Mini-review

Received: 30 January 2018 Revised: 14 February 2018 Accepted article published: 20 February 2018 Published online in Wiley Online Library: 3 April 2018

(wileyonlinelibrary.com) DOI 10.1002/jctb.5613

Trichoderma harzianum-based novel

formulations: potential applications for
management of Next-Gen agricultural
challenges
Leonardo F Fraceto,a* Cintia R Maruyama,a,b Mariana Guilger,b Sandhya
Mishra,c Chetan Keswani,d Harikesh Bahadur Singhd and Renata de Limab

Abstract
Fungi of the Trichoderma spp. genus, notably Trichoderma harzianum, are commonly used for biological management of
deleterious seed- and soil-borne pathogens. The global biopesticides market is booming with a major share of various
commercial formulations of T. harzianum. However, there are some major drawbacks associated with these commercial
formulations including short shelf life, low on-field stability and irregular performance in different agro-climatic regions. For
effectively resolving these issues, new strategies are urgently required for efficient management of pathogens. The present
review provides an overview of the use of Trichoderma spp., with special emphasis on T. harzianum, and discusses future
trends for biological control. Technologies are described for the microencapsulation of fungi and for the biogenic synthesis
of nanoparticles, with the aim of improving the biological control of pathogens and contributing to sustainable agricultural
practices.
© 2018 Society of Chemical Industry

Keywords: biological control; eco-friendly; sustainable agriculture; nanotechnology; microencapsulation; phytopathogens; Trichoderma
harzianum

INTRODUCTION due to their effectiveness against a wide range of seed- and

Biological control is considered as sustainable practice for the soil-borne phytopathogens.19–23 Their mechanism of action pri-
efficient management of notorious pathogens. Augmentation of marily includes mycoparasitism, antibiosis, competition and the
biopesticides involves control of pathogen populations using nat- induction of resistance in host plants.15,24–26
ural enemies and act as an promising alternative to chemical pest Trichoderma harzianum, like other species of the genus, hosts a
control.1–7 The use of organisms in biological control has increased hydrolytic enzyme complex composed of chitinases, 𝛽-glucanases
in popularity due to growing environmental concerns and the cellulases, proteases, etc. which decompose the cell wall of phy-
demand for organic produce.1,8–12 If the real cost of chemical topathogens, allowing the penetration of hyphae, colonization
pesticides were revealed, the market for biological control would and the initiation of mycoparasitic activity.22,27 In addition, T.
increase. This is due to the fact that chemical pesticides are subsi- harzianum is nonpathogenic towards humans, and can be easily
isolated, cultured and mass-produced at industrial scale.28
dized at the Government level and the damages (to human health
and environment) caused by them are not the responsibility of
the industry that produces them.13 According to Bourguet and
Guillemaud,14 if the values of the chemical pesticides involved the ∗ Correspondence to: LF Fraceto, São Paulo State University (UNESP), Institute
real costs of production, biological control agents would have a fair of Science and Technology, Avenida Três de Março, 511, Alto da Boa Vista,
18087-180, Sorocaba, São Paulo, Brazil. E-mail: [email protected]
competition in the market.
The Trichoderma genus of fungi belongs to the Hypocreaceae a São Paulo State University (UNESP), Institute of Science and Technology,
family (order: Hypocleales; class: Sordariomycetes; phylum: Avenida Três de Março, 511, Alto da Boa Vista, Sorocaba, São Paulo, Brazil
Ascomycota). The species of this genus are free-living soil microor-
b LABiToN – Laboratório de Avaliação de Bioatividade e Toxicologia de Nanoma-
ganisms colonizing and decomposing litter, and also have the teriais, University of Sorocaba, Rodovia Raposo Tavares, km 92.5, Sorocaba, São
ability to establish symbiotic relationships with plants.15,16 These Paulo, Brazil
filamentous fungi produce a rapidly growing white mycelium
that acquires a green coloration as it develops.17 Weindling 18 c Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical
Garden, Chinese Academy of Sciences, Mengla, Yunnan, China
reported for the first time that T. lignorum showed in vitro antag-
onistic potential against various soil-borne phytopathogens. d Department of Mycology and Plant Pathology, Institute of Agricultural Sci-
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Trichoderma spp. are now widely used as biological control agents ences, Banaras Hindu University, Varanasi, India

J Chem Technol Biotechnol 2018; 93: 2056–2063 www.soci.org © 2018 Society of Chemical Industry
Applications of Trichoderma harzianum-based novel formulations www.soci.org

the soil and irrigation, among others. Products based on Tricho-

Table 1. A representative list of target organisms and crops derma spp. are mainly aimed at controlling seed- and soil-borne
for which Trichoderma harzianum and other Trichoderma spp. phytopathogens.12 Examples of commercial products based on
have been used as biological control agents T. harzianum and the target pathogens are shown in Table 2.
Biological Considering the robust applications of Trichoderma spp. in agri-
control agents Target organisms Crops References culture, certain neglected aspects need to be urgently addressed,
namely the maintenance of satisfactory stability during transport
Trichoderma Sclerotinia Soybeans, potato, 27,107–116 and storage, high viability and development of formulations with
harzianum sclerotiorum, tomato, zucchini,
eggplant,
prolonged shelf life. In addition, for mass acceptance of these bio-
Phytophthora spp.,
tobacco, peanut formulations, they must be cost-effective and easy to apply.42 In
Meloidogine javanica,
this regard, further studies are required to minimize the shortcom-
Alternaria alternata,
Fusarium solani, ings of these products and to ensure their effectiveness in biolog-
Macrophomina ical control practices.
phaseolina In order to overcome the limitations of biological control tech-
Trichoderma Fusarium oxysporum, Olive 47,18,48 niques due to environmental factors, Singh et al. 43 suggested the
harzianum T16 Fusarium solani, Tomato
and T23
development of formulations based on consortia of synergistic
Fusarium oxysporum, biological control agents that act by means of different mecha-
Trichoderma nisms under both biotic and abiotic stresses. It was reported that
harzianum Ths97
a combination of three strains of T. harzianum provided successful
Trichoderma
harzianum Th908 control of an important plant pathogen affecting several crops in
Trichoderma spp. Phytium spp., Pea, peat moss 49,50,30,51–53 different climate zones.
Rhizoctonia spp., Melon, pepper, Another promising way to enhance the activity of biological con-
Bipolaris oryzae, tomato trol agents is to employ novel technologies involving the use of
Rice
Crinipallis perniciosa, Cocoa
active biomolecules such as antimicrobial proteins, lytic enzymes
Fusarium fujikuroi, Rice and bioactive secondary metabolites – that are primarily responsi-
Phytophthora ble for the management of phytopathogens. The development of
ramorum
such novel techniques to improve the results achieved using Tri-
choderma-based formulations, which have already demonstrated
strong activity against important phytopathogens, is a highly
During interaction with phytopathogens, T. harzianum produces promising area of research.
and secretes both volatile and nonvolatile secondary metabo- Micro- and nanotechnological interventions offer effective
lites with antibiotic properties.29 These substances act to inhibit methods which reduce environmental impacts and increase
mycelial development,30 prevent the release of spores, and alter agricultural productivity.44,45 These include techniques for the
the composition of the rhizosphere to make it unsuitable for the encapsulation of active ingredients of Trichoderma spp. in poly-
growth and development of the phytopathogens.31 Furthermore, meric nanoparticles, for targeted and subsequent controlled
T. harzianum releases peptides, proteins and low molecular weight release of the active biomolecules,46–48 nanoscale products for
compounds 16 that contribute to activation of the induced defense stimulation of the growth and development of plants 34,49 and
mechanisms of the host,32,33 thereby promoting its health, and the synthesis of metal nanoparticles for effective management of
enhancing root growth.34 phytopathogens.50–52
Different species and strains of Trichoderma spp. are used in agri- Two strategies involving the use of micro- and nanotechnology
culture, especially T. harzianum,35,36 and there are constant efforts to improve the performance of active ingredients are described
to identify superior strains that possess higher antagonistic activity below, together with discussion of the remaining challenges.
against phytopathogens. A representative list of the most popular
strains of Trichoderma spp. is presented in Table 1. Several species
of Trichoderma spp. have acquired industrial importance due to
USE OF MICROENCAPSULATION
their capacity to produce large quantities of lytic enzymes and TECHNOLOGIES TO IMPROVE THE ACTIVITY
antimicrobial secondary metabolites, especially antibiotics, and AND USE OF TRICHODERMA spp.
various commercial products based on Trichoderma spp. are now Micro- and nanotechnological interventions can be employed to
marketed worldwide.12,37 Over 50 Trichoderma-based biofungicide improve biological control techniques by enhancing the viability
formulations have been developed, and the genus now accounts of biological control agents.68 An increasing number of recent
for 60% of the fungi used as biological control agents globally, with methodologies have been studied for this purpose, including
new products constantly being registered.38 microencapsulation 40,53,54 and nanoencapsulation.50,55
Although, the biological control of phytopathogens using T. Microencapsulation consists of creating a physical barrier that
harzianum is a popular practice, there are certain limitations that provides protection of the active ingredient against external fac-
can reduce its effectiveness, including sensitivity to ultraviolet tors such as mechanical stress, ultraviolet radiation, oxidation and
light, and unsuitable humidity and/or temperature, which can lead high temperatures.39,56,57 As a result, the microorganism is able to
to low persistence in adverse environmental conditions.39,40 survive for a longer duration and its metabolic activity is main-
Following the promising results obtained on different crops, the tained, too.58 The microencapsulation methods used may be phys-
industrial scale production of T. harzianum using solid and liq- ical (spray drying), chemical (polymerization) or physico-chemical
uid substrates has increased substantially.38,41 Techniques used (coacervation and ionic gelation).
for application of live spores include foliar spraying, seed and Microencapsulation by spray drying is a simple, low cost and
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seedling treatment, application after pruning, incorporation in rapid technique, and the final product does not require multiple

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 www.soci.org LF Fraceto et al.

Table 2. Representative list of commercially available products based on Trichoderma harzianum

Product (Brand name) Target pathogens Manufacturer

Tricho™ Rhizoctonia, Sclerotinia, Fusarium, Botrytis, Sclerotium, Orius Biotecnologia (Colombia)

Rosellinia, Pythium, Arrmilaria, Alternaria
Natibiol™ Rhizoctonia Probiagro S.A. (Venezuela)
BioFungo™ Botrytis cinerea, Sphaerotheca pannosa Orius Biotecnología (Colombia)
ECO-77™ Botrytis, Eutypa Plant Health Products (South Africa)
Lycomax Soil pathogens Russell IPM (United Kingdom)
Trichodermil™ Fusarium, Rhizoctonia, Sclerotinia sclerotiorum, Itaforte BioProdutos (Brazil)
Phytophthora capsici, Phytophthora palmivora,
Botrytis ricini
Trianum™ Soil-borne pathogens Koppert BV (The Netherlands)
ECO-T™ Rhizoctonia, Pythium, Fusarium, Phytophthora Plant Health Products (South Africa)
Trichosav Soil-borne pathogens Centros de Reproducción de Medios
Biológicos (Cuba)
Agroguard WG™ Pythium, Rhizoctonia, Sclerotinia, Sclerotium, Phoma Life Systems Technology S.A. (Colombia)
Bioderma H Phytophthora, Fusarium, Pythium, Cercospora, Biotech International Ltd. (India)
Colletotrichum, Alternaria, Ascochyta,
Macrophomina, Myrothecium, Ralstonia
FoliGuard™ Botrytis cinerea, Sphaeroteca pannosa, Oidium, Live Systems Technology S.A. (Colombia)
Alternaria, Cladosporium
RootShield™/PlantShield™ Pythium, Rhizoctonia solani, Fusarium, Sclerotinia Bioworks (United States)
homeocarpa
Ecotrich ES™ Rhizoctonia solani, Pythium spp., Sclerotinia spp. Ballagro Agro Tecnologia Ltd. (Brazil)
VinevaxTM – Trichoprotection™ Armillaria, Eutypa lata, Chondrostereum purpureum, Agrimm Technologies Ltd. (New Zealand)
Phaeomoniella chlamydospora, Botryosphaeria
stevensii
Antagon WP™ Fusarium, Rhizoctonia, Pythium, Sclerotinia, Sclerotium, Bio Ecológico Ltda (Colombia)
Botrytis, Ceratocystis, Rosellinia
Trichoderma harzianum Fusarium, Rhizoctonia, Pythium IAB S.L. (Spain)
Trichosoil Fusarium spp. Lage S.A. (Uruguay)

washing steps to eliminate residues or solvents.59 Microparti- be encapsulated.67,68 The coacervation method provides effective
cle formation can be modified by adjusting the spray drying encapsulation and subsequent controlled release of the active
conditions such as the feed flow, air flow and inlet/ outlet agents by mechanical means or by changes in temperature or
temperatures.60,61 In the case of biological control agents, the pH.68 However, the use of this technique is limited by its high cost
spray drying method has several disadvantages. It produces and the need to control the kinetic conditions.69
microparticles of varying sizes and morphology, whereas high Ionic gelation can be performed using extrusion, electrostatic
temperatures and rapid rates of drying can prejudice the encap- deposition and atomization processes.70 Normally, a polymer solu-
sulation of conidia or viable spores.40 tion containing the active ingredient to be encapsulated is dripped
The polymerization method is based on the formation of into an ionic solution, under constant stirring. When the droplets
microparticles by polymerization of monomers and can be enter the ionic solution, there is an instantaneous formation of
achieved by several techniques.62 In situ emulsion polymerization spherical microparticles.71–73 This is a simple procedure that does
generally employs a medium, composed of water, monomer, not require the use of specialized equipment, organic solvents or
surfactant and an initiation agent.63 In situ polymerization in
high temperatures.72,74 However, a drawback of this method is the
suspension uses a system containing one or more water-insoluble
occurrence of heterogeneous microparticle gelation, due primarily
monomers and an initiator, soluble in the organic phase.64 In situ
to the diffusion mechanism.75
interfacial polymerization involves complementary monomers
Microencapsulation techniques can be used in various sec-
and immiscible phases.65 However, the limitations of this type
tors, including the pharmaceutical,76 food,77 and cosmetics
of polymerization are the toxicity of unreacted monomers, the
high permeability of the coating and the high fragility of the industries,78 and in agriculture.79
membranes.66 Muñoz-Celaya et al. 80 encapsulated the conidia of T. harzianum
The coacervation microencapsulation technique has been in polymeric carbohydrate matrices, as a way to increase the
used to encapsulate preservatives, oils, microbial cells and shelf life of formulations. Maltodextrin and gum arabic (in a 1:1
enzymes.67–69 This method involves the phase separation of ratio) were used for synthesis of microparticles by the spray dry-
one or more polymers immiscible with the polymer coating ing method, giving a high percentage of viable conidia (86%).
solution, under specific pH and temperature conditions. The The combination of these polymers resulted in a high activation
immiscible polymer is added to the coating polymer solution energy, which together with storage at low temperatures provided
under constant stirring, leading to the formation of microparticles stability for at least 8 weeks. Loss of viability of the conidia after this
due to separation of the incompatible polymer followed by depo- period could have been due to the oxidative stress and continued
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sition of the coacervation phase, which surrounds the material to metabolic activity at 4 ∘ C.

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In other work, Jin and Custis 79 used the spray drying method Devi et al. 92 evaluated the ability of 75 isolates of Trichoderma
for encapsulation, which prevented microbial contamination, spp., including T. harzianum, to synthesize silver nanoparticles
induced dormancy and increased the shelf life of the product; and obtained particles with sizes ranging from 8 to 60 nm. Singh
however, a major disadvantage was the small number of viable and Raja 93 synthesized silver nanoparticles using filtrates of the
encapsulated conidia. Therefore, a specific methodology was fungi F. oxysporum, Alternaria alternata, Phoma glomerata and T.
developed for the microencapsulation of T. harzianum, using harzianum, with use of the last microorganism resulting in the
sugars to increase the percentage of viable conidia. The inlet most stable nanoparticles.
and outlet temperatures used in the spray drying procedure Ahluwalia et al. 55 synthesized silver nanoparticles by the reduc-
also influenced the viability of the conidia. The average sizes of tion of silver nitrate using the filtrate of T. harzianum and observed
the microparticles obtained were in the range of 10–25 𝜇m and the adsorption of a layer of organic compounds derived from
could be adjusted by varying the atomization rate. The use of 2% the culture filtrate. The nanoparticles showed in vitro potential for
sucrose concentration with inlet and outlet temperatures of 60 ∘ C the control of both Gram-positive and Gram-negative bacteria.
and 31 ∘ C, respectively, resulted in an improved survival rate of Gherbawy et al. 94 also synthesized these nanoparticles and used
the conidia as compared to the free conidia. them together with triclabendazole to control the hatching of Fas-
Thus, the encapsulation of bioactive compounds through the ciola eggs in infestations of sheep and camels. It was found that
various encapsulation methods ends up being a cost-effective the nanoparticles significantly increased the effectiveness of the
method.81 The spray-drying method has ended up being the most pharmaceutical products.
used in the food industries 82,83 because it is a more economical
Singh et al. 95 synthesized bimetallic nanoparticles composed
and efficient technique, has high equipment availability and low
of gold and silver using T. harzianum and demonstrated their
process costs. In addition, encapsulated products require fewer
catalytic properties in the reduction of methylene blue dye in
applications 48 when compared to the common products on the
the presence of NaBH4 . Bhadwal et al. 96 synthesized biogenic
market, leading to lower costs.
nanoparticles of cadmium sulfide using T. harzianum and reported
their photocatalytic potential in the degradation of methylene
USE OF SYSTEMS BASED ON TRICHODERMA blue. Sundaravadivelan and Padmanabhan 97 evaluated the lar-
vicidal and pupicidal potentials of silver nanoparticles produced
SPP. FOR METALLIC NANOPARTICLE using T. harzianum for the control of mosquito Aedes aegypti, and
BIOSYNTHESIS obtained promising results. Shelar and Chavan 98 synthesized sil-
Due to their extremely small size, large surface area, and unique ver nanoparticles using T. harzianum and found that it significantly
chemical, physical, and biological properties, metallic nanopar- increased the germination index in sunflower and soybean.
ticles differ from those of the bulk materials of origin, which Guilger et al. 50 synthesized silver nanoparticles using T.
can provide them with the potential to control microorganisms.84 harzianum filtrate and observed its inhibitory activity against
These nanomaterials can be synthesized by chemical, physical and the in vitro germination of sclerotia of Sclerotinia sclerotiorum
biogenic methods. Biogenic synthesis uses extracts of living organ- (white mold). It was also found that the nanoparticles did not
isms as reducing and stabilizing agents, offering the advantages affect the growth of T. harzianum, enabling the fungus and the
of simplicity, cost-effectiveness and low environmental impact.85 nanoparticles to be used in combination.
In biogenic synthesis, the reduction of precursor salts is per- Nandini et al. 99 investigated the potential of six species of
formed using organic reducing agent molecules such as proteins, Trichoderma (T. asperellum, T. harzianum, T. atroviride, T. virens, T.
amino acids, sugars and enzymes. This process occurs naturally in
longibrachiatum and T. brevicompactum) for the biogenic synthesis
the environment, because microorganisms exposed to undesired
of selenium nanoparticles, and obtained positive results with all of
elements secrete metabolites and other compounds that convert
the species. The nanoparticles varied in size and surface charges,
metallic ions into elemental metal, as a form of protection.86,87
and demonstrated potential for control of the phytopathogen
Fungi are widely used in this type of synthesis due to their rapid
Sclerospora graminicola, which affects pearl millet crops.
growth, ease of handling and production of large quantities of
Despite the feasibility of using Trichoderma spp. for biogenic syn-
enzymes.88,89 However, the main disadvantage associated with the
thesis of nanoparticles, an important consideration is the need for
use of fungal biomolecules as reducing agents is their pathogenic
behavior while using phytopathogens such as Fusarium oxys- regulatory structures to govern the uses of these new nanomate-
porum.90 In this context, the advantage of using Trichoderma rials. The toxicity of nanoparticles can be significantly influenced
spp. is that they are used as biological control agent for the by their properties including size, surface charge, coating and crys-
management of various phytopathogens. Among the biogenic talline structure, as well as parameters of the environment in which
nanoparticles synthesized using Trichoderma spp., silver nanopar- they are released, such as temperature, pH, ionic strength and the
ticles are notable due to their known antimicrobial activity. amount of organic matter present.100,101
Additionally, an advantage of biogenic synthesis is that the Since 2004, the European Union has discussed issues related to
nanoparticles possess a stabilizing coating composed of proteins the safety of nanomaterials and the associated ethical questions,
and metabolites derived from the organism, and in the case of but there is still a lack of adequate legislation. There are recog-
Trichoderma spp., the proteins and metabolites are involved in bio- nized benefits of the development of nanotechnological prod-
logical control of phytopathogens, which can provide additional ucts, but the risks they may pose to humans and the environ-
biological activity, stability and augment the action (Figure 1). ment must also be considered.102 Rauscher et al. 103 described
Rodrigues et al. 91 evaluated the filtrate of the fungi used for some of the measures taken by the European Union to regu-
the synthesis of nanoparticles through SDS-PAGE and found the late products containing nanomaterials, including chemicals, bio-
same protein bands for the filtrate and the nanoparticles, which cides, cosmetics, foods, food packaging and medical equipment.
indicates that the proteins from the fungi are capped around the In order to translate the potential of new nanomaterials, such as
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nanoparticles. the biogenic nanoparticles synthesized using Trichoderma spp.,

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 www.soci.org LF Fraceto et al.

Figure 1. Schematic representation of the biogenic synthesis of silver nanoparticles using Trichoderma harzianum.

it will be necessary to regulate nanotechnology at an interna- drying enable the production of microparticles that present
tional level, establishing standardized practices for the use of relatively homogeneous size distributions, enabling them to be
nanomaterials.103,104 more easily used in existing application systems. Another impor-
Although nanoparticles synthesized by means of biogenic tant development is the synthesis of nanoparticles using biogenic
routes, such as those produced using Trichoderma spp., can routes. Molecules and macromolecules secreted by T. harzianum
present lower toxicity than those obtained using chemical act as a source of reducing power for the synthesis of metallic
methods because of the lack of toxic residues from the syn- particles, whereas macromolecules such as proteins, peptides,
thesis and the stabilization of the nanoparticles with organic and carbohydrates present as coatings on the particles can act
compounds,105 they can nonetheless exhibit potential cytotox- as additional agents for biological control. In this context, the
icity and genotoxicity.106 However, despite the possible toxicity association of the nanotechnology with the biologically active
risks, they can offer solutions in various sectors, including in fractions/moieties derived from T. harzianum on the surface of the
agriculture. nanoparticles can promote additional benefits for the efficient
Given the above considerations, further exploration is needed management of phytopathogens.
into the potential of biogenic nanoparticles combined with Tricho- However, as this is a new technology, it should be stressed that
derma spp. as biological control agents, focusing on elucidating detailed investigation should be conducted to confirm that these
the possible risks to human and environmental health. It is there- nanoparticles do not adversely affect nontarget organisms or
fore vital to continue studies in this area, aiming at the future estab- cause environmental contamination. Moreover, to commercialize
lishment of an appropriate regulatory framework. nanotechnological products obtained by the biogenic synthesis
route, it is necessary to establish protocols for standardization of
the preparation of these biocontrol agents, as well as methods for
FUTURE TRENDS, GAPS AND OBSTACLES scaling up production processes. There is tremendous potential for
Fungi of the genus Trichoderma spp. have been widely used for the development and commercialization of novel products for the
the biological control of phytopathogens, although their effective- biological control of pests and pathogens based on the fungi of
ness can be limited due to major concerns of shelf life and unreli- genus Trichoderma spp., especially considering their applications
able performance under field conditions.12,40 The development of in sustainable agriculture.
new strategies, such as those described in this review, can provide
ways to increase the efficacy of these fungi in biological control.
ACKNOWLEDGEMENTS
The use of microencapsulation techniques can help to protect the
The authors are grateful for the financial support provided by
fungi in the field and hence improve their effectiveness. Further-
the São Paulo State Research Foundation (FAPESP, grants #2014/
more, encapsulation can extend the shelf life of the commercial
2161-5, #2015/15617-9, #2015/18231-4, and #2016/13692-6).
products.
It is important to highlight that the development of biolog-
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