination of
Bacillus inoculation and rock phosphate fertilized with 50 % mineral production
has the same effect on plant growth and green biomass as the control fertilized with
100 % required mineral P fertilizer.
The beneficial microbes used for biofertilizer research and development in
Vietnam are listed in Table 12.10.
Different biofertilizers have been developed in Vietnam in the past. There are
Rhizobium inoculants for legumes (soybean, groundnut); free living or associate
N-fixing inoculant for rice, maize, and vegetables; P-solubilizing inoculants for
agricultural and forestry crops; and multifunctional biofertilizer for industrial crops.
12 Biofertilizer Research, Development, and Application in Vietnam 203
– Should be nontoxic
– Should have good moisture absorption capacity
– Should be easy to process and free of lump-forming materials
– Should be easy to sterilize by autoclaving or gamma irradiation
– Should be inexpensive
– Should have good pH buffering capacity
204 P. Van Toan
Table 12.13 Effect of different sterilizing methods on the survival of Bradyrhizobium japonicum
in the inoculants
Survival of Bradyrhizobium in the peat carrier-based inoculant in the
storage of 108 CFU/g
6
Sterilizing method 2h 1 month 2 months 3 months months
Control (non-sterilize) 2.5 24.0 0.1 – –
Dry hot gas 27 26.0 25.0 20.0 10.5
Saturated steam 2.5 25.0 25.5 23.0 11.5
Gamma irradiation 2.4 40.0 45.0 47.0 37.0
Note: Dry hot gas at 165 °C in 4 h and saturated steam of 1.5 Atm in 2 h
In addition the carrier should ensure the survival of microbes during the storage
period and survival of the inoculant bacteria in soil. In Vietnam peat is the most
popular carrier material.
Different methods are used for the carrier sterilization. Effects of irradiation in
the carrier processing and survival of Rhizobium can be seen in Tables 12.12 and
12.13. With the irradiation dosage of 25 kg, peat can be used as sterile carrier for the
rhizobial inoculant (Table 12.12). The density of Rhizobium japonicum in the peat
carrier based irradiated at 30 kg after 6 months of storage in polyethylene bags was
3.7 × 109/g, as compared with 1.05 × 109/g when sterilizing by dry hot gas and 1.15
× 109/g sterilizing by saturated steam (Table 12.13).
Liquid formulation of biofertilizers contains the desired microorganisms and
nutrients along with the substances that encourage longer shelf life and tolerance to
adverse conditions. The advantages of liquid biofertilizers over conventional carrier-
based biofertilizers are the following: (a) longer shelf life (12–24 months), (b) no
effect at high temperature, (c) no contamination and no loss of properties due to
storage, (d) high populations can be maintained more than 109 cells/ml up to 12–24
months, (e) easy to use by the farmers, (f) high export potential, and (g) dosages are
ten times less than carrier-based, quality control protocols that are easy and quick.
The data in Table 12.14 shows the differences in the rhizobial density in solid and
liquid formulation.
12 Biofertilizer Research, Development, and Application in Vietnam 205
The effect of liquid biofertilizer on nodule formation and yield of soybean was
tested, which showed no difference in the application of solid and liquid formula-
tion (Table 12.15).
Multifunctional biofertilizers are the formulation containing consortia of N-fixing
and P-solubilizing microbes and microorganism antagonistic to soilborne pathogens.
Different multifunctional biofertilizers have been developed in Vietnam. The list of
microbes used for multifunctional biofertilizer is presented in Table 12.16.
Effects of multistrain inoculant on target crops were tested in the greenhouse.
The data in Tables 12.17, 12.18, and 12.19 showed that multistrain inoculant has
positive effect on the growth of tested crops and is able to control the bacterial wilt
206 P. Van Toan
Table 12.17 The capability of multifunctional biofertilizer to control the bacterial wilt on tomato
Dry green biomass
% increase
Treatments (g/plant) to control
Control 3.207 –
Inoculation with R. solanacearum 2.660 −17.16
Inoculation with multifunctional biofertilizer 4.760 48.43
Inoculation with R. solanacearum and multifunctional 4.330 35.02
biofertilizer
CV% 8.2
LSD 0.4606
disease. Depending on the crop and location, the bacterial wilt control effect can be
more or less than 90 %.
Biofertilizers were tested in field condition following the regulation of the Ministry
of Agriculture and Rural Development in Vietnam. The effect of different biofertil-
izers is presented in Figures 12.3 and 12.4 showing that rhizobial inoculant can
increase grain yield of groundnut by 13.8–17.5 % in North Vietnam and 22 % in
South Vietnam. Experiments to evaluate the N fertilizer use efficiency showed that
12 Biofertilizer Research, Development, and Application in Vietnam 207
Table 12.18 The capability of multifunctional biofertilizer to control the bacterial wilt on
peanut
Dry green biomass
Nodule number % increase
Treatments (nod/plant) (g/pot) to control
Control 0.00 1.142 –
Inoculation with R. solanacearum 1.67 0.520 −54.47
Inoculation with multifunctional biofertilizer 32.54 1.578 38.18
Inoculation with R. solanacearum and 31.12 1.509 32.14
multifunctional biofertilizer
CV (%) – 10.50
LSD – 0.252
Table 12.19 The capability of multifunctional biofertilizer to control the bacterial wilt on potato
Dry biomass % increase
Treatments (g/plant) to control
Control 1.51 –
Inoculation with R. solanacearum 1.37 −9.27
Inoculation with multifunctional biofertilizer 2.20 45.69
Inoculation with R. solanacearum and multifunctional 2.09 38.41
biofertilizer
CV(%) 5.5
LSD 0.16
120
115
110
105 Control
100 Inoculation
95
90
Fertile Soil Infertile Soil Fertile Soil
Fig. 12.3 Effect of rhizobial inoculant to increasing groundnut yield in North Vietnam (%)
rhizobial inoculant has the same effect like mineral N dose of 60–69 kg N/ha (Table
12.20). Thus, rhizobial inoculation can efficiently save 20–30 kg mineral N/ha.
The economical benefits of rhizobial inoculation are used in Table 12.21 and
were calculated to about 442.000 VNĐ/ha.
N-fixing inoculant had positive effects on the growth and yield of rice. It depends
on the fertilizer status and nutrition content of soil. It can increase the rice yield by
4.07–19.59 % or can save 20 % of required N fertilizer (Fig. 12.5).
Application of N-fixing inoculant to maize and tea can increase the yield and
nutrient uptake in maize (Fig. 12.6) and tea (Fig. 12.7).
208 P. Van Toan
120
110
100
90
New cultivated soil Intercropping rice Intercropping vegetable
peanut soil peanut soil
Control Inoculation
Fig. 12.4 Effect of rhizobial inoculant to increasing peanut yield in South Vietnam (%)
Using the Azospirillum inoculation can save mineral N fertilizer. The data in
Table 12.22 showed that Azospirillum inoculation is equivalent to application of
14.15–17.36 kg mineral N fertilizer depending on the soil types and growth
season.
Azospirillum inoculant in maize and tea in Vietnam (Table 12.23) showed that
Azospirillum inoculant can increase yield by 9.42–10.17 % in maize and 17.98 % in
tea in comparison to the control without inoculation.
Phosphate-solubilizing biofertilizer is applied to rice and other food crops in
Vietnam. Tables 12.24 and 12.25 showed the result on the effects of phosphate-
solubilizing biofertilizer on the growth and yield of rice (Fig. 12.8).
12 Biofertilizer Research, Development, and Application in Vietnam 209
115
110
105
100
95
90
Fertile Soil Infertile Soil
Fig. 12.6 Effects of Azospirillum inoculation to increasing the maize yield (%)
200
150
100
50
0
Yield %
(tons/ha)
Basal fertilization
Basal fertilization+Azzospirillum
Azospirillillum+80% N of basal fertilization
Table 12.23 Effect of Azospirillum inoculant on the yield of maize and tea
Yield % increasing
Soil and crops Fertilization (tons/ha) to control
Maize on fertile soil Control: NPK,180.120.90 + 10 t FYM 4.14 –
(basal fertilization)
Basal fertilization + Azospirillum 4.53 9.42
inoculation
80 % N of basal fertilization + 4.03 −2.58
Azospirillum inoculation
Maize on infertile Control: NPK, 180.120.90 + 10 t FYM 2.95
soil (basal fertilization)
Basal fertilization + Azospirillum 3.25 10.17
inoculation
80 % N of basal fertilization + 2.87 −2.28
Azospirillum inoculation
Tea on fertile soil Control: NPK, 120.60.60 (basal 14.29 –
fertilization)
Basal fertilization + Azospirillum 16.86 17.98
inoculation
80 % N of basal fertilization + 15.10 5.66
Azospirillum inoculation
140
120
100 Farmer practis
80
Recommended
60 fertilization
40 Farmer
practis+inoculation
20
0
Plant hight (cm) Yield (%)
Fig. 12.8 Effect of P-solubilizing inoculation to increasing the plant height and yield (%) of
maize
Biofertilizer research and development began in Vietnam more than 20 years ago,
but the production and application of biofertilizer are limited. There is no factory
producing sterile inoculant in the country. They are produced only by research orga-
nizations in small scale. In general peat is used as carrier and sterilized by autoclave
before it is inoculated with microbial biomass. Radiation sterilization technique is
applied, but only for the research purpose and at laboratory scale. This kind of bio-
fertilizer has good quality, but lacking large-scale production facility, information,
and demonstration, their application is mostly in the experiment of some research
project. The inoculant using area changes from many thousand to hundred thousand
hectares per year, depending on the budget of the project. Most of biofertilizers
12 Biofertilizer Research, Development, and Application in Vietnam 213
At present, Vietnam does not away law directly for biofertilizer production and
commercialization. Indirectly there are laws on standardization and technical regu-
lation; the Law on Quality of Commercial Products, 2008, regulates the production
214 P. Van Toan
Table 12.32 Effect of multifunctional biofertilizer on crop yield and root disease control in large-
scale field trials
Yield (tons/ha) % root disease % decrease
Farmer % Farmer to farmer
Crops and location practice Biofertilizer increase practice Biofertilizer practice
Tomato – Vinh 15.52 1.85 19.27 1.25 0.00 100
Phuc Province
Potato – Hanoi 12.9 14.50 11.24 24.6 11.3 55.1
Province
Peanut – Namdinh 2.13 2.52 16.73 40.0 15.0 62.5
Province
Cabbage – 27.15 30.72 13.15 – – –
Namdinh Province
Watermelon – 17.5 20.00 13.0 12.0 3.3 72.5
Hoa Binh
Province
Pepper – Dak 2.28 3.50 53.51 6.7 4.7 29.99
Nong Province
Cotton – Dak Lak 1.48 1.86 25.96 8.6 0 100
Province
Coffee – Dak Lak 5.67 6.84 20.63 – – –
Province
Table 12.33 Economical effect of multifunctional biofertilizer to crop production (1000 VND)
Fertilizer input Benefit to farmer practice
Farmer From yield From decreasing Total
Crops practice Biofertilizer increase the input benefit
Tomato 6127 5527 6460 600 7060
Potato 5830 5630 4800 200 5.000
Peanut 3183 2383 3120 800 3920
Cabbage 6105 4905 7140 1200 8340
Pepper 9790 7790 4270 2000 6270
Cotton 1499 1967 1742 −468 1273
Coffee 7780 11,560 17,550 −3700 13,850
Vietnam Goverment
Fig. 12.9 Management system for the fertilizer production and commercialization in Vietnam
• Conduction of the field trial to evaluate the impact of biofertilizer on the growth,
the yield of crops, and the effectivity of tested fertilizer
• Declaration of biofertilizer to satisfy the national technical regulation and focus
on the main characters, the quality, and the application guideline of biofertilizer
• Acceptance of the declaration by the provincial Department of Crop Production
and Department of Crop Production of MARD
12.4 Conclusion
Biofertilizer has positive effect on plant growth and yield of most agricultural crops
in Vietnam. The research program of inoculant is well established. But its produc-
tion and application are limited. Problems and solutions can be faced as follows:
References
Forum of Nuclear Cooperation in Asia (FNCA) (2006) Biofertilizer Manual
Luong Huu Thanh, Pham Van Toan (2008) Complete the technology for production of multifunc-
tional Biofertilizer and its application. National Research project no KC04DA11
Nguyen Kim Vu (1995) Research on production technology and application of N-fixing inoculant
to increasing the yield of rice and upland crop in Vietnam. National Research project no
KC0801
Nguyen Van Bo (2014) Measures for improving fertilizer use efficiency in Vietnam. In: Proceeding
of national workshop on measures for improving fertilizer use efficiency in Vietnam, Hanoi on
28/3/2014, pp 9–32. Agricultural Publishing House
Nguyen Thu Ha (2015) Collection and preservation of microbial germbank used in agriculture
Pham Van Toan (2000) Research and development of combination biofertilizer from N. fixing and
P. solubilizing biofertilizer in Vietnam. National Research project no KHCN02.06b
Pham Van Toan (2005) Research and development of multifunctional Biofertilizer in Vietnam.
National Research project no KC0404
Biopesticides Research: Current Status
and Future Trends in Sri Lanka 13
R.H.S. Rajapakse, Disna Ratnasekera, and S. Abeysinghe
Abstract
Sri Lanka is a tropical country equally having rich diversity of arthropods includ-
ing natural enemies, economic pests, and indigenous plants majority with unique
chemical properties. Because of the substantial losses due to pests and diseases,
plant protection remains an essential issue in agriculture production in the coun-
try. There is increasing concern over synthetic pesticide usage due to their
adverse long-term effects on human health, environment, and natural pest man-
agement systems. As an eco-friendly alternate, the importance of biopesticides in
raising agricultural productivity is well recognized in Sri Lanka. Biopesticides
are quiet popular among farming community due to their unique features, viz.,
safety, limited host range or target specificity, the absence of toxic residues, eco-
friendly nature, and ease of application. Biopesticides have diverse modes of
action and hence resistance development in pests is slower/negligible. Currently,
plant powders, nonvolatile and volatile oils, and plant crude extracts are com-
mercially available for management of insect pests and nematodes. Further, sev-
eral bacterial and fungal biopesticides have shown promising results for the
efficient management of plant pathogens in Sri Lanka.
Keywords
Biopesticides • Biocontrol agents • Organic farming • Agriculturally important
microorganisms
13.1 Introduction
Sri Lanka has been known as an agricultural country since ancient times.
Approximately 30 % of the 6.5 million ha of land area in Sri Lanka are under culti-
vation. Due to the substantial crop losses caused by pests and diseases, plant protec-
tion remains an essential issue for improving agriculture production in the country.
The present annual synthetic pesticide consumption is estimated at 1700 t of active
ingredients amounting to approximately Rs 4.6 billion. There is an increasing con-
cern over synthetic pesticide usage due to their adverse long-term effects on human
health, environment, and natural pest management systems. As an alternate, the
importance of biopesticides in raising agricultural productivity is well recognized in
Sri Lanka.
The term biopesticides refer to those pesticides obtained from biological sources
such as microbes (fungi, bacteria, viruses, protozoa, and nematodes), semiochemi-
cals (insect sex pheromones), biochemicals (substances from biological sources
especially botanicals such as essential oils, nonvolatile oils, and extracts), and com-
mercially produced natural enemies (predators and parasitoids). Biopesticides are
distinguished from synthetic pesticides by their unique features: safety, limited host
range or target specificity (very low risk to nontarget organisms), the absence of
toxic residues on fruit and vegetables (easily degradable in nature), environment-
friendly, and easily applied using conventional spray equipment. Biopesticides offer
diverse modes of actions; hence, resistance development in pests is slower.
Therefore, there is immense scope in identifying and developing biopesticides as
alternative pest management strategies. The importance of novel and alternative
biopesticides and the necessity for research and development of novel, cost-
effective, environmentally friendly pesticide is well recognized in Sri Lanka.
Limitations and challengers of commercializing biopesticides are slow action
and low persistence when exposed to solar UV, high cost of production, and lack of
awareness among Sri Lankan farmers. The major constraints to biopesticide devel-
opment are poor awareness of decision-makers about opportunities offered by
biopesticides, lack of multidisciplinary expertise in the crucial later stages of devel-
opment, difficulty in conducting toxicological tests, and the long testing period of
bioactive compounds before registration and commercialization.
Biopesticides are broadly categorized into two groups, biochemical pest control
agents and microbial pest control agents.
Biochemical pest control agents: these chemicals are not directly toxic to target
organisms like nerve poisons and exhibit different mode of action like mating dis-
ruption, molt inhibition, and growth regulation. They are naturally occurring sub-
stances; if synthesized, they must be identical to the natural chemical. There are
four classes of compounds that fall into biochemical pest control agents:
13 Biopesticides Research: Current Status and Future Trends in Sri Lanka 221
Microbial pest control agents: these include formulations that occur in nature or
organisms effective as pest management agents. Microbial pest control agents
include:
Nearly 30 % of the 6.5 million ha of lands in Sri Lanka are cultivated to agricultural
crops. Of this, the food crop sector dominated by rice (730,000 ha), vegetables
(90,000 ha), root and tuber crops (100,000 ha), fruits, other field crops, and export
agricultural crops (200,000 ha) occupies around 12 million ha. The plantation crops
and other perennials occupy around 700,000 ha. Sri Lanka has a very rich natural
enemy complex of crop pests as compared to tropical Asian countries giving more
opportunities for the utilization of natural biocontrol. For instance, the egg parasit-
ism of brown plant hopper found to reach as high as 80 % in fields kept free of
pesticides during the early vegetative stage. Furthermore, rice land spiders fauna
were found very effective in managing hopper pests and also less affected by dia-
mondback moth damage when the wrapper leaves are kept free of insecticides to
allow the multiplication of parasitoids like Cotesia plutellae, Diadegma sp. The leaf
miner, Liriomyza sp., damage found to be low when the affected crops are sprayed
only with neem extracts as compared to other insecticides. These evidences suggest
the vast potential available for the application of biological pest control agents for
crop pest management in Sri Lanka (Table 13.3).
However, at present, only a few pesticides that qualify as biological pesticides
are marketed in Sri Lanka (Tables 13.1 and 13.2). Furthermore, farmer accep-
tance and applicability of these products at field level are found to be very low.
This could be due to the inherent features of the biopesticides that require careful
planning in the treatment schedules than the conventional pesticides and the low
profile given for promotional activities including education of farmers and exten-
sion staff.
222 R.H.S. Rajapakse et al.
Table 13.1 Commercially available biopesticides and other related compounds in Sri Lanka
Trade name/mode
Common name Origin of action Toxicity Remarks
Bacillus Bacillus Bt 85 % WG Stomach Acute oral
thuringiensis Bt thuringiensis poison. The LD50 for rats
subsp. kurstaki endotoxin > 2.67 g/kg, 1
crystals are × 1011spores/kg
solubilized,
and the
epithelial
cells of the
gut are
damaged,
insects stop
feeding, and
eventually
starve to
death
Abamectin Isolated from Acts by stimulating Acute oral
fermentation the release of LD50 for rats
of r-aminobutyric acid, 10s
Streptomyces an inhibitory
avermitilis. neurotransmitter,
Avermectin thus causing
(Vertimec) paralysis
Azadirachtin/neem Principal Neemasal and Ecdysone Acute oral
seed water extract insecticidal Neemgrow antagonist. LD50 for rats
ingredient of Disrupts >5000 mg/kg
neem seed insect
extract – molting.
contains Fungicidal
limonoids and miticidal,
antifeedant
and repellent
Many plant species with pest control properties were identified and used at village
level in small scale, and Sri Lankan farmers have sound knowledge about the indig-
enous practices of utilization of plant products for pest management. Neem prod-
ucts are highly utilized in pest management, and many researches have been
conducted to estimate their possible effects with fulfilled results. A number of
neem-based commercial products are available in Sri Lankan market for agricul-
tural uses in different trade names, and it is recommended to use for the control of
13 Biopesticides Research: Current Status and Future Trends in Sri Lanka 223
Table 13.2 Commercially available insect growth regulators, chitin synthesis inhibitors, molt-
accelerating compounds in Sri Lanka
Trade
Common name Origin name Mode of action Toxicity Remarks
Tebufenozide Molt- Mimic Ecdysone Acute Recommended
accelerating agonist, binds oral for the control of
compound to the receptor LD50 – leaffolder of rice
sites of rats 5000 and leaf-eating
molting mg/Kg caterpillar on
hormone, vegetables
ecdysone,
lethal molting
Methoxyfenozide Molt- Runner Accelerate Acute Recommended
accelerating molting oral for the control of
compound LD50 – leaffolder of rice
rats and leaf-eating
>5000 caterpillar on
mg/Kg vegetables
Chlorfluazuron Chitin Atabron Anti-molting Acute Recommended
synthesis agent oral for the control of
inhibitor LD50 – leaffolder of rice
rats 5000 and leaf-eating
mg/Kg caterpillar on
vegetables
Cyromazine Inhibits Trigard IGR with Acute Effective against
molting and contact action oral leaf miners
pupation interferes with LD50 –
molting and rats 3387
pupation mg/Kg
systemic in
plants
Buprofezin Chitin Applaud Chitin Acute Recommended
synthesis synthesis and oral against BPH and
inhibits prostaglandin LD50 – whiteflies
inhibition rats 2000
mg/Kg
Novaluron Chitin Rimon Affect molting, Acute Recommended
synthesis abnormal oral against BPH,
inhibits endocuticular LD50 – fruit borer, and
deposition, and rats 5000 leafhopper
abortive mg/Kg
molting
Table 13.3 Biopesticides with potential use in Sri Lanka for crop pest management
Trade Mode of
Common name Origin name action Toxicity Remarks
Spinosad Actinomycetes Success Nicotinic Acute No
(spinosyn C Saccharopolyspora acetylcholine oral residues,
and D) spinosa receptor, LD50 – rapidly
different rats 5000 degraded,
from nicotine mg/kg for leaf
or miner
imidacloprid control
leads to
paralysis
Metarhizium Entomopathogenic – – LC50 for BPH
anisopliae local fungus rats > control in
cultures 4850 rice
mg/m3
Predatory mites – – – – For the
control of
coconut
mites
Spodoptera Baculoviridae: “Spod-X” Active by – For
exigua NPV Nucleopolyhedrovirus Ness A ingestion. vegetable
WA, LC Caterpillars caterpillar
cease feeding control
after 4 days
and die after
5–10 days
Essential oils Bioactive compounds – Act as a – Stored
of plants repellents, grain pests
like- growth of rice,
Cinnamomum inhibitors cereals,
zeylanicum, legumes,
Cymbopogon and
nardus, C. potatoes
citratus, etc.
The postharvest losses and quality deterioration caused by storage pests are major
problems throughout the world and more vulnerable in tropical countries like Sri
Lanka. Traditional methods of pest control in grains by mixing them with neem,
citrus and maduruthala leaves, plant oils, and powdered plant materials have been
utilized by farmers for many years.
Continuous research has been performed systematically to evaluate plant-derived
biochemicals using various indigenous plant species in different forms such as
13 Biopesticides Research: Current Status and Future Trends in Sri Lanka 225
crude ethanol (CE) extracts, vegetable oils, dry powders, and combinations of plant
materials with insecticides against Callosobruchus spp. in laboratory conditions
(Rajapakse et al. 1998, 2002; Rajapakse and Ratnasekera 2009; Ratnasekera and
Rajapakse 2012).
Mixing plant oils with stored seeds is common among farmer communities.
Nonvolatile oil is used as a coating for seeds and effectively protects seeds against
insect pests during stage. The film of oil prevents the attachment of the egg to the
seed coat. Most of the oils are very effective and retain their effectiveness over a
long period. The non-bitter taste of plant oil used is an added advantage over neem
oil, which is known for its bitter taste. The relatively small amounts of oils required
their effectiveness, and the simple technology of extraction will make these plant
oils a better candidate for seed dressing purposes for cowpea storage. Nonvolatile
oils can have negative effects on adult beetles through contact toxicity or through
deterrence. With increase of prevailing prices of insecticides, the application of
plant oils would be an inexpensive control method against C. maculatus and C.
226 R.H.S. Rajapakse et al.
Table 13.4 Toxicity of the ethanol extracts of the leaves of 20 plants to C. maculatus and C.
chinensis
Corrected mortality
Day-1 Day-2 Day-3
Plant species C.c. C.m. C.c. C.m. C.c. C.m.
Capsicum frutescens 35 ± 1.0 40 ± 2.6 45 ± 1.2 48 ± 3.1 60 ± 6.6 60 ± 6.7
Myristica fragrans 00 00 00 00 00 00
Piper nigrum 35 ± 1.2 40 ± 1.8 48 ± 2.1 50 ± 2.7 55 ± 2.1 60 ± 6.5
Citrus reticulata 10 ± 0.8 12 ± 1.0 10 ± 1.0 08 ± 0.7 12 ± 1.1 14 ± 1.0
Cymbopogon citratus 50 ± 3.5 60 ± 5.2 50 ± 4.6 60 ± 4.3 65 ± 3.7 67 ± 4.9
Artocarpus 05 ± 0.5 08 ± 0.7 12 ± 1.0 11 ± 1.0 15 ± 1.1 17 ± 1.1
heterophyllus
Gliricidia sepium 00 00 00 00 00 00
Eugenia caryophyllata 50 ± 4.8 60 ± 5.1 60 ± 4.8 60 ± 4.0 71 ± 5.6 70 ± 6.2
Ricinus communis 00 00 00 00 00 00
Dillenia retusa 40 ± 2.8 44 ± 3.0 50 ± 3.7 56 ± 3.8 50 ± 4.2 58 ± 4.7
Azadirachta indica 50 ± 3.5 60 ± 4.2 60 ± 4.0 65 ± 4.6 80± 6.7
Cajanus cajan 00 00 00 00 00 00
Cassia occidentalis 06 ± 0.4 09 ± 0.6 12 ± 0.8 13 ± 1.1 15 ± 1.3 18 ± 1.5
Annona reticulata 80 ± 5.7 80 ± 5.6 80 ± 6.3 90 ± 6.0 90 ± 6.8 91 ± 7.1
Mangifera indica 00 00 00 00 00 00
Eupatorium odoratum 00 00 00 00 00 00
Ocimum sanctum 80 ± 5.7 80 ± 5.6 80 ± 6.3 90 ± 6.0 90 ± 6.8 91 ± 7.1
Capsicum annuum 30 ± 1.8 35 ± 2.1 43 ± 3.8 50 ± 4.6 48 ± 3.9 45 ± 3.2
Dioscorea polygonoides 00 00 00 00 00 00
wild
Hibiscus rosainensis 00 00 00 00 00 00
Source: Rajapakse and Ratnasekera (2008)
Volatile oils can be extracted mostly by aromatic plants. The yield volatile oil is
usually low, but due to repellence or toxicity, even small amounts of the concen-
trated essential extract can be very effective in airtight or hermetic storage
13 Biopesticides Research: Current Status and Future Trends in Sri Lanka 227
Table 13.5 The effect of standard chemical control, pirimiphos-methyl, and nonvolatile vegeta-
ble oil against C. maculatus adults
Treatments Dosage Mean (1 DAT) % mortality (7 DAT)
Control 0.00g 2.700g
Pirimiphos-methyl Full dose 100.00a 100.000a
¾ dose 90.367c 95.300b
½ dose 73.267e 85.700e
¼ dose 65.000f 81.000f
Groundnut oil 10 ml/kg 95.000b 85.700e
Coconut oil 10 ml/kg 91.000c 92.000c
Soybean oil 10 ml/kg 85.000d 87.700d
Source: Rajapakse 2002
structures. Nayanathara and Ratnasekera (2010) reported that volatile fumes of cin-
namon and citronella oil could effectively repel stored pests in bulk storages.
According to their findings, no losses on viability and no off-flavors are detected.
Hence, volatile oils have broad consumer preference in seed storage both for con-
sumption and store as planting material as oils are not mix with seeds.
Further, the repellent effects of ten oils, Domba (Calophyllum inophyllum L.),
Batu (Solanum indicum L.), leaf oil and bark oil of cinnamon (Cinnamomum verum
Presl.), mustard oil (Brassica juncea Cross.), neem oil (Azadirachta indica A. Juss.),
mee oil (Madhuca longifolia Koenig.), castor oil (Ricinus communis L.), citronella
oil (Cymbopogon nardus L.), and sesame oil (Sesamum indicum L.) were tested for
pulse beetle (Callosobruchus maculatus L.) in the laboratory conditions, and they
reported that citronella oil, neem oil, cinnamon leaf oil, and cinnamon bark oil
vapors showed significantly control in pulse beetles after infestation indicating the
highest repellent action and toxic effects (Rajapakse and Ratnasekera 2009).
Mixing plant materials as powder form is a simple technique that can be easily
adopted among farmers. Many plants have been tested in the laboratory as powders
to evaluate their possible effects. Clove powder was the most effective among the
four powders tested for adult mortality followed by root dust of papaya. Among the
plant powders tested, maduruthala (O. sanctum) was the most effective for sup-
pressing oviposition significantly followed by geta thumba (Leucas zeylanica).
Same study reported that enhanced toxicity and mortality to Callosobruchus spp.
persistence of the insecticide in causing significant reduction when combined with
vegetable oils. Further, these results revealed the potential applicability of some
indigenous plant materials as stored grain protectants. The modes of action of these
substances are not yet known, and further studies must be carried out especially to
clarify how it is involved in the physiology of reproduction.
228 R.H.S. Rajapakse et al.
13.5.1 Bacteria
13.5.2 Fungi
Mortality of insects due to fungal attack is considerably reported in the natural con-
ditions but did not utilized one of them as a biopesticide in commercial level up to
now. Recently, the Tea Research Institute of Sri Lanka focusing research on utiliza-
tion of locally isolated Beauveria bassiana against shot hole borer, and it shows
promising results under laboratory conditions (Pavithrani et al. 2009). Metarhizium
anisopliae has a potential use to manage coconut black beetle, Oryctes rhinoceros
(Fernando et al 1995), and the Coconut Research Institute of Sri Lanka has recom-
mended it to manage this beetle in local conditions. Metarhizium anisopliae and
entomopathogenic nematodes (Nematoda: Heterorhabditidae) were bioassayed
against Kalotermitidae termites of tea by the Tea Research Institute of Sri Lanka,
and promising strains are identified and further experiments are continuing yet.
A fungus Trichoderma viride normally colonizes near the rhizosphere and para-
sitize on pathogenic fungi such as Phythium, Rhizoctonia, and Fusarium.
Commercial formulations of T. viride are available in the local market, and it pro-
vides resistance to rot and wilt diseases of many crops.
Insect viruses have long been considered as advantageous agents for management
of insect pests of agricultural important crops, and among them, baculoviruses pay
a vital role in pest management in the world. The scientist in the Coconut Research
Institute at Sri Lanka did several attempts to use baculoviruses against the coconut
black beetle, Oryctes rhinoceros, and it is recommended to use Oryctes rhinoceros
virus (Orv) for control of coconut black beetle. Commercial formulation of
Spodoptera exigua NPV is available in the local market and is recommended for
management of caterpillars on vegetables.
The economy of Sri Lanka is mainly agriculture-based. It has two sectors, namely,
domestic and plantation sector. The domestic sector, which forms the dominant part
of agriculture, accounts for 1.7 million farming families in a population of around
19 million. Both sectors jointly contribute 20 % to gross domestic product (GDP)
and 34 % to employment (Central Bank of Sri Lanka Report 2002). In management
of pests, the plantation sector approaches in a more organized manner, whereas in
the domestic sector, it is more complicated due to large number of farmers, crops,
and the pests involved. Pest management in Sri Lanka is mostly synthetic pesticide-
dependent, and the annual imports of pesticides cost around 0.1 % of the gross
domestic production in 2012. Apart from pests and weeds, plant diseases caused by
various groups of plant pathogens are often a big challenge in agriculture in
230 R.H.S. Rajapakse et al.
Sri Lanka. Currently, protection of plants from diseases has also been largely based
on the use of synthetic chemical pesticides. Applications of pesticides can have
drastic negative effects on the environment, consumer, applicant, and appearance of
pesticide resistance strains of the target organisms. Therefore, reduction or elimina-
tion of chemical pesticides in agriculture is highly important. One of the most desir-
able means to achieve this goal is by the use of new tools based on biological control
agents (BCAs) and natural antimicrobial chemicals for disease control. Many stud-
ies have been devoted in Sri Lanka to the identification of microorganisms and
antimicrobial botanicals that can be able to reduce activity and/or kill plant patho-
gens during the past two decades. When a particular country is concerned, it is
important to develop its own BCAs as many quarantine procedures are imposed in
almost all countries. On the other hand, BCAs are very specific in action, and many
factors are involved in their mode of actions. Therefore, BCA developed for a par-
ticular plant-pathogen interaction in a country may not be suitable or not effective
in another country. However, most experiences were on laboratory scale, and dis-
ease control trials were confined to experimental greenhouses or small field plots.
Thus, large-scale mass production, formulations, and storage of biopesticides must
be initiated, and research leading for these aspects should be given more priorities.
Up to now, BCAs against several important soilborne pathogens, foliar patho-
gens, and postharvest pathogens in different crops have been tested in different
laboratories in Sri Lanka. For soilborne pathogens, Trichoderma, Bacillus, and
Pseudomonas spp. have been identified for Rhizoctonia, Sclerotium, and Fusarium
spp. on rice, chili, bean, cucumber, and banana. Soe and Costa (2012) reported that
Bacillus megaterium, Bacillus subtilis, and Aspergillus niger isolated from the rice
sheath were antagonistic to Rhizoctonia solani, the causal agent of sheath blight
disease of rice. The talc-based formulations of these antagonists were effective as
biopesticides on sheath blight pathogen. Abeysinghe (2009a) showed that Bacillus
subtilis was effective on Sclerotium rolfsii on chili and Pseudomonas spp. intro-
duced to the root system of bean was able to induced systemic resistance to bean
rust caused by Uromyces appendiculatus has also reported (Abeysinghe 2009b).
Trichoderma harzianum has also been identified as a biocontrol agent against bean
rust, and induced systemic resistance (ISR) was identified as a possible mode of
action (Abeysinghe 2009c). In the case of postharvest diseases Wijesinghe et al.
(2011) has shown that Trichoderma asperellum was effective against black rot
pathogen, Thielaviopsis paradoxa on pineapple. Jayasuriya and Thennakoon (2007)
reported that one of the important soilborne fungal pathogens of rubber, Rigidoporus
microporus, could be controlled by Trichoderma spp. Sivakumar et al. (2000)
reported that Trichoderma harzianum was effective on Nephelium lappaceum
against Botryodiplodia, Colletotrichum, and Gliocephalotrichum. Adikaram et al.
(2002) reported that Aureobasidium pullulans is effective against Botrytis cinerea of
strawberry fruits, and ISR has been identified as the mode of action. Gunasinghe
et al. (2009) reported that Flavobacterium spp. and Pantoea agglomerans have been
identified for controlling Colletotrichum musae and Lasiodiplodia theobromae, the
postharvest pathogens of banana.
13 Biopesticides Research: Current Status and Future Trends in Sri Lanka 231
Apart from these microorganisms, some botanical extracts have also been tested
against nematodes and fungal pathogens. Root nematodes in tomato have been suc-
cessfully controlled by leaf extract of Piper betle (Premachandra et al. 2014). Field
trials conducted by using Trichoderma bioformulation against Panama wilt patho-
gen Fusarium oxysporum f.sp. cubense have been successful in initial trials, and
further testing is in progress (Abeysinghe et al. unpublished). Moreover, biofilm
formation is one of the important aspects of root-colonizing bacteria, and efficacy
of such biofilms formation is known as an important factor for success of biocontrol
agents (Seneviratne et al. 2008). Therefore, numbers of research topics are currently
being focused into these aspects in Sri Lanka.
As mentioned above, biological control research toward screening, formulation,
and testing them under greenhouse as well as in field conditions are being con-
ducted. However, very limited biocontrol agents are evaluated to make a successful
transition from the laboratory to field. A good formulation is the key to the com-
mercial success of biocontrol agents. Understanding the biocontrol agent can lead
to innovative ways of improving the efficacy of the biocontrol product because
many physical and biological parameters could be influenced to the efficacy of the
product.
However, more efficient and effective ways of growing and formulating BCAs
are needed in many cases in order to make biocontrol economically viable. Research
on safety and environmental fate of BCAs is lacking at present. Therefore, regula-
tory criteria that are essential for safety have to be worked out. Additional funding
is needed for biocontrol research to be progressed in Sri Lanka. In this context,
importance of private/public partnerships with academia should be highlighted.
The Sri Lanka National Agricultural Policy clearly mentions the importance of pro-
moting production and utilization of organic and biofertilizer and gradually reduces
the use of chemical fertilizers through integrated plant nutrition system.
In 2002, the National Engineering Research and Development Center (NERD)
established the “Biogas and Biofertilizer Project at Muthurajawela in Sri Lanka,”
and this project was due to misunderstanding between the NERD and local
authorities.
Sarvodaya Economic Enterprise Development Service (Guarantee) Limited has
established effective microorganisms unit in 1996, and presently, this is the only one
producer with sole agent right to produce and market effective microorganism prod-
ucts in Sri Lanka. They marketed several EM products for crop production.
The search of new innovative crop management programs to reduce the health
risk and environmental pollution should have focused in the future research and
biofertilizer, and biological pesticides will ensure these criteria in food safety and
sustainability of agriculture in Sri Lanka. But there are several technological and
policy gaps to promote biofertilizer and biopesticide in Sri Lanka. The major prob-
lems are lack of funds for research and fewer facilities in research laboratories with
232 R.H.S. Rajapakse et al.
no novel equipment for research. It is necessary to train the research scientists with
no novel equipment for research, and also, it is necessary to develop international
linkages of local scientist with other scientists worldwide to easily exchange their
knowledge. The Sri Lanka National Agricultural Policy clearly mentions the impor-
tance of promoting production and utilization of organic and biofertilizer and grad-
ually reduces the use of chemical fertilizers through integrated plant nutation
system. But fertilizer subsidy is one of the major benefits enjoyed by farmers espe-
cially small holders and rice farmers. Rice is the staple food crop grown in Sri
Lanka and very difficult to cut down the fertilizer subsidy without proper low-cost
alternatives because of the political influence of the country. Fertilizer subsidy is
one of the barriers to promote biofertilizer, and meanwhile more research is required
to develop low-cost technology for utilization of biofertilizer as an alternative to
inorganic fertilizer. More field of research on biofertilizer is still in its infancy;
therefore, both laboratory and field experiments are required to fully explore poten-
tial use of biofertilizer for crop production in the future.
Sri Lanka’s agriculture have been practicing organic farming over centuries and use
of biofertilizer and biopesticides has been a traditional practice followed by many
rural farmers to increase the soil fertility, soil aggregate stability, water infiltration,
and soil water-holding capacity. Commercial formulation of biochemical pest con-
trol agents such as semiochemicals, hormones like ecdysteroids and juvenile hor-
mones, natural plant regulators, and enzymes is available in the pesticide market of
Sri Lanka today. As well, effective microorganisms such as bacteria, fungi, viruses,
nematodes, or genetically modified microorganisms effective as pest and pathogen
management agents are also available in the pesticide market.
References
Abeysinghe S (2009a) The effect of mode of application of Bacillus subtilis CA32r on control of
Sclerotium rolfsii on Capsicum annuum. Arch Phytopathol Plant Protect 42(9):835–846
Abeysinghe S (2009b) Induced systemic resistance (ISR) in bean (Phaseolus vulgaris L.) mediated
by rhizobacteria against bean rust caused by Uromyces appendiculatus under greenhouse and
field conditions. Arch Phytopathol Plant Protect 42(11):1079–1087
Abeysinghe S (2009c) Systemic resistance induced by Trichoderma harzianum RU01 against
Uromyces appendiculatus on Phaseolus vulgaris. J Nat Sci Found Sri Lanka 37(3):203–207
Adikaram NKB, Joyce DC, Terry LA (2002) Biocontrol activity and induced resistance as a pos-
sible mode of action for Aureobasidium pullulans against grey mould of strawberry fruit.
Australas Plant Pathol 31:223–229
Central Bank of Sri Lanka Report (2002) Economic and Financial Reports 2002 – Central Bank of
Sri Lanka. www.cbsl.gov.lk/htm/english. Accessed on 22 Aug 2016
Fernando LCP, Kanagaratnam P, Narangoda K (1995) Studies on the use of Metarhizium aniso-
pliae (Metsch) Sor. For the control of Oryctes rhinoceros in Sri Lanka. Cocos 10:46–52
Gunasinghe WKRN, Karunaratne AM (2009) Interactions of Colletotrichum musae and
Lasiodiplodia theobromae and their biocontrol by Pantoea agglomerans and Flavobacterium
sp. in expression of crown rot of “Embul” banana. BioControl 54:587–596
Jayasekara APDA, Seneviratne G, De Silva MSDL, Jayasinghe LASP, Prematunga P (2008)
Preliminary investigations on the potential applications of biofilmedbiofertilizers for tea nurs-
eries. In: Nainanayake NPAD, Everard JMDT (eds) Proceedings of the second symposium on
plantation crop research-export competitiveness through quality improvements. Coconut
Research Institute, Lunuwila, pp 170–175
Jayasinghearachchi HS, Seneviratne G (2006) Fungal solubilization of rock phosphate is enhanced
by forming fungal-rhizobial biofilms. Soil Biol Biotechnol 38(2):405–408
Jayasuriya KE, Thennakoon BI (2007) Biological control of Rigidoporus microporus, the cause of
white root disease in rubber. Ceylon J Sci (Biol Sci) 36(1):9–16
Kulasooriya SA, de Silva RSY (1981) Multivariate interpretation of the distribution of nitrogen-
fixing blue-green algae in rice soils in central Sri Lanka. Ann Bot 47:31–52
Mannakkara A and Rohan Rajapakse (2002) Host susceptibility of rice weevil, Sitophilusoryzae L.
(Coleoptera: Curculionidae) and effect of neem based pesticide for its control. In: Proceedings
of the Sri Lanka association for the advancement of science, 58th Annual Session, p 52
Nayanathara KHG, Ratnasekera D (2010) Efficacy of Cinnamon and Citronella oil vapours in the
control of Callosobruchus chinensis L. in bulk stored green gram. J Ent Res 34(3):201–211
Pavithrani YLB, Walgama RS, Mannakkara A, Nugaliyadda L (2009) Influence of temperature on
vegetative growth and pathogenicity of Beauveriabassiana on short hole borer Xyleborus for-
nicates (Coleoptera..Scolytidae) in tea. Proceedings of the second national symposium, Faculty
of Agriculture University of Ruhuna Sri Lanka, 10th September 2009
Premachandra WTSD, Mampitiyarchchi H, Ebssa L (2014) Nemato-toxic potential of Betel (Piper
betle L.) (Piperaceae) leaf. Crop Prot 65:1–5
Rajapakse RHS (1990) The effect of four botanicals on the oviposition and adult emergence of
Callosobruchus maculatus. Entomon India 21(3&4):211–215
Rajapakse RHS (2000) Pesticidal potential of tropical plants- insecticidal activity of some selected
botanicals against Callosobruchus maculates (F.) and C.chinensis L. (Coleoptera: Bruchidae).
Proc Abstracts Entomo Congress Asso Adv Ento Kerala India 11
Rajapakse RHS, Van Emden HF (1997) Potential of four vegetable oils and ten botanical powders
for reducing infestation of cowpeas by Callosobruchus maculatus, C. chinensis and C. rhode-
sianus. J Stored Prod Res 33(1):59–68
Rajapakse RHS, Ratnasekera D (2008) Pesticidal potential of some selected tropical plant extracts
against Callosobruchus maculates (F) and Callosobruches chinensis (L) (Coleptera: Bruchidae).
Trop Agric Res Ext Sri Lanka 11:69–71
234 R.H.S. Rajapakse et al.
Rajapakse RHS, Ratnasekera D (2012) The potential use of indigenous plant materials against
Callosobruchus chinensis L. and Callosobruchus maculatus L. (Coleoptera, Bruchidae) in
stored legumes in Sri Lanka. J Biopest 5(Supplementary): 88–94
Rajapakse RHS, Senanayake SGJN, Disna Ratnasekera (1998) The effect of five botanicals on
oviposition, adult emergence and mortality of Callosobruchus maculatus on cowpea. J Entomol
Res 22(2):117–122
Rajapakse Rohan, Rajapakse HL de Z and Ratnasekera Disna (2002) Effect of botanicals on ovi-
position, hatchability and mortality of Callosobruchus maculates L. (Coleoptera: Bruchidae).
Entomon, India 27(1): 93–98
Ratnasekera D, Rajapakse R (2009) Repellent properties of plant oil vapours on pulse beetle
(Callasobruchus maculatus L.) (Coleoptera: Bruchidae) in stored Green gram (Vigna radiata
Walp.). Trop Agric Res Ext Sri Lanka12(1):13–16
Senevirathna G, Zavahir JS, Bandara WMMS, Weerasekara MLMAW (2009) Fungal-bacterial
biofilms: their development for novel biotechnological applications. World J Microbiol
Biotechnol 24:739–743
Sivakumar D, Wijeratnam RSW, Wijesundara RLC, Marikar FMT, Abeysekara M (2000)
Antagonism effect of Trichoderma harzianum on postharvest pathogens of rambutan
(Nephelium lappaceum). Phytoparasitica 28(3):240–247
Soe KT, De Costa DM (2012) Development of a spore-based formulation of microbial pesticides
for control of rice sheath blight. Biocontrol Sci Tech 22(6):633–657
Wijesinghe CJ, Wijeratnam RSW, Samarasekara JKRR, Wijesundera RLC (2011) Development of
a formulation of Trichoderma asperellum to control black rot disease on pineapple caused by
(Thielaviopsis paradoxa). Crop Prot 30:300–306
Part IV
Biopesticide and Biofertilizer Regulatory
Requirements in North Asia
Commercialization and Regulatory
Requirements of Biopesticides in China 14
Tao Tian, Bingbing Sun, Hongtao Li, Yan Li, Tantan Gao,
Yunchao Li, Qingchao Zeng, and Qi Wang
Abstract
The development of modern agriculture systems, based on the scientific manage-
ment of water and fertilizers and excellent crop varieties, has played an impor-
tant role in ensuring supplies of agricultural products and increased farmers’
income in China. However, the continuous cropping of single crops and intense
management of water and fertilizer has actually promoted the prevalence of plant
diseases and insect pests, resulting in excess application of chemical pesticides.
Currently, the problem of pesticide residue pollution is becoming a critical threat
to food security and environmental health in China. To reduce the application of
chemical pesticides and fertilizers, “the zero-growth plans to 2020 for the appli-
cation of chemical pesticides and fertilizers” were proposed by the Chinese
Ministry of Agriculture on March 18, 2015. As partial substitutes for chemical
pesticide, biopesticides are receiving increasing attention from the government
and the public in China. In this chapter, the current status of biopesticides in
China is introduced, including an overview of the progress in developing biopes-
ticides, the current status of the biopesticide industry, the registration status of
biopesticide products, the application status of biopesticides, current registration
management of biopesticides, future innovations in the registration management
T. Tian • B. Sun
Institute of Plant Protection, Tianjin Academy of Agricultural Sciences,
Tianjin 300384, China
H. Li • Y. Li
Institute of Genetics and Physiology, Hebei Academy of Agricultural and Forestry Sciences,
Shijiazhuang 050051, China
Y. Li • T. Gao • Q. Zeng • Q. Wang (*)
Department of Plant Pathology, China Agricultural University, Beijing 100193, China
e-mail: [email protected]
Keywords
Biopesticides • Registration • Chemical pesticides • Fertilizer
As the largest developing country, China has a population of about 1.37 billion and
a cultivated land area of about 135 million hectares (National data 2014). The devel-
opment of modern agriculture systems, based on the scientific management of
water, fertilizers, and excellent crop varieties, has played an important role in ensur-
ing supplies of agricultural products and increased farmers’ income in China.
However, the long-term, continuous cropping of single crop and the intense man-
agement of water and fertilizers have promoted the prevalence of plant diseases and
insect pests, resulting in a huge demand for chemical pesticides. In 2015, the amount
of chemical pesticides applied in China reached 320,000 t (The ministry of agricul-
ture 2015). Currently, the problem of pesticide residue pollution is becoming a criti-
cal threat for the food security and environmental health in China. To reduce the
application of chemical pesticides and fertilizers, “the zero-growth plans to 2020 for
the application of chemical pesticides and fertilizers” were proposed by the Chinese
Ministry of Agriculture on March 18, 2015 (Start-up chemical fertilizers zero
growth plan 2015). As partial substitutes for chemical pesticides, biopesticides are
receiving increasing attention from the government and the public, thereby increas-
ing the opportunity to develop and commercialize biopesticides in China.
At present, there is no clear definition of a biopesticide in China. In “pesticide
registration and management terms” (NY/T1667.1_l667.8_2008), a biopesticide is
defined as a pesticide that controls plant pathogens, insect pests, mice, and weeds
that uses directly a living organism or bioactive substances extracted from the meta-
bolic processes of an organism, as well as synthetic substances that have the same
structure compared with the natural compounds. According to their composition
and sources, biopesticides are divided into six categories: microbial pesticides,
botanical pesticides, agro-antibiotics, natural enemy organism, biochemical pesti-
cides, and transgenic organisms (Ji and Wang 2010).
The study and development of biological pesticides in China began almost 70
years ago and can be divided into three stages.
In 1959, the insecticide Bacillus thuringiensis (Bt) was introduced in China from
the former Soviet Union, which marked the beginning of the modern Chinese pesti-
cide industry. Since then, Chinese biopesticide factories have developed several
types of biopesticide products including Bt insecticide, jinggangmycin, gongzhul-
ingmycin, and povamycin M. Notably, as the first agro-antibiotic with completely
14 Commercialization and Regulatory Requirements of Biopesticides in China 239
By 2014, there were more than 260 biopesticide production enterprises in China,
which accounted for about 10 % of pesticide production enterprises. The total out-
put of biopesticide production was nearly 130,000 t, and its output value was about
US$ 440 million, which accounted for about 9 % of the output and output value of
pesticide production (Fig. 14.1) (China biological pesticide market 2014). Today,
240 T. Tian et al.
12
4000
10
3000 8
6
2000
4
1000
2
0 0
2008 2009 2010 2011 2012 2013 2014 2008 2009 2010 2011 2012 2013 2014
Years Years
the product level of the Chinese biopesticide industry has achieved significant
progress because of continuous research efforts on biopesticides. For example, the
formulation of biopesticides is more stable and diverse, and some products show
slow release and highly efficient effects. On the whole, the Chinese biopesticide
industry has already mastered the key technology for product development at a tech-
nical level, even reaching leading international levels in some areas, such as the
artificial breeding technology of Trichogramma predatory mites and biocontrol
agents for phytopathogenic nematodes (Qiu 2015).
With farmers’ increasing demands for safe pest control products and the encourage-
ment of policies related to the application of biopesticides, in general, the overall
application area of biopesticides has shown a steadily increased trend (about 10 %)
14 Commercialization and Regulatory Requirements of Biopesticides in China 241
Table 14.1 Registration of products and active ingredients of biological pesticides in China (as
of December 2014)
Kinds of
active Total
S. no. Category ingredients products Active ingredients of commodities
01 Microbial 30 281 Bacillus (Bt and other species of
pesticides Bacillus), pseudomonad, Empedobacter
brevis, Agrobacterium radiobacter,
Beauveria, Metarhizium, Trichoderma,
Paecilomyces lilacinus, Conidiobolus
thromboides, Aureobasidium pullulans,
Pythium oligandrum, Verticillium
chlamydosporium, nuclear polyhedrosis
virus, cytoplasmic polyhedrosis virus,
granulosis virus
02 Botanical 29 157 Eucalyptol, star anise oil, pyrethrins,
pesticides pyrethrin (I + II), physcion, nucleotide,
baicalin, santonin, matrine, swainsonine,
celangulin, capsaicin, chamaejasmine,
triptolide, vertrine, toosendanin,
hyoscyamine, cnidiadin, carvacrol,
berberine, nicotine, allicin, azadirachtin,
d-camphor, rotenone, camphor, methyl
eugenol, sanguinarine, soybean lecithin
03 Biochemical 26 231 Oligosaccharins, brassinolide,
pesticides 6-benzylaminol-purine, harpin protein,
gibberellic acid, trimedlure, heteroauxin,
fatty acid mixed, ethephon, plant
activator protein, chitosan, muscalure,
torula yeast, triacontanol, oxyenadenine,
fungus proteoglycan
04 Natural enemies 5 7 Amblyseius cucumeris, Microsporidia,
Harmonia axyridis, Trichogramma,
Anastatus sp.
05 Agro-antibiotics 27 2200 Jinggangmycin, gongzhulingmycin,
povamycin, kasugamycin, agro-antibiotic
120, wuyimycin, zhongshengmycin,
ningnanmycin, abamectin, ivermectin,
streptomycin, liuyangmycin, spinosad,
cycloheximide, blasticidin S, tetramycin,
nikkomycin, phenazino-1-carboxylic
acid
06 Genetically 0 0
modified
organisms
Total 117 2876
(Communication from Lin Rong-hua and Yang Jun)
242 T. Tian et al.
in the last decades in China. It is notable that the application area of biopesticides in
2012 had increased by about 40 % compared to that in 2011, because of the promo-
tion policies of the Chinese government. At present, the total area of the application
of biopesticides in China is about 26 m h annually (Yang et al. 2014). The detailed
application areas of all types of biopesticide categories in 2010 and 2011 are listed
in Table 14.2.
14 Commercialization and Regulatory Requirements of Biopesticides in China 243
At present, there are 12 units of laws, rules, and regulations related to biopesticide
registration and management in China, including the Code of Primary Products
Quality Security, Administrative Permit Law, Standardization Law, Trademark Law,
Patent Law, Law on Protection of the Rights and Interests of Consumers, Agricultural
Chemicals Administrative Protection Regulations, Dangerous Chemicals
Management Regulations, Regulations on the Control of Pesticides, Measures for
Implementation of Pesticide Management Regulations, and Data Requirement on
Pesticide Registration.
In China the registration and management of biopesticides adhere to the follow-
ing four principles:
At present, there are some issues with the registration and management of biopesti-
cides in China. The main problems include (a) lack of detection techniques and
qualified testing units for the quality of biopesticide products, (b) the definition of
244 T. Tian et al.
Table 14.3 Data requirements of new product registration of regular chemical pesticide (techni-
cal material)
S.
no. Categories Requirements
01 Physical and chemical Active ingredient concentrations, name and content of major
properties impurities, physical and chemical parameters of products
(appearance, melting point, boiling point, density or bulk
density, specific optical rotation, etc.), analytical methods for
active ingredients
02 Toxicological Acute oral, dermal and inhalation toxicity; irritation test of skin
information and eyes, skin sensitization test
03 Efficacy data Efficacy test reports of the plot experiment of products in
multiple regions for 2 years or more
04 Environmental test Acute toxicity test of birds, fish, water flea, algae, bees (oral and
data contact), and silkworms. If it is highly toxic or virulent to these
organisms, the environmental impact experiment report must be
provided
05 Residual test data Reports of residual tests for 2 years or more
Table 14.4 Data requirements of new product registration of regular chemical pesticide
(formulation)
S.
no. Categories Requirements
01 Physical and chemical Formulation, content of active ingredients, the specific name and
properties concentration of other ingredients, physical and chemical
parameters of the product, items and index of quality control,
classification (according to purpose), analytical methods of
active ingredients
02 Toxicological Tests of acute oral, dermal, and inhalation toxicity, toxic
information symptoms, and methods of emergency treatment
03 Efficacy data Efficacy test reports of the plot experiment of products in
multiple regions for 2 years or more (influence on the quality of
harvested items, resistance research, and shelter setting)
04 Environmental test Acute toxicity test report of bees, birds, fishes, and silkworms
data (in some cases it could be relieved or exempted); if the
formulation is released slowly, test reports of soil degradation
and adsorption are required
05 Residual test data Reports of residual tests for 2 years or more
biochemical pesticides which is not clear, (c) lack of scientific and systematic prod-
uct quality standards (strain identification and analysis methods of microorganism,
active ingredient analysis methods, quality control and storage of products), (d) the
registration of technical materials and technical concentrates, and (e) relieving or
exempting the required registration data on a scientific and reasonable basis.
Therefore, efforts should be made to improve the management of biopesticide reg-
istration in the future, for instance, augmenting the qualified biopesticide testing
14 Commercialization and Regulatory Requirements of Biopesticides in China 245
Table 14.5 Data requirements of new product registration of microbial pesticides (technical
concentrate)
S.
no. Categories Requirements
01 Physical, chemical, and The data form of microbial pesticides is different from that of
biological properties chemical pesticides and the specified requirements including
analytical methods, storage stability, contamination by
microbes, taxonomic status, strain authentication report, and
strain code
02 Toxicological Proof materials that the active ingredient does not belong to
information known pathogens of humans or other mammals must be
provided; acute oral, dermal, and inhalation toxicity tests;
irritation and infectivity test of eyes; sensitization;
pathogenicity (oral, inhaling, injection); other toxicological
information (if necessary)
03 Environmental test data Acute toxicity test of birds, fish, water flea, algae, bees (oral
and contact), and silkworms; if it is highly toxic or virulent to
these organisms, the experimental report of environmental
multiplication ability must be provided
04 Other required data The same as that of chemical pesticides
Table 14.6 Data requirements of new product registration of microbial pesticides (formulation)
S.
no. Categories Requirements
01 Physical, chemical, and The data form of microbial pesticides is different from that of
biological properties chemical pesticides, and the specified requirements include
analytical methods and storage stability
02 Efficacy data Toxicological information
03 Toxicological Proof materials that the active ingredients do not belong to
information known pathogen of human or other mammals should be
provided; additionally, six tests, including acute oral, dermal,
and inhalation toxicity, irritation test of the eyes and skin, and
sensitization of skins, should be provided
04 Environmental test data The acute toxicity test report about bees, birds, fishes, and
silkworms (in some cases it could be relieved or exempted)
05 Residual test data Could apply for relief or exemption depending on the decision
of temporary committee of registration
units, improving the techniques for product quality control, and eventually
establishing a profound evaluation, registration, and management system of
biopesticides.
The future direction of the development of biopesticide registration and manage-
ment should apply to different product categories: (a) according to the properties of
every category of biopesticides, specific management system should be developed
for them, and (b) as for the management of agro-antibiotic pesticides, they should
be carefully treated based on their properties. For those antibiotics that are accepted
worldwide, the registration requirements should be simplified; however, for those
246 T. Tian et al.
Table 14.7 Data requirements of new product registration of botanical pesticides (technical
concentrate)
S.
no. Categories Requirements
01 Physical, chemical, and The same as that of chemical pesticides. However, if the
biological properties analysis report of total fractions could not be provided for
specific reasons, the proof issued from a testing agency
authorized by the Ministry of Agriculture should be provided,
and the one or more active ingredients must be identified
02 Toxicological The required information is the same as that of chemical
information pesticides. However, if it has been registered as a food additive,
pharmaceutical, or healthcare product, the information about
reproduction, teratogenesis, chronic effect, and carcinogenicity
could be relieved or exempted by application
03 Environmental test data Environmental behavior (soil degradation test, hydrolysis test,
photolysis test in water), environmental toxicology (acute
toxicity test of birds, fish, water flea, algae, bee, and silkworm)
04 Other required data The same as chemical pesticides
Table 14.8 Data requirements of new product registration of botanical pesticides (formulation)
S.
no. Categories Requirements
01 Physical and chemical The same as that of chemical pesticides
properties
02 Efficacy data The same as that of chemical pesticides
03 Toxicological Six test items, including acute oral, dermal, and inhalation
information toxicity, irritation test of the eyes and skin, and sensitization
of the skin, should be conducted
04 Environmental test data Acute toxicity test of birds, fish, water flea, algae, bee, and
silkworm; if the formulation is released slowly, test reports of
soil degradation and adsorption are required
05 Residual test data Report of residual test in multiple plots for 2 years or
applying for the decision of the registration committee
antibiotics that could affect humans and animals, the registration requirements
should be strengthened to prevent their injudicious use.
To promote the development of biopesticide industry, the registration and man-
agement policies of biopesticides should be adjusted to better match China’s
national conditions. The revised data requirements for biopesticide registration
should follow the principles of shortened registration process and the simplified
data requirements to streamline the registration policy. For example, the original
registration process (field trials-temporary registration phase-officially registered
phase) should be adjusted to field trials followed by the officially registered phase.
The regulation of pesticide registration data requirement should incorporate some
noteworthy changes including the following: (a) agro-antibiotics should be
14 Commercialization and Regulatory Requirements of Biopesticides in China 247
Table 14.9 Data requirement of new product registration of biochemical pesticides (technical
concentrate)
S.
no. Categories Requirements
01 Physical, chemical, The same as that of chemical pesticides. Could apply for relief or
and biological exemption of the data of residue and environmental behavior
properties tests if products are derived from biological fermentation and
have been safely applied in agro-production for years
02 Toxicological Six tests, including acute oral, dermal, and inhalation toxicity,
information irritation test of the eyes and skin, and sensitization of the skin,
are required
03 Environmental test Acute toxicity tests of fish, water flea, algae, bee (oral and
data contact), and silkworm; unless it is highly toxic, data of
environmental behavior could be exempted
04 Other required data The same as that of chemical pesticides
Table 14.11 Data requirement of new product registration of natural enemies (formulation)
S.
no. Categories Requirements
01 Quality of products Reports concerning biological characteristics, index and test
methods of quality control, and verifying report of test methods
02 Efficacy data Efficacy test reports of the plot experiments of products in
multiple regions for 2 years (control objects, scope of
application, control efficacy, economical efficiency)
03 Toxicological Information concerning bioactivity and biosecurity, influence on
information crops
04 Environmental test Influence on national protected species, beneficial organisms,
data untargeted organisms; possibility of hybridization with
indigenous species or races and its influence
248 T. Tian et al.
Table 14.12 Data requirement of new product registration of genetically modified organisms
S.
no. Categories Requirements
01 Physical and Category of the genetically modified organism (plant, animal, or
chemical properties others), recipient organism, target genes, vectors, types of
genetic manipulation
02 Toxicological Acute oral, dermal, and inhalation toxicity to mammals (rat),
information sensitization of the skin, safety of agricultural products
03 Efficacy data Efficacy test reports of the plot experiment of products in
multiple regions for 2 years or more
04 Environmental test The influence of the residual body of the genetically modified
data organism on the environment, including the influence of gene
flow to the ecosystem, the genetic makeup, and the stability of
the gene; decomposition property; influence on environmental
organisms (soil microbes, birds, bees, and aquatic organisms)
05 Residual test data Residue examination of toxicants in agro-products
Source: Tables 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 14.10, 14.11, and 14.12 are modified from
Lin Huarong
In the last decade, more than 20 major, national scientific projects had been deployed
by the Chinese government to accelerate the research and industrialization of
biopesticides. As a result, the Chinese biopesticide industry has achieved significant
progress in the fields of key technology and product development. The present tech-
nological level of biopesticide products, such as Bt, agro-antibiotics, cotton boll-
worm nuclear polyhedrosis virus (NPV), and fungal insecticides, is very advanced.
Several new activator proteins originating from microbes have been identified, and
related products have been developed, which laid a foundation for the commercial-
ization of such biopesticides. Nosema locustae and several new types of agro-anti-
biotic products have been developed and applied successfully (Qiu 2013). The “gas
dual-dynamic solid-state fermentation” technique and apparatus have been devel-
oped, which overcome the shortcomings of traditional solid-state fermentation pro-
cess, such as the high rate of contamination and uncontrollable fermentation
parameters (Chen and Qiu 2010). Meanwhile, certain new botanical pesticides and
natural enemies have been identified and developed.
14 Commercialization and Regulatory Requirements of Biopesticides in China 249
According to the action targets, microbial pesticides could be divided into four
types: microbiocides, microbial insecticides, microbial herbicides, and microbial
growth regulators. At present, more than 100 different bacteria have been screened
as insecticides in China. Among them four Bacillus species, B. thuringiensis, B.
popilliae, B. sphaericus, and B. lentimorbus, were developed as products and have
been applied practically. Recently, scientific interest in screening synergistic fac-
tors, fermentation technology, and the genetic recombination of broad-spectrum Bt
is in focus. The control efficiency of the pathogenicity-enhanced Bt strain CAB109
against beet armyworm reached 75.3 %, which exceeds the control efficiency of
commonly used chemical pesticides and microbial insecticides. Two hundred
twenty virus strains have been isolated from 188 species of insects, of which 110
were first reported by Chinese researchers. As a candidate microbiocide, Bacillus is
the most extensively studied genus, and applied research using Bacillus reached
internationally advanced levels in China. Certain excellent strains have been regis-
tered as biopesticides for disease control in various crops. B. subtilis strain Bs-916
was registered for the control of rice sheath blight disease with stable control effi-
ciency in the field of 60–80 %. B. subtilis strain B908 was registered to control
Panax notoginseng root rot, tobacco black shank, and rice sheath blight disease. The
control efficiency in the field of both B. subtilis strains BL03 and XM16 against
apple core mold and cotton anthracnose disease reached a remarkable 90 %. The
control efficiency of B. pumilus strain TW2 against rice neck blast was about 80 %,
which was effective for the disease control. Fusarium and Godronia were reported
to be highly effective for the control of wild oats and semen cuscutae, respectively
(Zhu and Yin 2012; Liu et al. 2014).
The systematic study of botanical pesticide research in China started in the 1950s.
To date, more than 2000 kinds of plants have been scrutinized for agro-active com-
pounds. Plants with insecticidal activities have been identified, such as Sabina vul-
garis Antoine, Cynanchum hancockianum (Maxim.) Al. Iljinski, Senecio palmatus,
the flower bud of lily magnolia, and Macleaya cordata (Willd.) R. Br. In addition,
many plants with insecticide or microbiocide activities have been reported. Spinacia
oleracea L. and Albizia julibrissin Durazz. effectively repressed tobacco mosaic
virus (TMV). Cephalotaxus hainanensis and other plants were allelopathic to some
kinds of weeds (Zhang et al. 2015). In the last decade, the scope for screening plant
candidates has expanded, and more attention has been paid to the identification of
biologically active plant substances. Zhang et al. (2015) reported that some sub-
stances extracted from plants such as cinnamon, the root of red-rooted salvia, leaves
of clove, lotus, and tea seeds had the potential for industrial exploitation because of
their obvious preservation effects on kiwi fruit, apple, and medlar. Meanwhile, cer-
tain essential oils were reported to have insecticidal or antimicrobial activities,
250 T. Tian et al.
and some components of these oils were identified and verified functionally
(Hu et al. 2011).
The finiteness of natural resources is a bottleneck, limiting the industrialization
of botanical pesticides. Based on the totipotency of plant cells, different kinds of
bioreactors have been developed. At present, the applications of bioreactors include
plant cell suspension culture, adventitious root development, and hairy root culture.
Using these techniques, more than 100 different kinds of plants have been cultivated
artificially, including pyrethrum, Tripterygium wilfordii, Tagetes patula, ginseng,
Melia azedarach, and Celastrus angulatus. In addition to the target compounds,
many new compounds have been discovered during the in vitro cultivation of plant
tissues. For example, the new taxane compounds named sinenxans A, B, and C were
identified from callus of Taxus chinensis. New diterpenoid and triterpenoid com-
pounds were separated from calli and hairy roots of T. wilfordii. New sesquiterpenoid-
like compounds were isolated from the hairy roots of Artemisia apiacea (Zhang
et al. 2015). These discoveries illustrate the new approach for seeking new botanical
candidates of biopesticides.
Since the 1950s, research employing natural enemies for management of agricul-
tural pests has been conducted in China. In the 1980s, natural enemies of insect
pests of important crops were investigated systematically nationwide. According to
14 Commercialization and Regulatory Requirements of Biopesticides in China 251
the statistical data, more than 1000 natural enemy species for rice pests, more than
960 natural enemy species for corn pests, and more than 840 natural enemy species
for cotton pests were identified. According to the category of natural enemies, more
than 900 species of parasitoid wasps of Ichneumonidae, more than 380 species of
predaceous ladybird, more than 400 species of tachinid, more than 400 species of
200 phytoseiid mites, and more than 150 species of farmland spiders were
identified.
Presently, with breakthroughs in artificial diets and propagation techniques, a
group of natural enemies could be produced commercially on a large scale, includ-
ing Trichogramma spp., Anastatus spp., Chrysopa spp., Encarsia formosa Gahan,
Orius sauteri (Poppius), Eretmocerus sp., Thyscus fulvus Compore et Annecke,
Chouioia cunea Yang, and predator mites. For example, using C. cunea Yang to
control a worldwide quarantine object (fall webworm) represents a significant
achievement in China. This successful control exploited the advantages of C. cunea
Yang, such as high parasitic rate, strong reproductive capacity, high ratio of females,
and year-round convenient alternative hosts. This showed that C. cunea Yang could
be an excellent candidate for management of fall webworm. In the C. cunea Yang
release area, from the second year to the fifth year, the rate of attack by fall web-
worm was lower than 0.1 %, and the parasitic rate of C. cunea Yang was more than
92 %, which indicated a sustainable effect (Yang et al. 2014).
14.7.5 Agro-antibiotics
China has become the world’s largest producer of jinggangmycin and abamectin,
which are among the best-selling and most applied fungicide and insecticide prod-
ucts in China. From the viewpoints of industrial scale and technological level, three
agro-antibiotics, jinggangmycin, abamectin, and gibberellin, are the leading prod-
ucts. Some agro-antibiotics, such as agricultural streptomycin, agro-antibiotic 120,
povamycin, and zhongshengmycin, were listed as staple and backbone products of
the microbial pesticide industry in China (Ren 2014).
In the last decade, some new varieties of agro-antibiotics have been developed in
China, such as wanlongmycin reported by the Institute of Plant Protection of
Guangdong Province, zuelamycin reported by the Northwest Agriculture and
Forestry University, antimycin reported by the Microbiology Institute of the
Zhejiang Academy of Agricultural Sciences, aureonucleomycin and phoslactomy-
cin reported by the Shanghai Pesticide Research Institute, meilingmycin reported
by the Jiangxi Agricultural University, and polaramycin reported by the Chinese
Academy of Medical Sciences (Qiu 2013). With the progress in bioengineering
technology, some important properties, such as efficiency and safety, of agro-
antibiotics were improved by remodeling the existing agro-antibiotics via biological
synthesis and chemical semi-synthesis. Meanwhile, significantly improved yields
of antibiotics were realized using genetic engineering (Chen et al. 2014).
252 T. Tian et al.
To promote the application of biopesticides, a financial subsidy policy was put into
practice by the Chinese Ministry of Agriculture in 2014. To establish a standard
system of financial subsidies, pilot schemes of the first batch of financial subsidies
were conducted in some counties of Beijing City, Shandong Province, and Shanghai
City (Qiu 2015). The exploratory system of financial subsidies of Beijing City will
be introduced in detail.
Beijing is not only the capital of China but also the economical center and one of
the biggest cities. Currently, Beijing is suffering from pollution from different
sources including industrial emission, vehicle exhaust, and pesticide residues. To
relieve the pressure from environmental pollution, large amounts of money have
been invested to seek feasible methods to counter environmental pollution. Among
them, promoting the application of biopesticides by providing allowances to grow-
ers has accelerated the application of biopesticides. So far, four different approaches
have been established to subsidize the use of biopesticides.
The first model is the subsidized sale model that was trialed in Renshou Town,
Changping District. Customers buy the biopesticide products listed in the subsidy
catalogue from agricultural materials stores at a subsidized price. Only growers who
hold debit cards of agricultural capital subsidies for strawberry planting are quali-
fied for the allowance. Biopesticide products are subsidized by 80 %, and other
chemical pesticides with high efficiency, low toxicity, and low-residue properties
are subsidized by 50 %. The second model is the chain delivery model, which was
trialed in Tongzhou District. The chain delivery system, comprising agricultural
sales agencies and local agricultural services, distributes subsidized biopesticides to
growers directly. The third model is the village committee delivery system, which
was trialed in Fangshan District. This system comprises the plant protection station
of Fangshan District, the agricultural offices of local towns, and local village com-
mittees. Subsidized biopesticides are distributed from the plant protection station of
Fangshan District to the local village committees via agricultural offices of local
towns. The fourth model is the specialized service organization delivery system
trialed in Daxing District. Basing on the local situation of plant diseases and insects,
the subsidized biopesticides were distributed to technicians of local specialized crop
cooperation services, and these technicians provide a unified service for pest control
(Yue et al. 2014).
14.9 Conclusion
Through long-term efforts of the government, scientists, and industries, the Chinese
biopesticide industry has made great achievements. However, there are still some
obstacles to the ongoing development of the Chinese biopesticide industry. Firstly,
some innate disadvantages, including relatively higher controlling cost, unstable
controlling effects, and higher knowledge background requirement for users
14 Commercialization and Regulatory Requirements of Biopesticides in China 253
compared with that of chemical pesticides, have meant that, in practice, many farm-
ers consider biopesticides as the last choice for pest control. Secondly, it is very
difficult to detect pesticide residues of agricultural products widely in such a large
market as China, and it is impossible for consumers to distinguish harmless agricul-
tural products from their harmful equivalents. Agricultural products subjected to
improperly applied chemical pesticides for pest control may be produced at a rela-
tively lower cost; therefore, they are more competitive in the market. Thirdly, in the
pursuit of maximum profits, many big pesticide enterprises are reluctant to recom-
mend biopesticides to the farmers. By contrast, although some small pesticide
enterprises wish to develop biopesticides to survive in the fiercely competitive mar-
ket, they seldom possess adequate financial and technological capacity to support
the development of new biopesticide products. Biopesticides could be a powerful
aid to sustainable agriculture. They are the most likely source of alternatives to
some of the most problematic chemical pesticides that may severely affect the
health of consumers and environment. Therefore, the prospects of biopesticides are
bright, because they represent an ultimate solution to the problems including pest
resistance to traditional chemical pesticides and the side effects of pesticides on the
surrounding environment and on human health (Qiu 2015).
Promoting the development of the biopesticide industry to guarantee the safety
of the environment and the food chain has become an important concern of the
Chinese government and the public. To alleviate this concern, some measures
should be taken or enhanced, such as the support of policy and finance, intensified
supervision of pesticides, and decreased registration cost (time and capital). It must
be noted that although it is important to encourage the application of biopesticides
to aid the development of sustainable agriculture, biopesticides are not absolutely
safe: some biopesticide products still have potential hazards to the environment and
human health. Therefore, risk assessment of biopesticide products should be
enhanced in the future to establish a rigorous risk assessment system for biopesti-
cide products (Yang et al. 2014).
References
Chen HZ, Qiu WH (2010) Key technologies for bioethanol production from lignocelluloses.
Biotechnol Adv Sinica 28:556–562
Chen Y, Zhang XL, Wang Y et al (2014) Research progress of insecticidal antibiotics. J Agr
Biotechnol Sinica 22:1455–1462
China Biological Pesticide Market (2014) http://www.51report.com/free/3043302.html. Accessed
15 Mar 2014
Hu LF, Xu ML, Zhu HX (2011) Advances in antifungal activity of plant essential oil. Nat Prod Res
Dev 23:384–391
Huang JA (2002) The biological pesticide and its application status. Chinese J Microb Ecol
6:365–368
Ji Y, Wang YY (2010) Current status and registration information provision of biopesticides. J Agr
Chem Market Sinica 13:29–30
Liu ZH, Luo YC, Zhang DJ et al (2014) Research progress and prospects of microbial pesticide
formulation for plant disease control. Chinese J Pesticide Sci 16(5):497–507
254 T. Tian et al.
Abstract
In Taiwan, the biopesticides are divided into three categories including natural
products, biochemical agents, and microbial agents. A total of 52 agro-pesticide
permits of biopesticide have been registered in Taiwan. Most of biopesticides are
classified as microbial agents, and the majority components of microbial agents
are Bacillus thuringiensis and Bacillus subtilis which have 24 and 10 agro-
pesticide permits, respectively. According to the statistics of agro-pesticide pro-
duction report, the biopesticide sales volume in Taiwan was 0.401, 0.295, and
0.225 % of total pesticide sales volume in 2009, 2010, and 2011, respectively.
The amount of sales volume was far lower compared to the global biopesticide
sales volume (6.46 %). Besides, the requirements of food safety from consumers
and the booming of organic farming contribute to accelerating the development
of biopesticides. In dealing with the need of more biopesticides, the competent
authority for agro-pesticide management, Bureau of Animal and Plant Health
Inspection and Quarantine (BAPHIQ), Council of Agriculture (COA), has
decreased the requirements of biopesticide registration on the basis of product
safety; if the biopesticide is developed by using local microorganisms as active
ingredients, the testing data of toxicology required for product registration has
been reduced to only three items, including acute oral toxicity/pathogenicity,
acute dermal toxicity/pathogenicity (conditional, depends on the formulation of
biopesticides), and acute pulmonary toxicity/pathogenicity data. In addition, it
does not need to provide the data of wettability, stability test, and preheat
treatment for heat tolerance. Furthermore, BAPHIQ continued to revise two key
regulations on agro-pesticide registration in 2013, such as “standards for physi-
cochemical property tests and toxicology testing of agro-pesticides” and “stan-
dards for agro-pesticide field test,” which is a step further to reduce technical
barriers in achieving the goal of promoting biopesticide production. However,
biopesticide registrants encounter several problems in Taiwan. First, the complex
biological characteristics of microbes are difficult to fit the unique regulation of
product specifications. Second, the quality control data is related to the business
confidentiality. Third, the requirements of constructing biopesticide factory need
to be clarified. Finally, shall biopesticides be listed as least-regulated pesticides?
To resolve the abovementioned problems, it is necessary to establish a single-
desk unit to provide the preregistration consultation and regulation advice for
industry and to set up the standard procedures of research, invention, and regis-
tration for enhancing the development of biopesticides.
Keywords
Biopesticide • Agro-pesticide Management Act • Microbial agent • Agro-
pesticide registration
15.1 Introduction
Taiwan is located on the junction of tropical and subtropical areas; the warm and
humid climate and intensive multiple cropping system have facilitated the develop-
ment of plant diseases and pests, which are important limiting factors to the agricul-
tural production. With the agricultural trade liberalization and accession of Taiwan
to the World Trade Organization (WTO), the imports of agricultural products to
Taiwan have been increasing vastly, resulting in the high risk of invasion by exotic
plant diseases and pests. In view of ensuring the yield and quality of crops, the easy
and simple way for control of pests and diseases is by using synthetic pesticides.
In 1962, Rachel Carson noticed that the use of chemical pesticides was causing
damage to the nontarget organisms and ecological systems (Carson 1962).
Thereafter, farmers gradually realized the importance of environmental protection
and initiated to reform the cultivation methods such as ecological farming, nontoxic
agriculture, and organic farming. By this trend, biopesticides had been attracting
researchers’ attention. Biopesticides in general are considered as safer and more
specific and pose less residual problem as compared to chemical pesticides. With
these characteristics, the biopesticide has gained public attention because it fits the
consumers’ demand for food safety.
According to the “Agro-pesticide Act” Article 5 of Taiwan, the formulated agro-
pesticides including the chemicals or biologically based formulations are listed
below: (1) those used for preventing and eliminating pests of crops and forest or the
15 The Registration and Regulation of Biopesticides in Taiwan 257
Fig. 15.1 Role of central competent authority for agro-pesticide management in Taiwan
products thereof, (2) those used for regulating the growth of crops and forest or for
influencing the physiological functions thereof, (3) those used for regulating the
growth of beneficial insects, and (4) any other chemicals designated by the central
competent authority for protecting plants that belong to the scope of pesticide man-
agement by the government (Agro-pesticide Act 2007). The competent authority for
pesticide management is the Bureau of Animal and Plant Health Inspection and
Quarantine (BAPHIQ), Council of Agriculture (COA), at the central level and the
local government at the regional level. Taiwan Agricultural Chemicals and Toxic
Substances Research Institute (TACTRI), COA, is in charge of pharmacology and
toxicology tests of agro-pesticide. Agriculture and Food Agency (AFA), COA,
monitors the pesticide residues and harmful substances in crops. The district agri-
cultural research and extension stations are responsible for counseling the safe use
of agro-pesticides to farmers (Fig. 15.1).
Biopesticides are divided into three categories including natural products, micro-
bial agents, and biochemical agents. The natural products refer to those natural
ingredients which are not chemically refined or synthesized, e.g., pyrethrum. The
microbial agents are used for controlling the pests or diseases of crops, e.g., Bacillus
thuringiensis and Bacillus subtilis or their active ingredients. The biochemical
agents mean biological ingredients are extracted with chemical solvents or synthe-
sized; the control methods do not directly kill pests but attract them, e.g., as phero-
mones (The standards for physico-chemical property tests and toxicology testing of
agro-pesticides 2013).
Owing to food safety requirements of consumers, the development of biopesti-
cides is more important and urgent with each passing day. In order to accelerate the
development and registration of biopesticides in Taiwan, it is necessary to under-
stand the “Agro-pesticide Act” and registration procedures for biopesticides.
258 T.-C. Lin et al.
3.74% 6.46%
Chemical pesticides
Bio-pesticides
96.26% 93.54%
2009 2014 (estimated value)
Fig. 15.2 The global sales volume of biopesticides and chemical pesticides
Fig. 15.3 The domestic sales volume of biopesticides and chemical pesticides in Taiwan
15 The Registration and Regulation of Biopesticides in Taiwan 259
TACTRI
Application Data Re-examination in Safety,
Pesticide Toxicological & Field
(Applicants) Physical, Chemical &
Test Data Examination
Residue Tolerance
Official Budget (Toxic Group of P.A.C.)
Physical &
(Government Units) Chemical Test
Specification
Tests Toxicological
Issue License Test
(BAPHIQ)
Efficacy Test
Draft of Field Test Phytotoxicity Test
Pay Fee, License and Report (TACTRI)
Submit Obtaining (Applicants) Residue Test
Prior to implementing the field trials, biopesticides shall confirm the specification
inspection, and the design of their domestic field test shall be approved by the cen-
tral competent authority (BAPHIQ). The field test of the biopesticides (including
efficacy test, hazard test, and residue test) shall be implemented in more than three
different locations, and at least one of them shall be conducted domestically.
Although the reports of foreign field tests are acceptable in Taiwan, the efficacy of
biopesticides should be confirmed again, domestically. The efficacy test is divided
into the complete test and verification test. The control treatment with the similar
type of registered biopesticides is required for both tests. If the candidate biopesti-
cide is a brand-new active ingredient and the same type of product is not available
for comparison, the efficacy test report with untreated group as control is accept-
able. In the case of hazard test of biopesticides, it shall be carried out on at least two
different varieties of the same crop.
Notes
1. Applicants for the registration of technical grade agro-pesticide shall provide testing on pathogenicity studies. Applicants for the registration of formulated
agro-pesticide shall provide testing on acute toxicity studies (LD50)
2. Intravenous (IV) injection pathogenicity studies are applicable for bacterial and viral products; intraperitoneal (IP) injection pathogenicity studies are
applicable for fungal and protozoan products
3. Acute eye and dermal irritation testing is required on an animal testing, preferable with albino rabbits. Products that have a pH value <2 or >11.5 are
exempted from this testing, but are required to label on the product with warnings of eye and dermal corrosive toxicity and severe acute eye and dermal irrita-
tion. Applicants for the registration of technical grade agro-pesticide shall provide the testing results of the technical grade agro-pesticide. Applicants for the
registration of formulated agro-pesticide shall merely provide the testing results of acute eye irritation
4. Required for products that contain genetically modified microorganisms or for those with frequent contact toward the skin, inhalation, and human body
5. Results of products that contain genetically modified viruses are required
6. Results of products that contain genetically modified microorganisms or are to be used in aquatic environment (including rice paddy field) are required. If
other ingredients are highly toxic or have synergistic effect, the results of aquatic acute toxicity testing of the formulated agro-pesticide shall be provided
7. Results of products that contain genetically modified microorganisms or microbial herbicides are required. The plant species for testing depend on each
individual case
8. Products that contain genetically modified microorganisms or microbial insecticides used in parasite release sites are required to provide the results of toxic-
ity/pathogenicity test on the parasite
9. Unless the method of use poses low risk to pollinators (such as indoor usages), all other methods of use in the fields for nectar plants shall provide honey bee
pathogenicity testing results of technical grade agro-pesticide. Other ingredients added in formulated agro-pesticide known to be toxic to honey bees are
required to provide the testing results of acute contact toxicity to honey bee adults of the formulated agro-pesticide. Nectar plants include: buckwheat, citrus,
longan, lychee, melons, orange, pomelo, wax apple, loquat, starfruit, guava, plum, Chinese plum, peach, pear, strawberry, tea, camellia, canola, Astragalus
sinicus, Sesbania cannabina, garden cosmos, Bidens pilosa var. radiata, Bidens pilosa var. pilosa, sunflower, kumquat (fortunella), lemon, asparagus, broccoli,
The Registration and Regulation of Biopesticides in Taiwan
cabbage, cauliflower, orange jessamine, melaleuca, leucaena, and Chinese tallow tree. Pollen plants: rice, maize, sorghum, millet, wheat, barley, garden cos-
mos, sunflower, Bidens pilosa var. radiata, Bidens pilosa var. minor, Bidens pilosa var. pilosa, asparagus, broccoli, cabbage, cauliflower, grape, kumquat
(fortunella), lemon, loquat, orange jessamine, melaleuca, leucaena, Chinese tallow tree, and nutgall tree
10. According to the provided results, further results of toxicological testing shall be required if there are safety concerns
265
266 T.-C. Lin et al.
The researchers and inventors dealing with the registration of biopesticides lack the
understanding of “Agro-pesticide Act.” Thus, the documents or information required
for registration are usually not ready for reviewing. For example, the active ingredi-
ents of the microbial agents will be differed with microorganisms cultured in differ-
ent media, resulting in difficulties in registration. Prior to registration, the applicants
can consult experts at single window of TACTRI to ensure the validity and integrity
of the required documents. Basically, to confirm the active ingredients of biopesti-
cides (including microorganisms themselves and its metabolites), the applicants
should establish the procedures for identification of the active ingredients of biopes-
ticides, and the microorganisms themselves should be preserved at the Bioresource
Collection and Research Center (BCRC) of the Food Industry Research and
Development Institute in Taiwan for further examination.
Although biopesticides are usually concerned with characteristics of high secu-
rity, low toxicity, zero residue, and low environmental hazard, the individual prod-
ucts have yet to assess whether or not to classify as least-regulated agro-pesticides.
To avoid the side effects of “bad money drives out good” and to ensure the quality
of the products, biopesticides are still registered in accordance with the regulations
of the “Agro-pesticide Act.”
15.6 Conclusion
References
Agricultural Chemicals And Toxic Substances Research Institute, COA, R.O.C. (Taiwan) http://
www.tactri.gov.tw. Accessed 10 Mar 2016
Agro-pesticide Act (2007) http://law.moj.gov.tw. Accessed 10 Mar 2016
15 The Registration and Regulation of Biopesticides in Taiwan 269
Agro-pesticides information, Bureau of Animal and Plant Health Inspection and Quarantine,
Council of Agriculture (COA), R.O.C. (Taiwan) http://pesticide.baphiq.gov.tw. Accessed 10
Mar 2016
BCC Research (2010) Biopesticides: the global market. BCC Research, Wellesley
Carson R (1962) Silent spring. New Yorker, lnc, New York
Standards for Physico-Chemical Property Tests and Toxicology Testing of Agro-Pesticides (2013)
http://law.moj.gov.tw. Accessed 10 Mar 2016
The Taiwan Accreditation Foundation (TAF) http://www.taftw.org.tw. Accessed 10 Mar 2016
Part V
Biopesticide and Biofertilizer Regulatory
Requirements in West Asia
Biorational, Environmentally Safe
Methods for the Control of Soil 16
Pathogens and Pests in Israel
Abstract
Since 1992, 74 % of the plant protection products have been removed from the
European market. While this process resulted in the withdrawal of most of the
pesticides applied to the soil, alternative nontoxic solutions to these chemicals
are lacking. Clearly there is an acute need for biorational control strategies for
the management of soil pathogens and pests. Here we review the use of compost,
biofumigation, and bacterial biological control. While these measures were first
intended for sustainable and organic agriculture, we believe they will be adopted
in the near future by the conventional sector. The pathogen- and pest-suppressive
capability of composts is associated with microbial activity of bacterial and fun-
gal populations in the rhizosphere and the interactions between microbials and
macrobials in the soil. Biofumigation is a cultural method to treat soilborne
pathogens and pests, using green manures, as well as by crop rotation, intercrop-
ping, and pure compound amendments as seed meal, dried plant material, or soil
mulching. Biofumigation technology demonstrates the potential to reduce envi-
ronmental pollution through the replacement of toxic synthetic pesticides with
biodegradable plant secondary metabolites. While there is considerable literature
on the use of biofumigation on soil pathogens and nematodes, much less can be
found on the control of soilborne insect pests. Here the emphasis will be on field
L. Shaltiel-Harpaz • S. Masaphy
Migal Galilee Research Institute, Kiryat Shmona, Israel
Tel Hai College, Upper Galilee, Tel Hai, 12210, Israel
L. Tsror (Lahkim)
Department of Plant Pathology and Weed Research, Gilat Research Center, Institute of Plant
Protection, Agricultural Research Organization (ARO), Negev, Israel
E. Palevsky (*)
Department of Entomology, Institute of Plant Protection, Newe-Ya’ar Research Center,
Agricultural Research Organization (ARO), P.O. Box 1021, Ramat Yishay 30095, Israel
e-mail: [email protected]
Keywords
PGPR • Fusarium wilt • Biofumigation • Sustainable agriculture
16.1 Introduction
16.2 Compost
produces CH4, CO2, and many intermediate organic compounds (Golueke 1972).
Optimal microbial activity for substrate decomposition requires that the tempera-
tures of compost piles be maintained below 60°C. Careful temperature control also
allows for the survival and increase of pathogen antagonists in compost such as
actinomycetes and Bacillus spp. (Hoitink and Fahy 1986). Composts can vary
according to the initial raw material used, the chemical composition of the wastes
(especially the lignin and cellulose content), the presence of beneficial microflora,
and the conditions during the composting process (Hoitink and Boehm 1999; Noble
and Coventry 2005). The disease-suppressive capability of composts is associated
with microbial activity of bacterial and fungal populations in the rhizosphere and
the interactions between antagonists and pathogens. Composts contribute to the
suppressive activity of the amended soils through one or a combination of (a) com-
petition for nutrients, (b) antibiosis, (c) production of lytic and other extracellular
enzymes and compounds, (d) hyperparasitism and predation, and (e) host-mediated
induction of resistance or abiotic factors such as pH and C-source (Hoitink et al.
1997; Whipps 1997; Steyaert et al. 2003; El-Masry et al. 2002; Keswani et al. 2014;
Stoffella and Kahn 2001; Malandraki et al. 2008; Hoitink and Fahy 1986; Alfano
et al. 2007; Yogev et al. 2010; Borrero et al. 2004; Bisen et al. 2015, 2016). As a
result, it is often difficult to determine the exact suppression mechanisms, especially
in compost due to the complex structure of the microbial community in compost
(Boulter et al. 2000, 2002).
The potential of composts to suppress soilborne pathogens such as Pythium spp.,
Rhizoctonia solani, and Fusarium spp. has been demonstrated in many studies
(Erhart et al. 1999; Hoitink et al. 1997; Borrero et al. 2006). Under normal produc-
tion circumstances, when inoculum pressure is not drastic, or when inoculum is
weakened (Freeman and Katan 1988), compost-derived pathogen suppression may
serve as a practical control tool. Suppression of Fusarium diseases by composts was
reported for a single combination of compost and pathosystem, viz., coffee-waste
composts for controlling Fusarium wilt in melon, tomato residues to control
Fusarium crown and root rot in tomato, and separated cattle manure for controlling
Fusarium root and stem rot in cucumber and Fusarium crown and root rot in tomato
(Ros et al. 2005; Cheuk et al. 2005; Kannangara et al. 2004; Raviv 2005). Yogev
et al. (2006) demonstrated suppression of diseases caused by different formae spe-
ciales of Fusarium oxysporum by composts based on plant-waste residues. Composts
derived from plant residues suppressed diseases caused by Fusarium pathogens, as
compared with disease development in the highly conducive peat. The composting
of plant residues not only provides suppressive composts but also has high sanita-
tion value, since composting eliminates surviving pathogens from the infected tis-
sues (Hoitink and Fahy 1986). Composting of plant residues is also of significant
environmental value as it avoids the need for landfills (Yogev et al. 2006).
In addition to the suppressive effect of compost on plant pathogens, long-term
compost amendments supported higher levels of mites belonging to the suborders
Mesostigmata and Oribatida (Leroy et al. 2007; Minor and Norton 2004). Mites
(Acari) of these taxa perform important ecological functions in the soil, serving as
predators (mostly from Mesostigmata) and decomposers (Oribatida) (Manu and
276 L. Shaltiel-Harpaz et al.
Honciuc 2010; de Groot et al. 2016). Acarine soil predators feed on insects, mites,
nematodes, and fungi (Gerson et al. 2003). One acarine soil pest of economic
importance of flowers, vegetables, and cereals is the bulb mite Rhizoglyphus robini
(Gerson et al. 1983, 1985; Diaz et al. 2000). Biological control of this pest in lily
began with the identification and evaluation of the generalist predator Gaeolaelaps
aculeifer (Lesna et al. 1995, 1996; Conijn et al. 1997; Lesna et al. 2000). Another
avenue of research that was pursued was the association between Fusarium oxyspo-
rum and R. robini. Plant damage and mite populations were significantly higher
when both pathogen and mites were present (Okabe and Amano 1991). Additionally,
the mite was attracted to alcohols secreted by this fungus, on infested bulbs (Shinkaji
et al. 1988; Okabe and Amano 1990). Recently it was demonstrated that R. robini is
attracted to feed on plant tissue infested with F. oxysporum but will not attack
healthy onion seedlings (Ofek et al. 2014; Lebiush-Mordechai et al. 2014). For
future research testing, the hypothesis that onions and garlic grown in suppressive
soils, antagonistic to F. oxysporum, created by applying mature compost will harbor
lower levels of R. robini and have reduced plant damage (Yogev et al. 2006).
Another contributing factor to onion and garlic damage associated with R. robini
and plant pathogens is the plant-parasitic nematode Ditylenchus dipsaci.
Interestingly, compost amendments have been shown to reduce plant-parasitic nem-
atodes while enhancing nonpathogenic, free-living nematodes (Thoden et al. 2011;
Korthals et al. 2014). This in turn becomes relevant for the biocontrol of R. robini,
other arthropod pests, and plant-parasitic nematodes because these free-living bac-
teriophagous nematodes are an excellent food source for generalist and specialized
predatory mites (Heidemann et al. 2014; Britto et al. 2012; Read et al. 2006).
16.3 Biofumigation
Different mechanisms have been proposed for GLS mode of action against pests.
These include inactivating the thiol group of essential enzymes of the pest or alkyl-
ating the nucleophilic groups of biopolymers like DNA or as uncouplers (Tsao et al.
2002). Due to uncoupling between the respiratory chain and phosphorylation, respi-
ration is accelerated, requiring more adenosine triphosphate (ATP) as an energy
source, and at the same time, ATP production is blocked. This causes exhaustion of
stored energy sources which finally leads to the death of the pest (Vig et al. 2009).
The effect of GLS on an insect pest may vary depending on the type of ITCs pro-
duced, for example, the contact toxicities of methyl, propyl, allyl, phenyl, benzyl,
and 2-phenylethyl ITCs were tested in the laboratory on eggs of the black vine
weevil Otiorhynchus sulcatus. This weevil is a serious economic pest of high-value
nursery, greenhouse, and field crops, where the larvae feed on the roots. All ITCs
tested were toxic to the weevil eggs; however, ITCs containing an aromatic moiety
were considerably more toxic than aliphatic (methyl, propyl, and allyl) ITCs. These
results suggest that soil amendments of Brassica spp. tissues producing aromatic
ITCs may have a greater insecticidal potential than those producing aliphatic ITCs
(Borek et al. 1995). When Brassica juncea or Sinapis alba seed meals were evalu-
ated in field experiments against O. sulcatus, B. juncea meal treatments resulted in
100 % weevil mortality, while S. alba meal had no effect on weevil mortality (Brown
et al. 2004). Methyl-ITCs were also toxic to the soil-inhabiting white-fringed wee-
vil larvae, Naupactus leucoloma (Matthiessen and Shackleton 2000). Brassica jun-
cea seed meal incorporated into the top potting medium was also found efficient in
278 L. Shaltiel-Harpaz et al.
controlling the fly Bradysia impatiens (fungus gnats) (Diptera: Sciaridae), known as
a serious pest in nurseries and greenhouses, as the females lay eggs in moist organic
matter or potting media and the larvae feed on the plants (Main et al. 2014).
Wireworms, a widespread and important soilborne crop pest, can also be con-
trolled with allyl isothiocyanate (AITC). Soil amended with allyl glucosinolate,
present in relatively high concentrations in Brassica oleracea, B. juncea, B. cari-
nata, and B. nigra, was found to have acute toxicity to the wireworm Limonius cali-
fornicus (Williams et al. 1993). On the other hand, meal of B. napus amended (3 %
on a weight basis) to soil repelled wireworms L. californicus but in uncovered con-
tainers did not kill them (Brown et al. 1991). Other species of wireworms, Agriotes
brevis, A. sordidus, and A. ustulatus, were also affected by chopped fresh plants
from B. juncea sel. ISCI 99 and biofumigant meals derived from defatted seeds of
B. carinata sel. ISCI 7 in both laboratory and field trials. Defatted seed meals,
applied at a rate to release 160 μ moles of glucosinolate L−1 of soil, caused very high
larval mortality and prevented wireworms from damaging seedlings. The insecti-
cidal effect of the chopped whole plants of B. juncea was less consistent (Furlan
et al. 2010). Masked chafer beetle Cyclocephala spp. larvae is a common soil-
inhabiting pest of ornamental plants, turfgrasses, corn, and soybean. Soil amended
with B. juncea tissue was also effective against larvae of this beetle. AITC levels
were positively correlated to larval mortality, with 8 % B. juncea treatment resulting
in 100 % larval mortality with an average AITC concentration of 11.4 mg L−1 of soil
(Noble et al. 2002). Indole-3-acetonitrile found in B. oleracea inhibited the growth
of moths of the Pyralidae family, of the European corn borer Pyrausta nubilalis, and
of the honeycomb moth Galleria mellonella (Smissman et al. 1961).
Endophytic bacteria are known to enhance plant health in crops through various
mechanisms: phosphate solubilization, nitrogen fixation, siderophore production,
phytohormone and enzyme production, induction of host resistance, and biological
control of plant pests and diseases (Ryan et al. 2008; Rosenblueth and Martínez-
Romero 2006). Ho et al. (2015) used Burkholderia cenocepacia 869 T2, and Tan
et al. (2015) used Serratia marcescens ITBB B5-1 for the control of banana
Fusarium wilt. Six endophytic bacteria isolated from cotton, Aureobacterium saper-
dae, Bacillus pumilus, Phyllobacterium rubiacearum, Pseudomonas putida, P.
putida, and Burkholderia solanacearum, reduced disease severity when inoculated
to cotton seedlings (Chen et al. 1995).
16.4.2 PGPRs
PGPRs are beneficial rhizospheric bacteria that directly or indirectly affect plant
growth and fitness (Saraf et al. 2014). The rhizobacteria Pseudomonas fluorescens
was reported to synthesize antifungal antibiotics, such as 2,4-diacetylphloroglucinol,
which inhibits the growth of phytopathogenic fungi (Nowak-Thompson et al. 1994),
while other PGPR strains drastically reduced fusaric acid produced by F. udum, the
causal agent of Fusarium wilt disease in pigeon pea (Dutta et al. 2008). P. fluores-
cens showed direct antagonistic activity against F. oxysporum found in rice and
sugarcane by the production of antifungal metabolites (Kumar et al. 2002).
Pseudomonas stutzeri produced extracellular chitinase and laminarinase which
lysed the mycelia of Fusarium solani (Lim et al. 1991). Soil inoculation with phos-
phate-solubilizing bacteria managed the wilt of tomato caused by F. oxysporum f.
sp. lycopersici (Khan et al. 2007). Bacillus amyloliquefaciens W2, isolated from the
soil of Crocus sativus fields in India, successfully reduced the wilt disease rate of
Crocus sativus in vitro and in a potted plant (Gupta and Vakhlu 2015). Paenibacillus
16 Biorational, Environmentally Safe Methods for the Control of Soil Pathogens… 281
sp. 300 and Streptomyces sp. 385 suppressed Fusarium wilt of cucumber caused by
F. oxysporum f. sp. cucumerinum in a nonsterile, soilless potting medium due to
their chitinolytic activity (Singh et al. 1999).
In a study conducted by Omar et al. (2006), soil inoculation of BBCA combined
with a fungicide showed synergistic activity. Two antagonistic bacteria, Bacillus
megaterium C96 and Burkholderia cepacia C91, were examined for the control of
F. oxysporum f. sp. radices, the causal agent of crown and root rot of tomato alone
or combined with the fungicide carbendazim. The fungicide had almost no effect at
concentrations lower than 50 μg ml−1, and each bacterial isolate showed 20 % reduc-
tion in the disease symptoms. However, combining 1 μg ml−1 of carbendazim with
B. cepacia C91 and 10 μg ml−1 carbendazim with B. megaterium C96 reduced dis-
ease symptoms by 46 % and 84 %, respectively. de Boer et al. (2003) showed syn-
ergistic activity using combinations of P. putida strains. When WCS358 and RE8
strains were mixed into the soil, disease suppression was significantly enhanced to
approximately 50 % as compared to 30 % reduction for the single-strain treatments.
The authors suggested that the synergistic effect was the result of the combination
of different disease-suppressive mechanisms. The combination of the two chitino-
lytic bacteria Paenibacillus sp. 300 and Streptomyces sp. 385 provided better sup-
pression of Fusarium wilt of cucumber than when each was applied separately
(Singh et al. 1999). Similarly Park et al. (1988) reported improved suppression of
Fusarium wilt on cucumber by the combination of P. putida and nonpathogenic
isolates of F. oxysporum.
Formulated BBCAs usually provided better disease suppression than the unfor-
mulated BBCAs. Singh et al. (1999) showed that of several formulations tested for
Paenibacillus sp. 300 and Streptomyces sp. 385, the zeolite-based chitosan-amended
formulation provided the best protection against the Fusarium disease. El‐Hassan
and Gowen (2006) tested different formulations for BBCA B. subtilis used for pro-
tecting lentil against the wilt disease caused by F. oxysporum f. sp. lentis. Seed
treatments with formulations of B. subtilis based on glucose, talc, and peat signifi-
cantly enhanced biocontrol activity against Fusarium compared with a treatment in
which spores were applied directly to seed. Bora et al. (2004) used talc-based seed
treatment formulations of two strains of P. putida (strain 30 and strain 180) to sup-
press the development of Fusarium wilt of muskmelon, caused by F. oxysporum
f. sp. melonis. In field trials, control efficacy of P. putida strains 30 and 180 were
63 % and 50 %, respectively, while the fungicide benomyl used as a commercial
reference was less effective. Zacky and Ting (2015) evaluated sodium alginate,
kaolin clay, and alginate-kaolin formulations for the BBCA Streptomyces griseus.
Results indicated that formulated cells of S. griseus, irrespective of the materials
used, were generally more effective in inhibiting growth of F. oxysporum than non-
formulated cells.
To summarize BBCAs play an important role in preventing and suppressing
plant diseases. BBCAs isolated from ecological niches, suitably formulated, have
successfully reduced Fusarium disease in short-term experiments conducted in
282 L. Shaltiel-Harpaz et al.
potted plants, in greenhouses, and in the field. Further BBCAs research is needed to
attain long-term effects on Fusarium disease reduction.
The effects of growing Brassica as green manure crops preceding tomato produc-
tion were investigated in two field trials (commercial-size greenhouses) in the B’sor
region in the southwest of Israel. Green manure cover crops including two mustard
Brassica juncea cvs. (“red giant” and “99”), arugula Eruca sativa, and broccoli B.
oleracea were compared to fallow treatment. Fusarium wilt incidence in 2007 was
reduced from 20.8 % in fallow treatment to 8.3–17.7 % in the green manure crops
and, in 2008, from 19.4 % in fallow to 7.4–13.9 % in the green manure crops, but
with no significant differences (Table 16.1). Yields were not statistically affected by
the cover crops. These results are in accordance with Hartz et al. (2005), who also
reported that mustard crops were ineffective in suppressing soilborne disease or
improving tomato yield, apparently due to the high level of pathogen density.
Biocontrol agents have the potential to manage various soilborne diseases including
Fusarium wilt, crown rot, and root rot of tomato. Among the antagonists that have
shown satisfactory degrees of control against these diseases are Trichoderma
Table 16.1 Effect of green manure crops preceding tomatoes on Fusarium wilt incidence (90
days after planting) and yield
2007 2008
Yield (kg/
Fusarium wilt (%) Yield (kg/plot)* Fusarium wilt (%) plot)**
Broccoli 11.5 43.1 13.9 1.39
Arugula 8.3 44.8 8.3 1.13
Mustard cv red giant 17.7 44.3 12 1.21
Mustard cv 99 10.4 44.9 7.4 1.1
Fallow 20.8 41.1 19.4 1.27
Trials were conducted in a randomized block design with four replications (24–27 plants/
replicate)
Green manure crop sowed in October 2006, November 2007; incorporation of biomass into the
soil: January 2007, February 2008; cherry tomato (cv. 139) planting dates: February 2007, April
2008
* 9 harvests; ** 2 harvests
Disease symptoms were analyzed by analysis of variance (ANOVA). Means were compared with
student’s multiple range test at a significance level of P < 0.05. Percentages were arcsine trans-
formed before analysis
16 Biorational, Environmentally Safe Methods for the Control of Soil Pathogens… 283
Mealybugs are considered serious pests of various crops feeding on plant sap on
roots and foliage (Kaydan et al. 2015). In an experiment conducted in 2010 in north-
ern Israel, researchers evaluated the effect of biofumigation with three Brassicaceae
species, B. oleracea, B. napus, and Eruca sativa, with fresh and dried plants com-
pared to an untreated control, in two cropping systems: Planococcus citri on Mentha
spicata and Phenacoccus solani on Artemisia dracunculus.
The experiments were conducted in a 400 m2 screenhouse, and the herbs were
grown in 10 L flowerpots in local basalt soil, mixed with the respective amendments
described below. Brassicaceae plants used as dry amendments were harvested and
dried in the shade to 20 % of their fresh weight and applied at 0.2 % of soil weight.
The fresh amendments were harvested just before incorporating into the soil and
applied at 1 % of soil weight. The herbs were planted on two dates, on the day the
amendments were added to the soil and a month later, replicated ten times per treat-
ment in both systems. Each plant was inoculated with ten adult mealybugs.
Mealybug populations (adults and nymphs) were monitored on each plant every
week for 2 months until harvest. At harvest each plant was uprooted and all the
mealybugs from root to shoot were counted and the condition of each plant was
rated from zero (dead plants) to five (foliage in excellent condition).
Biofumigation treatments had a negative effect on the establishment and devel-
opment of the mealybugs in the two systems. On A. dracunculus the effect on
284 L. Shaltiel-Harpaz et al.
Table 16.2 Effect of hypovirulent Fusarium strains application to tomato prior to inoculation
with F. oxysporum f. sp. radicis-lycopersici (FORL) on disease symptoms
Isolate Site/host Symptoms on tomato plants x % disease reduction y
Trial 1
FORL II* Tomato 2.71 a z –
FORL I** Tomato 2.62 a –
AZ Noncultivated field 1.51 b 44
BE-10 Nursery 0.86 c 68
NI-2 Garlic field 0.80 c 71
KI-32 Radish field 0.80 c 71
NZ-3 Netzer 0.74 c 73
TY-5 Celery field 0.70 c 74
NZ-11 Netzer 0.69 c 75
TY-19 Celery field 0.66 c 76
G-11 Hatzeva 0.65 c 76
B-10 Unknown 0.62 c 77
BE-35 Nursery 0.60 c 78
NO-18 Carrot field 0.58 c 79
Check*** 0.45 c 86
Trial 2
FORL II* Tomato 2.50 b –
FORL I** Tomato 2.35 b –
TY-18 Celery field 1.89 c 25
NT-21 Onion field 1.47 cd 41
KI-29 Radish field 1.14 d 55
NT-11 Corn field 0.71 e 72
NZ-8 Netzer 0.69 e 73
SU-4 Ein-Yahav 0.68 e 73
B-4 Unknown 0.59 e 73
JE-6 Tzomet 0.48 ef 81
MO-10 Hatzeva 0f 100
Check*** 0.28 ef 86
Two experiments in a completely randomized design were conducted in climate chambers. In each
treatment 45–60 plants. Conidial suspension of hypovirulent Fusarium isolates was applied to
tomato seedlings (cv. M-82; 14-days old), and inoculation with a virulent F. oxysporum f. sp.
radicis-lycopersici (FORL) was performed a week later
* Inoculated with the virulent FORL 7 days after hypovirulent Fusarium application
** Inoculated with the virulent FORL at time of hypovirulent Fusarium application
*** Non-inoculated control
x
Disease symptoms were determined 10 weeks after inoculation with FORL on a scale of 0–5,
where 0 = no symptoms, 1 = low infection with lesions smaller than 1 mm diameter, 2 = moderate
infection with lesions size above 1 mm, 3 = high infection with large lesions, 4 = full girdling at
stem base, and 5 = death of plant
y
Reduction in plant symptoms as compared with FORL II
z
Disease symptoms were analyzed by analysis of variance (ANOVA). Means were compared with
student’s multiple range test at a significance level of P < 0.05. Different letters within a column
indicate a significant difference
16 Biorational, Environmentally Safe Methods for the Control of Soil Pathogens… 285
2000
a a a
nymphs per plant
ab
1500 b b
b
1000
Phenacoccus solani
500
0
Control B.oleracea B.oleracea B.napus B.napus E.sativa E.sativa
fresh dried fresh dried fresh dried
Fig. 16.1 Total numbers of Phenacoccus solani nymphs (average ± S.E.) on tarragon Artemisia
dracunculus plants at harvest in different biofumigation treatments incorporated a month before
the herbs were planted
Note: Columns marked with different letters differ significantly at p < 0.05 (Tukey-Kramer HSD
means comparison test)
P. solani lasted for 2 months until harvest (Fig. 16.1), while on M. spicata, the effect
on P. citri lasted only 1 month (Fig. 16.2), and at harvest there was no difference in
P. citri adult population size between the treatments (F6.63 = 0.8; p = 0.59).
The effect of biofumigation with canola in the A. dracunculus system on P. citri
was not significantly different from the control (Fig. 16.1), while in the M. spicata
system, it was as good as B. oleracea and E. sativa (Fig. 16.2).
Drying B. oleracea before application to the soil improved its ability to control
P. solani (Fig. 16.1), while in the other Brassicaceae, the condition at application did
not affect the results (Figs. 16.1 and 16.2). In addition, differences were found in
herb plant response: M. spicata plants benefited from the biofumigation at the
beginning compared to control (2 weeks after planting chi-square = 14.92; p =
0.02), and at harvest there was no difference between the plant condition in the dif-
ferent treatments (chi-square = 3.26; p = 0.78). On the other hand, there was 30 %
mortality of the A. dracunculus plants in the fresh cabbage treatment and 20 % in
the dry B. oleracea treatment.
286 L. Shaltiel-Harpaz et al.
Fig. 16.2 Total numbers of Phenacoccus citri nymphs (average ± S.E.) on mint Mentha spicata
plants in different biofumigation treatments incorporated on the same day the herbs were planted
Note: Dates with one, two, or three asterisks indicate a significant difference between treatments
and the non-treated control at p < 0.05, <0.01, <0.001, respectively (one-way ANOVA)
16.5.4 Conclusion
Our results along with the cited literature demonstrate the potential of biofumiga-
tion for the control of soilborne insect pests. It is important to consider that the
efficacy of the same biofumigant plant on similar pests in different planting systems
can vary. Additionally the phytotoxicity of the biofumigant plant on the crop and its
compatibility to natural enemies need to be determined before implementing this
technology in an integrated pest management (IPM) regime.
References
Alfano G, Ivey ML, Cakir C, Bos J, Miller S, Madden L, Kamoun S, Hoitink H (2007) Systemic
modulation of gene expression in tomato by Trichoderma hamatum 382. Phytopathology
97:429–437
Baker KF, Snyder WC (1965) Ecology of soil-borne plant pathogens: prelude to biological control.
University California Press, Berkeley
Bisen K, Keswani C, Mishra S, Saxena A, Rakshit A, Singh HB (2015) Unrealized potential of
seed biopriming for versatile agriculture. In: Rakshit A, Singh HB, Sen A (eds) Nutrient use
efficiency: from basics to advances. Springer, New Delhi, pp 193–206
Bisen K, Keswani C, Patel JS, Sarma BK, Singh HB (2016) Trichoderma spp.: efficient inducers
of systemic resistance in plants. In: Chaudhary DK, Verma A (eds) Microbial-mediated induced
systemic resistance in plants. Springer, Singapore, pp 185–195
16 Biorational, Environmentally Safe Methods for the Control of Soil Pathogens… 287
Block E, Putman D, Zhao SH (1992) Allium chemistry: GC-MS analysis of thiosulfinates and
related compounds from onion, leek, scallion, shallot, chive, and Chinese chive. J Agric Food
Chem 40:2431–2438
Bora T, Özaktan H, Göre E, Aslan E (2004) Biological control of Fusarium oxysporum f. sp. melo-
nis by wettable powder formulations of the two strains of Pseudomonas putida. J Pathol
152:471–475
Borek V, Elberson LR, McCaffrey JP, Morra MJ (1995) Toxicity of aliphatic and aromatic isothio-
cyanates to eggs of the black vine weevil (Coleoptera: Curculionidae). J Econ Entomol
88(5):1192–1196
Borrero C, Trillas MI, Ordovás J, Tello JC, Avilés M (2004) Predictive factors for the suppression
of Fusarium wilt of tomato in plant growth media. Phytopathology 94:1094–1101
Borrero C, Ordovás J, Trillas M, Avilés M (2006) Tomato Fusarium wilt suppressiveness. The
relationship between the organic plant growth media and their microbial communities as char-
acterised by Biolog®. Soil Biol Biochem 38:1631–1637
Boulter J, Boland G, Trevors J (2000) Compost: a study of the development process and end-
product potential for suppression of turfgrass disease. World J Microb Biot 16:115–134
Boulter JI, Boland GJ, Trevors JT (2002) Assessment of compost for suppression of Fusarium
Patch (Microdochium nivale) and Typhula Blight (Typhula ishikariensis) snow molds of turf-
grass. Biol Control 25:162–172
Britto EPJ, Gago E, Moraes GJ (2012) How promising is Lasioseius floridensis as a control agent
of Polyphagotarsonemus latus? Exp Appl Acarol 56:221–231
Brown PD, Morra MJ (1997) Control of soil-borne plant pests using glucosinolate-containing
plants. Adv Agron (USA) 61:167–231
Brown PD, Morra MJ, McCaffrey JP, Auld DL, Williams L III (1991) Allelochemicals produced
during glucosinolate degradation in soil. J Chem Ecol 17:2021–2034
Brown J, Hamilton M, Brown D (2004) Using Brassicaceae seed meal as an alternative to highly
toxic soil fumigants in strawberry production. In: Proceedings of the first international sympo-
sium biofumigation: a possible alternative to Methyl Bromide, 2004, pp 14–15
Chen C, Bauske E, Musson G, Rodriguezkabana R, Kloepper J (1995) Biological control of
Fusarium wilt on cotton by use of endophytic bacteria. Biol Control 5:83–91
Cheuk W, Lo KV, Copeman R, Joliffe P, Fraser BS (2005) Disease suppression on greenhouse
tomatoes using plant waste compost. J Environ Sci Heal B 40:449–461
Conijn CGM, Lesna I, Altena K, Lilien KH, Borochov A, Halevy AH (1997) Biological control of
the bulb mite Rhizoglyphus robini by the predatory mite Hypoaspis aculeifer on lilies: imple-
mentation in practice. Proceedings of the seventh international symposium on flower bulbs,
Herzliya, Israel, 10 16 March 1996 II: 619–624
Davis J, Huisman O, Westermann D, Hafez S, Everson D, Sorensen L, Schneider A (1996) Effects
of green manures on Verticillium wilt of potato. Phytopathology 86:444–453
de Boer M, Bom P, Kindt F, Keurentjes JJ, van der Sluis I, Van Loon L, Bakker PA (2003) Control
of Fusarium wilt of radish by combining Pseudomonas putida strains that have different
disease-suppressive mechanisms. Phytopathology 93:626–632
de Groot GA, Jagers OP, Akkerhuis G, Dimmers W, Charrier X, Faber J (2016) Biomass and diver-
sity of soil mite functional groups respond to extensification of land management, potentially
affecting soil ecosystem services. Frontiers in Environmental Science 4. doi:10.3389/
fenvs.2016.00015
Diaz A, Okabe K, Eckenrode CJ, Villani MG, Oconnor BM (2000) Biology, ecology, and manage-
ment of the bulb mites of the genus Rhizoglyphus (Acari: Acaridae). Exp Appl Acarol
24:85–113
Dold SE (2010) Impact of mustards (Brassicaceae) grown as cover crops on non-target arthropod
communities. MSc dissertation, University of Illinois, Urbana, Illinois
Dugravot S, Sanon A, Thibout E, Huignard J (2002) Susceptibility of Callosobruchus maculatus
(Coleoptera: Bruchidae) and its parasitoid Dinarmus basalis (Hymenoptera: Pteromalidae) to
sulphur-containing compounds: consequences on biological control. Environ Entomol
31:550–557
288 L. Shaltiel-Harpaz et al.
Kabouw P, van der Putten WH, van Dam NM, Biere A (2010) Effects of intraspecific variation in
white cabbage (Brassica oleracea var. capitata) on soil organisms. Plant Soil 336:509–518
Kannangara T, Utkhede R, Bactawar B (2004) Compost effect on greenhouse cucumbers and sup-
pression of plant pathogen Fusarium oxysporum. Compost Sci Util 12:308–313
Kaydan MB, Kozar F, Hodgson C (2015) A review of the phylogeny of Palaearctic mealybugs
(Hemiptera: Coccomorpha: Pseudococcidae). Arthropod Syst Phylogeny 73:175–195
Keswani C, Mishra S, Sarma BK et al (2014) Unraveling the efficient application of secondary
metabolites of various Trichoderma. Appl Microbiol Biotechnol 98:533–544
Khan M, Khan S, Mohiddin F (2007) Effect of certain fungal and bacterial phosphate solubilizing
microorganisms on the fusariam wilt of tomato. In: First international meeting on Microbial
Phosphate Solubilization, Springer, Dordrecht, pp 357–361
Kirkegaard J, Sarwar M (1998) Biofumigation potential of brassicas. Plant Soil 201:71–89
Korthals GW, Thoden TC, van den Berg W, Visser JHM (2014) Long-term effects of eight soil
health treatments to control plant-parasitic nematodes and Verticillium dahliae in agro-
ecosystems. Appl Soil Ecol 76:112–123
Kortnizki-Shapira N (1998) Biocontrol of Fusarium crown and root rot in tomato caused by
Fusarium oxysporum f. sp. radicis-lycopersici with hypovirulent isolates of Fusarium. MSc
dissertation, Tel Aviv University
Kumar NR, Arasu VT, Gunasekaran P (2002) Genotyping of antifungal compounds producing
plant growth-promoting rhizobacteria, Pseudomonas fluorescens. Curr Sci 82:1463–1466
Lebiush-Mordechai S, Erlich O, Maymon M, Freeman S, Ben-David T, Ofek T, Palevsky E, Tsror
(Lahkin) L (2014) Bulb and root rot in lily (Lilium longiflorum) and onion (Allium cepa) in
Israel. J Pathol 162:466–471
Leroy BLMM, Bommele L, Reheul D, Moens M, De Neve S (2007) The application of vegetable,
fruit and garden waste (VFG) compost in addition to cattle slurry in a silage maize monocul-
ture: effects on soil fauna and yield. Eur J Soil Biol 43:91–100
Lesna I, Sabelis MW, Bolland HR, Conijn CGM (1995) Candidate natural enemies for control of
Rhizoglyphus robini Claparede (Acari: Astigmata) in lily bulbs: exploration in the field and
preselection in the laboratory. Exp Appl Acarol 19:655–669
Lesna I, Sabelis MW, Bolland HR, Conijn CGM (1996) Biological control of the bulb mite,
Rhizoglyphus robini, by the predatory mite, Hypoaspis aculeifer, on lilies: predator-prey inter-
actions at various spatial scales under storage conditions. J Appl Ecol 33:369–376
Lesna I, Conijn CGM, Sabelis MW, van-Straalen NM (2000) Biological control of the bulb mite,
Rhizoglyphus robini, by the predatory mite, Hypoaspis aculeifer, on lilies: predator-prey
dynamics in the soil, under greenhouse and field conditions. Biocontrol Sci Technol
10:179–193
Lim H-S, Kim Y-S, Kim S-D (1991) Pseudomonas stutzeri YPL-1 genetic transformation and
antifungal mechanism against Fusarium solani, an agent of plant root rot. Appl Environ
Microbiol 57:510–516
Main M, McCaffrey J, Morra M (2014) Insecticidal activity of Brassica juncea seed meal to the
fungus gnat Bradysia impatiens Johannsen (Diptera: Sciaridae). J Appl Entomol 138:701–707
Malandraki I, Tjamos SE, Pantelides IS, Paplomatas EJ (2008) Thermal inactivation of compost
suppressiveness implicates possible biological factors in disease management. Biol Control
44:180–187
Manu M, Honciuc V (2010) Ecological research on the soil mites populations (Acari: Mesostigmata-
Gamasina, Oribatida) from forest ecosystems near Bucharest city. Rom J Biol Zool 55:19–30
Matthiessen J, Shackleton M (2000) Advantageous attributes of larval whitefringed weevil,
Naupactus leucoloma (Coleoptera: Curculionidae) for bioassays soil fumigants, and responses
to pure and plant-derived isothiocyanates. Bull Entomol Res 90:349–355
Minor MA, Norton RA (2004) Effects of soil amendments on assemblages of soil mites (Acari:
Oribatida, Mesostigmata) in short-rotation willow plantings in central New York. Can J For Res
34:1417–1425
290 L. Shaltiel-Harpaz et al.
Muehlchen A, Rand R, Parke J (1990) Evaluation of crucifer green manures for controlling
Aphanomyces root rot of peas. Plant Dis 74:651–654
Muslim A, Horinouchi H, Hyakumachi M (2003) Control of Fusarium crown and root rot of
tomato with hypovirulent binucleate Rhizoctonia in soil and rock wool systems. Plant Dis
87:739–747
Nammour D, Auger J, Huignard J (1989) Insecticidal effect of sulfur compounds (disulfides and
trisulfides) on Bruchidius atrolineatus (Pic) (Coleoptera, Bruchidae). Insect Sci Appl
10:49–53
Nelson H, Ouchi S, Shiraishi T, Oku H (1992) Induced resistance to Fusarium wilt of tomato and
cucumber: symptoms and pathogen proliferation. Ann Phytopathol Soc Jpn 58:659–663
Noble R, Coventry E (2005) Suppression of soil-borne plant diseases with composts: a review.
Biocontrol Sci Technol 15:3–20
Noble RR, Harvey SG, Sams CE (2002) Toxicity of Indian mustard and allyl isothiocyanate to
masked chafer beetle larvae. University of Tennessee. 10.1094/PHP-2002-0610-01-RS.
Accessed 25 Apr 2016
Nowak-Thompson B, Gould SJ, Kraus J, Loper JE (1994) Production of 2,4-diacetylphloroglucinol
by the biocontrol agent Pseudomonas fluorescens Pf-5. Can J Microbiol 40:1064–1066
Odukkathil G, Vasudevan N (2013) Toxicity and bioremediation of pesticides in agricultural soil.
Rev Environ Sci Biotechnol 12:421–444
Ofek T, Gal S, Inbar M, Lebiush-Mordechai S, Tsror L, Palevsky E (2014) The role of onion asso-
ciated fungi in bulb mite infestation and damage to onion seedlings. Exp Appl Acarol
62:437–448
Okabe K, Amano H (1990) Attractancy of Alcohols isolated from culture filtrates of Fusarium
fungi for the robine bulb mite, Rhizoglyphus robini CLAPAREDE (Acari: Acaridae), in sand.
Appl Entomol Zool 25:397–404
Okabe K, Amano H (1991) Penetration and population growth of the robine bulb mite, Rhizoglyphus
robini Claparede (Acari: Acaridae), on healthy and Fusarium-infected rakkyo bulbs. Appl
Entomol Zool 26:129–136
Omar I, O’neill T, Rossall S (2006) Biological control of Fusarium crown and root rot of tomato
with antagonistic bacteria and integrated control when combined with the fungicide carben-
dazim. Plant Pathol 55:92–99
Park I-K, Shin S-C (2005) Fumigant activity of plant essential oils and components from garlic
(Allium sativum) and clove bud (Eugenia caryophyllata) oils against the Japanese termite
(Reticulitermes speratus Kolbe). J Agric Food Chem 53:4388–4392
Park C-S, Paulitz T, Baker R (1988) Biocontrol of Fusarium wilt of cucumber resulting from inter-
actions between Pseudomonas putida and nonpathogenic isolates of Fusarium oxysporum.
Phytopathology 78:190–194
Ploeg A (2008) Biofumigation to manage plant-parasitic nematodes. In: Integrated management
and biocontrol of vegetable and grain crops nematodes. Springer, pp 239–248
Raviv M (2005) Production of high-quality composts for horticultural purposes: a mini-review.
HortTechnology 15:52–57
Read DS, Sheppard SK, Bruford MW, Glen DM, Symondson WOC (2006) Molecular detection of
predation by soil micro-arthropods on nematodes. Mol Ecol 15:1963–1972
Reddy PP (2013) Biofumigation. In: Recent advances in crop protection. Springer, New Delhi,
pp 37–60
Ros M, Hernandez M, Garcia C, Bernal A, Pascual J (2005) Biopesticide effect of green compost
against Fusarium wilt on melon plants. J Appl Microbiol 98(4):845–854
Rosenblueth M, Martínez-Romero E (2006) Bacterial endophytes and their interactions with hosts.
Mol Plant Microbe Interact 19:827–837
Ryan RP, Germaine K, Franks A, Ryan DJ, Dowling DN (2008) Bacterial endophytes: recent
developments and applications. FEMS Microbiol Lett 278:1–9
Saraf M, Pandya U, Thakkar A (2014) Role of allelochemicals in plant growth promoting rhizo-
bacteria for biocontrol of phytopathogens. Microbiol Res 169:18–29
16 Biorational, Environmentally Safe Methods for the Control of Soil Pathogens… 291
Abstract
A considerable part of agricultural products are destroyed in Iran by various
pests such as insects, weeds, fungi, nematodes, viruses, and bacteria. Application
of chemical pesticides has improved agricultural productivity, but they have also
caused adverse effects on the environment and human health. Microbial pesti-
cides (biopesticides) are often considered as low-risk substitute for conventional
chemical pesticides that can play a key role in resolving the problem in a sustain-
able way. Though reducing agrochemical usage is a public demand in Iran, the
adoption of biopesticides by growers has been much slower than expected.
However, growing social awareness about the advantages of biocontrol promises
bright prospects for the business of microbial pesticides in Iran. In this chapter
we reviewed the history of biocontrol of pest in Iran and its future prospects. We
also discussed the issues that restrain the interest of Iranian companies to fund
research and development of biopesticides and hinder their acceptance by Iranian
growers. Available biopesticides and the list of producing/distributing companies
in Iran are also included.
Keywords
Biopesticide • Agrochemicals • Biocontrol • Pest
17.1 Introduction
Iran is placed in West Asia and is adjacent to the Caspian Sea, Persian Gulf, and
Gulf of Oman. It is the 18th largest country of the world with an area of 1,633,189
square kilometers (Kiani Haftlanf 2003), with only one-third of its total surface area
suitable for farming. However, water deficiency in recent years reduces its cultiva-
ble land area (arable land, orchards, and vineyards) to only 10.4 % (FAO Stat
2015a).
Many insects, weeds, and pathogens (such as fungi, nematodes, viruses, bacteria,
and so on) adversely affect agriculture in Iran, either by yield loss or quality loss.
Application of chemical pesticides has been a routine management method for con-
trolling these pests; however, their hostile impact on the environment and human
life is undeniable (Moosavi and Zare 2012). The use of chemical pesticides has
increased tremendously in the last few decades because of the need for more food
from the diminishing natural resources.
Although the application of pesticides has improved agricultural productivity,
they have also exhibited unfavorable effect on human, soil, and water health as well
as developing crises such as pest resistance, pest resurgence, outbreak of secondary
pests, poisonous residues in food or feed, and genetic variation in plants (Al-Zaidi
et al. 2011). Human chronic diseases (such as cancers; diabetes; neurodegenerative
disorders like Parkinson, Alzheimer, and amyotrophic lateral sclerosis; birth defects;
and reproductive disorders) have increased abnormally in Iran, and there are many
evidences that correlate with the rapid increase in incidence of these diseases with
exposure to pesticides (Mousavi et al. 2009; Mostafalou and Abdollahi 2013). There
is an urgent need to lessen the use of these damaging compounds by finding some
alternative methods like biocontrol of pests (Vincent et al. 2007; Helyer et al. 2014).
Application of biopesticides can play a key role in resolving the problem in a sus-
tainable way. They provide a significant potential for implementation in sustainable
agriculture by simultaneously satisfying the consumers, farmers, and industries.
The main focus of this chapter is to review the current status and prospects of
research, development, and use of microbial biopesticides in Iran and address the
issues that limit the production and implementation of these products in routine
agriculture.
Though yearly precipitation averages 250 mm or less in most parts of the country,
Iran is one of the few countries in the world that have all four distinguished seasons.
About 64.9 % of the total area of Iran cannot be used for agricultural purposes
because of deserts, salt flats and bare-rock mountains, cities, towns, villages, indus-
trial areas, and roads. An additional 6.6 % is covered by forest and 18.1 % of the
total area is exploited for grazing and small fodder production. The remaining cul-
tivable land area is composed of 9.3 % arable land and 1.1 % for permanent crops
(FAO Stat 2015a). In spite of the large number of workforce employed in agricul-
tural activities, the farmlands are hardly economically viable in consequence of
17 Present Status and the Future Prospects of Microbial Biopesticides in Iran 295
Table 17.1 List of the most common products in Iran with their world ranking
World ranking1 Commodity
1 Berries, stone fruit, pistachios
2 Apricots, dates, walnuts, watermelons
3 Cherries, cucumbers and gherkins, eggplants (aubergines), melons (inc.
cantaloupes)
4 Pumpkins, squash, and gourds
5 Almond, anise, badian, fennel, coriander, figs, quinces
6 Sour cherries, hazelnuts, lettuce, chicory, plums and sloes, tomatoes
7 Chick peas, peaches and nectarines, spinach, tangerines, mandarins,
clementines, satsumas, tea
8 Kiwi fruit
9 Apple, lemons, and limes
10 Asparagus, grapes, persimmons
11 Lentils
13 Oranges, safflower seed
14 Carrots and turnips
15 Cabbages and other brassicas, citrus fruit, potatoes, sugar beet
16 Garlic, wheat
17 Beans
19 Grapefruit (inc. pomelos), pears, rapeseed
Ranking is ordered by quantity of production in 2013
1
small size of the farms (less than 10 h), poor soil, water deficiency, low seed quality,
and antiquated farming techniques. According to the latest FAO ranking for the
largest global producer rankings (FAO Stat 2015b), the most important crops in Iran
and their world production ranking are shown in Table 17.1.
The farmers try to increase their yield by any available measures, and one of the
simplest ways is to intensify the agrochemical inputs. Only in the year 2013, more
than 11,000 tons of active ingredients of pesticides (5261.8 tons of active ingredi-
ents of insecticides, 4144.77 tons of active ingredients of herbicides, and 1737.78
tons of active ingredient of fungicides and bactericides) were applied to agricultural
products of Iran (FAO stat 2015a). These chemicals can produce many problems
such as health concerns, unsafe food supplies, and environmental degradation
(Partap 2010). Though reducing agrochemical usage is extensively appreciated by
consumers as well as extension workers and researchers (Malek-Saeidi et al. 2012),
the adoption of organic agriculture by growers has been much slower than expected.
Area under organic agriculture in Iran was 11,601 hectares in 2014 that was equal
to 0.02 % of the total arable land (Willer and Lernoud 2016). About 95 % of organic
produce in Iran was exported in 2011 to European countries due to their high price
for internal market (Kledal et al. 2012).
Biocontrol in Iran can be divided into three eras. It was initiated in 1931 by
importing several vedalia beetles (Rodolia cardinalis) for control of cottony cush-
ion scale (Icerya purchasi) on citrus orchards. Application of mealybug ladybird
296 M.R. Moosavi and R. Zare
The lack of complete profile of biopesticides is a main reason that microbial pesti-
cides have not yet been considered as alternative to agrochemicals. The lack of
17 Present Status and the Future Prospects of Microbial Biopesticides in Iran 297
society awareness, distrust in farmers, and weakness of the supporting policy net-
work are the main causatives for this flaw (Mishra et al. 2015; Kumar and Singh
2015; Keswani et al. 2013). However, it must be known that biological control alone
will seldom provide complete control and it should be used in an integrated system
with other measures. Raising the profile of microbial biopesticides among the com-
munity and policy makers may help in promoting their adoption by the farmers.
Government patronage is also vital for microbial biopesticide usage. The Iranian
policy makers have started to modify the regulation to ease the process and decrease
the registration costs. As well, new rules such as banning the excessive application
of chemical pesticides and preventing trade of the products containing chemical
residues more than specified are needed to encourage the use of biopesticides.
Development of a novel formulation of biopesticide requires a lot of money and
time. It means that a corporation needs to invest millions of dollars which can only
be sold after the product is registered. The market usually accepts the new products
slowly and 5–8 years is required to reach to a break-even point. Lack of quick profit
makes the venture unattractive to many investors. Government support will be of
help to persuade investment in this sector.
The registration directive for biopesticide products is available on the Iranian
Plant Protection Organization’s website. The directive was adapted from FAO and
USEPA guidelines for registration of biocontrol agents and updated according to
quality control and mass production standards of biocontrol agents that were pro-
vided by the Biological Control Department, Iranian Research Institute of Plant
Protection. The result was published on December 29, 2004, as Directive
2339/220/25 and came into effect on April 23, 2007, after final modifications made
by faculty members of Iranian universities and the Iranian Research Institute of
Plant Protection.
17.3.2.2 Market
The market share of biopesticides in the global crop protection market was 5 %
(US$ 3 billion) in 2013. Biopesticide sales have grown with a compound annual
growth rate of 8.64 %, and this is expected to continue increasing at the same rate
until 2023, where the market will reach more than 7 % (US$ 4.5 billion) of the total
global pesticide market (Olson 2015).
The market share of different biocontrol agents is not similar. It was estimated
that about 74 % of the total biopesticide market was occupied by bacteria-based
products and the remaining was occupied by fungal (10 %), predator (8 %), viral (5
%), and other (3 %) biopesticides (Thakore 2006). About 357 microbial biopesti-
cides (105 in Australia, 101 in the USA, 39 in Europe, 33 in India, 30 in Africa,
21 in China, 19 in Iran, and 11 in Japan that include multiple registrations in differ-
ent countries) were registered and sold all around the world (Mishra et al. 2015).
While the USA, Canada, and Mexico used approximately 45 % of the biopesti-
cides, Asia only used 5 % of biopesticides sold throughout the world (Bailey et al.
2010). The biopesticide market’s growth will partly come from gaining the market
share of chemical pesticides and partly from new biopesticides that manage the
pests which are not controlled with chemicals.
No official data on Iran’s biopesticide market was available to be analyzed; how-
ever, its status is deducible from other information. Area under organic agriculture
was exponentially decreased from 2011 (43,332 ha) to 2014 (11,061 ha) (Willer and
Lernoud 2016). It was clear that higher price of organic products was deterrent to
consumers. Limited domestic market and difficulty in exporting agricultural prod-
ucts may be other reasons.
300 M.R. Moosavi and R. Zare
However, like other countries the future of the business is very bright. The grow-
ing belief that correlates the chronic human diseases with chemical pesticides makes
the consumers to seek for safer products with lower chemical residues. Though
many people are now willingly ready to pay more money for safer food, the domes-
tic market is still small. Government and media patronage is essential to orient the
food demands to safer products. However, government subsidy can be of help in
promoting the investment of companies on development of biopesticides and in
decreasing the final price of organic products. Government support will assist the
small businesses to survive in the market and supply biopesticides to niche
markets.
Table 17.2 List of microbial biopesticides and their producer/distributor companies in Iran
Company name Product name Active ingredient Use against
Gyah Corporation Mycotal Lecanicillium lecanii White fly and thrips
larvae
Trianum-P Trichoderma Soilborne fungi
harzianum strain T 22
Kara Industrial BasioKara Beauveria bassiana Heteroptera and
Biotechnology Co. Coleoptera
TrichoKara Trichoderma Plant pathogenic fungi
harzianum
MetaKara Metarhizium anisopliae Wide range of insect
pests
Afrasam Co. Naturalis Beauveria bassiana Sucking pests
XenTari and B. thuringiensis subsp. Armyworms and
FlorBac aizawai diamondback moth
larvae
BioBit and B. thuringiensis subsp. Lepidoptera larvae
DiPel kurstaki
Biorun BioBeet Bacillus thuringiensis Coleoptera larvae
subsp. san Diego
BioLep B. thuringiensis subsp. Lepidoptera larvae
kurstaki
MehrAsia Bithurin B. thuringiensis subsp. Lepidoptera larvae
Biotechnology kurstaki
Company Bithiran B. thuringiensis subsp. Coleoptera larvae
morrison
Biosubtil Bacillus subtilis Plant pathogenic fungi
Greenlife Biotech Co. Tricomix HV Trichoderma Plant pathogenic fungi
harzianum
Orocide Fusarium oxysporum Broomrape
Foroughe Dasht Co. RootGard Paecilomyces lilacinus Plant pathogenic
nematodes
BotaniGard Beauveria bassiana Wide range of insect
pest
17.5 Conclusion
get more farmers acquainted, especially those who work with traditional farming
systems.
The active chemical industries are well equipped to launch novel products in the
market, but the small- and medium-scale companies must be trained in this venture.
They must learn how to invest on which kind of biocontrol agent, how to estimate
the market size, and the hurdles in commercialization processes. A biocontrol agent
will be successful if it has an extensive host range and high efficiency, easily inhab-
its the soil and compote with soil microbiota, and manages important pests in prin-
ciple crops. However, complete success will be achieved if a potent biocontrol agent
can emulate the traits of conventional chemicals.
Establishing powerful international linkage to receive academic, promotional,
technical, and developmental support from international agricultural research and
related institutes can help to increase the knowledge of biocontrol of pests. Several
potent and indigenous biocontrol agents have been isolated from Iran that can
potentially act as biopesticide. However, insufficient knowledge on formulation and
commercial process hinder their introduction to market. There is an urgent need to
bridge the gaps between research studies, commercialization, and application of
microbial biopesticides.
Finally, in spite of bright prospects of biopesticide business in Iran, it should be
considered that successful adoption relies on the complete cooperation of all partici-
pants (society, farmers, NGOs, government authorities, pesticide companies, stake-
holders, retailers, and distributors).
Acknowledgment The authors thank Dr. R. Marzban (Iranian Research Institute of Plant
Protection, Tehran) for his constructive comments on the use of microbial biopesticides in Iran.
SARV Agricultural Complex (Shiraz-Iran) is gratefully acknowledged for gathering information
on microbial biopesticide market of Iran.
References
Al-Zaidi AA, Elhag EA, Al-Otaibi SH, Baig MB (2011) Negative effects of pesticides on the envi-
ronment and the farmers awareness in Saudi Arabia: a case study. J Anim Plant Sci
21:605–611
Bailey KL, Boyetchko SM, Längle T (2010) Social and economic drivers shaping the future of
biological control: a Canadian perspective on the factors affecting the development and use of
microbial biopesticides. Biol Control 52:221–229
Bailey A, Chandler D, Grant WP, Greaves J, Prince G, Tatchell M (eds) (2011) Biopesticides, pest
management and regulation. CABI, Wallingford
Cumagun CJR, Moosavi MR (2015) Significance of biocontrol agents of phytonematodes. In:
Askary TH, Martinelli PRP (eds) Biocontrol agents of phytonematodes. CABI, Wallingford,
pp 50–78
Damani Zamani F, Moosavi MR, Asadi R (2015) Efficacy of entomopathogenic nematode in bio-
logical control of tomato leafminer, Tuta absoluta in laboratory and greenhouse conditions.
Biocontrol Plant Prot 3:19–32
Ehlers R-U (2010) REBECA – EU-Policy support action to review regulation of biological control
agents. In: Gisi U, Chet I, Gullino ML (eds) Recent developments in management of plant
17 Present Status and the Future Prospects of Microbial Biopesticides in Iran 303
diseases, Plant pathology in the 21st century. Springer Science + Business Media, Dordrecht,
pp 147–161
Etehadi M, Rusta K, Gholi-Nia MJ (2011) Investigating the effectiveness of FFS approach in dis-
seminating IPM practices from farmers’ overview, case study Sistan and Baluchestan Province.
Iran Agr Exten Edu J 7:41–52
FAO stat (2015a) Food and agriculture organization of the United Nations, statistics division.
http://faostat3.fao.org/browse/area/102/E. Accessed 25 Mar 2016
FAO stat (2015b) Food and agriculture organization of the United Nations, statistics division, food
and agricultural commodities production/Countries by commodity http://faostat3.fao.org/
browse/rankings/countries_by_commodity/E Accessed 25 Mar 2016
Gifani A, Marzban R, Safekordi A, Ardjmand M, Dezianian A (2015) Ultraviolet protection of
nucleopolyhedrovirus through micro encapsulation with different polymers. Biocontrol Sci
Technol 25:814–827
Goudarzi M, Moosavi MR, Asadi R (2015) Effects of entomopathogenic nematodes,
Heterorhabditis bacteriophora and Steinernema carpocapsae, in biological control of Agrotis
segetum. Turk Entomol Derg-TU 39:239–250
Helyer N, Cattlin ND, Brown KC (2014) Biological control in plant protection: a color handbook,
2nd edn. CRC Press, Boca Raton
Kalantari M, Marzban R, Imani S, Askari H (2013) Effects of Bacillus thuringiensis isolates and
single nuclear polyhedrosis virus in combination and alone on Helicoverpa armigera. Arch
Phytopathol Plant Prot 47:1–9
Karimi E (2011) Investigating the barriers of organic agriculture development. J Iran Agr Econ
Dev 42:231–242
Keswani C, Singh SP, Singh HB (2013) Beauveria bassiana: status, mode of action, applications
and safety issues. Biotech Today 3:16–20
Khorramvatan S, Marzban R, Ardjmand M, Seifkordi A, Askary H (2014) The effect of polymers
on the stability of microencapsulated formulations of Bacillus thuringiensis subsp. kurstaki
(Bt-KD2) after exposure to ultra violet radiation. Biocontrol Sci Technol 24(4):462–472
Kiani Haftlanf K (2003) The book of Iran, a survey on the geography of Iran. The Ministry of
Culture and Islamic Guidance, Tehran
Kledal PR, Mahmoudi H, Mahdavi Damghani AM (2012) Organic food and farming in Iran. In:
Willer H, Kilcher L (eds) The world of organic agriculture – statistics and emerging trends
2012. Research Institute of Organic Agriculture (FiBL), Frick, and International Federation of
Organic Agriculture Movements (IFOAM), Bonn, pp 184–189
Kumar S, Singh A (2015) Biopesticides: present status and the future prospects. J Fertil Pestic 6:2.
doi:10.4172/jbfbp.1000e129
Malek-Saeidi H, Rezaei-Moghaddam K, Ajili A (2012) Professionals’ attitudes towards organic
farming: the case of Iran. J Agr Sci Tech 14:37–50
Marrone PG (2009) Barriers to adoption of biological control agents and biological pesticides. In:
Radeliffe EB, Hutchison WD, Cancelado RE (eds) Integrated pest management. Cambridge
University Press, Cambridge, pp 163–178
Mishra J, Tewari S, Singh S, Arora NK (2015) Biopesticides: where we stand? In: Arora NK (ed)
Plant microbes symbiosis: applied facets. Springer India, New Delhi, pp 37–75
Moosavi MR, Askary TH (2015) Nematophagous fungi- commercialization. In: Askary TH,
Martinelli PRP (eds) Biocontrol agents of phytonematodes. CABI, Wallingford, pp 187–202
Moosavi MR, Zare R (2012) Fungi as biological control agents of plant-parasitic nematodes. In:
Merillon JM, Ramawat KG (eds) Plant defence: biological control, vol 12, Progress in biologi-
cal control. Springer Science + Business Media, Dordrecht, pp 67–107
Moosavi MR, Zare R (2015) Factors affecting commercial success of biocontrol agents of phyto-
nematodes. In: Askary TH, Martinelli PRP (eds) Biocontrol agents of phytonematodes. CABI,
Wallingford, pp 423–445
Moosavi MR, Zare R, Zamanizadeh HR, Fatemy S (2010) Pathogenicity of Pochonia species on
eggs of Meloidogyne javanica. J Invertebr Pathol 104:125–133
304 M.R. Moosavi and R. Zare
Moosavi MR, Zare R, Zamanizadeh HR, Fatemy S (2011) Pathogenicity of Verticillium epiphytum
isolates against Meloidogyne javanica. Int J Pest Manage 57:291–297
Moosavi MR, Shakeri S, Mohammadi S (2015) The ability of separate and combined application
of five nematopathogenic fungi against Meloidogyne javanica. Iran J Plant Prot Sci
46:179–190
Mostafalou S, Abdollahi M (2013) Pesticides and human chronic diseases: evidences, mecha-
nisms, and perspectives. Toxicol Appl Pharmacol 268:157–177
Moumeni-Helali H, Ahmadpour A (2013) Impact of farmers’ field school approach on knowledge,
attitude and adoption of rice producers toward biological control: the case of Babol Township,
Iran. World Appl Sci J 21(6):862–868
Mousavi SM, Gouya MM, Ramazani R, Davanlou M, Hajsadeghi N, Seddighi Z (2009) Cancer
incidence and mortality in Iran. Ann Oncol 20:556–563
Munjanja B, Chaparadza A, Majoni S (2015) Biopesticide residue in foodstuffs. In: Nollet LML,
Rathore HS (eds) Biopesticides handbook. CRC Press, Boca Raton, pp 71–92
Naraghi L, Heydari A, Askari H, Pourrahim R, Marzban R (2014) Biological control of Polymyxa
betae, fungal vector of rhizomania disease of sugar beets in greenhouse conditions. J Plant Prot
Res 54:109–114
Nollet LML (2016) Biopesticides: regulatory schemes. In: Nollet LML, Rathore HS (eds)
Biopesticides handbook. CRC Press, Boca Raton, pp 141–152
Olson S (2015) An analysis of the biopesticide market now and where it is going. Outlooks Pest
Manag 26:203–206. doi:10.1564/v26_oct_04
Osku T, Chizari M, Rasuli F (2007) The impact of FFS approach on the knowledge and attitude of
rice farmers about biologic management of pests, case study: Mazandaran Province. J Iran Agr
Sci 38:109–119
Partap T (2010) Emerging organic farming sector in Asia: a synthesis of challenges and opportuni-
ties. In: Partap T, Saeed M (eds) Organic agriculture and agribusiness: innovation and funda-
mentals. Asian Productivity Organization Publishing, Tokyo, pp 8–20
Ravensberg WJ (2011) Registration of microbial pest control agents and products and other related
regulations. In: Ravensberg WJ (ed) A roadmap to the successful development and commer-
cialization of microbial pest control products for control of arthropods, vol 10, Progress in
biological control. Springer Science + Business Media, Dordrecht, pp 171–233
Razzaghi-borkhani F, Rezvanfar A, Shabanali Fami H (2010) The role of educational and com-
municational factors on the knowledge of Integrated Pest Management (IPM) among Paddy
farmers in Sari County. J Agr Edu Manag Res 13:2–17
Regnault-Roger C (2012) Trends for commercialization of biocontrol agent (biopesticide) prod-
ucts. In: Merillon JM, Ramawat KG (eds) Plant defence: biological control, vol 12, Progress in
biological control. Springer Science + Business Media, Dordrecht, pp 139–160
Rezvanfar A, Eraktan G, Olhan E (2011) Determine of factors associated with the adoption of
organic agriculture among small farmers in Iran. Afr J Agric Res 6:2950–2956
Romero-González R, Frenich AG, Martínez-Vidal JL (2015) Biopesticide residue in soil. In:
Nollet LML, Rathore HS (eds) Biopesticides handbook. CRC Press, Boca Raton, pp 119–138
Salehi Jouzani G, Abbasalizadeh S, Moradali MF, Morsali H (2015) Development of a cost effec-
tive bioprocess for production of an Iranian anti-coleoptera Bacillus thuringiensis strain. J Agr
Sci Tech 17:1183–1196
Sanganyado E, Munjanja B, Nyakubaya VT (2015) Biopesticide residue in water. In: Nollet LML,
Rathore HS (eds) Biopesticides handbook. CRC Press, Boca Raton, pp 93–117
Sharifi-Moghaddam M (2006) Report of the activities in IPM/FFS sites in Iran. Ministry of
Agriculture, Agricultural Extension and Farming System Department, Tehran
Soltani S, Azadi H, Mahmoudi H, Witlox F (2014) Organic agriculture in Iran: farmers’ barriers to
and factors influencing adoption. Renew Agr Food Syst 29:126–134
Thakore Y (2006) The biopesticide market for global agricultural use. Ind Biotechnol 2:192–208
17 Present Status and the Future Prospects of Microbial Biopesticides in Iran 305
Tormala T (1995) Economics of biocontrol agents: an industrial view. In: Hokkanen HMT, Lynch
JM (eds) Biological control: benefits and risks, vol 4, Plant and microbial biotechnology
research series. Cambridge University Press, Cambridge, pp 277–282
Veisi H, Mahmoudi H, Sharifi-Moghaddam M (2010) Identifying farmers’ adoption of integrated
pest management technologies. J Iran Agr Econ Dev 41:481–490
Vincent C, Goettel MS, Lazarovlts G (eds) (2007) Biological control: a global perspective. CABI,
Wallingford
Whipps JM, Davies KG (2000) Success in biological control of plant pathogens and nematodes by
microorganisms. In: Gurr G, Wratten S (eds) Biological control: measures of success. Kluwer
Academic Publishers, Dordrecht, pp 231–269
Willer H, Lernoud J (eds) (2016) The world of organic agriculture – statistics and emerging trends
2016. Research Institute of Organic Agriculture (FiBL), Frick, and IFOAM – Organics
International, Bonn