Keywords

12.1 Introduction

Biological control of plant disease provides an alternative to synthetic pesticides due to increasing public concern over environmental changes. Soil-borne fungi can survive in a highly competitive environment. Natural antagonism between fungi has been observed in every fungal ecosystem (Wicklow 1998; Ghisalberti 2002). Trichoderma species are free-living fungi; they easily colonize the root, soil, and foliar environment. Being invasive in nature they can easily work against fungal phytopathogens directly (mycoparasitism) or indirectly (competition for food and space, plant growth promotion, antibiosis, modification of environment). Trichoderma species secrete a wide range of lytic enzymes (chitinase, xylanase, cellulases, etc.) (Gloer 1997) and secondary metabolites. The antagonism behavior of Trichoderma toward other fungi is due to the combined action of lytic enzymes and secondary metabolite (Mishra et al. 2020a). Induction of systemic and local defense response in many agricultural crops (cotton, lettuce, ball pepper, and tobacco) by Trichoderma has been reported (Harman et al. 2004; Yedidia et al. 2003).

Pulses are cultivated all over the world and around 50% of pulse production occurs in Asia. Canada is the major producer and exporter of pulses all over the world (Hoover and Ratnayake 2002). The annual global pulse production is around 904 kg/ha. Pulse consumption is higher in Africa and lower in Europe (FAOSTAT 2011). In several countries of the world, pulses are the major component of the diet. In India, pulse consumption is highest followed by Kenya and Turkey (FAOSTAT 2011). Pulses are high in protein, dietary fibers, micronutrients, and various other substances. Due to their high nutritional content, pulses have been described as a future major source of nutritional and health benefits by the Indian Pulses and Grains Association (FAO/WHO 2009). Pulses are rich in nutrition as compared to vegetables and are a cheap source of proteins as compared to animal protein. For pulse, production water is not required in higher amounts. So, the cultivation of pulses can be done in rain-deprived areas. Pulses are commonly known as “poor man’s meat” (Iriti and Varoni 2017). The combination of essential amino acids with high protein content and fiber makes it useful for consumption. Pulse production is influenced by various abiotic and biotic factors. Among biotic factors, phytopathogens are the main cause of yield loss.

Trichoderma species are the most common and popular bioagents. It is commonly used as a biofertilizer for promoting plant health and antagonizing soil-borne pathogens (Mishra et al. 2020b). They can be applied directly to the plants in the form of talc-based formulation, or their secondary metabolites can be packed into a liquid form and applied to the stem of plants (Harman et al. 2004). Trichoderma applications made plants more resistant and increase their productivity for a safe agroecosystem (Vinale et al. 2008; Howell 2003).

12.2 Diversity of Different Trichoderma Isolates

Trichoderma spp. like T. ressei secrete cellulase enzymes which are used for the recycling of cellulosic wastes. Many Trichoderma strains are commercially used to control plant diseases like Fusarium wilt disease in pulses, Sclerotium rolfsii in tobacco and bean, Botrytis cinerea in apple, etc. (Cutler et al. 1999). Some commercial formulations are available in the market and are used for the control of several soil-borne plant diseases (Cardoza et al. 2005) (Table 12.2). Trichoderma metabolites can be classified into two categories: volatile and non-volatile. Volatile metabolites are low molecular weight compounds; these include simple aromatic compounds, polyketides, butenolides, volatile terpenes, and isocyanate metabolites. These are relatively non-polar substances with high vapor pressure. Non-polar metabolites are high molecular weight polar metabolites. They induce phytopathogenic action through direct interaction with a pathogen (Fig. 12.1). Here we will describe the different types of metabolites produced by Trichoderma species and their biological role (Table 12.1).

Fig. 12.1
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Approaches used for the identification of metabolites from Trichoderma spp

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Table 12.1 Some common Trichoderma metabolites with their physiological activities
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12.3 Metabolites Present in Trichoderma spp. Are Associated with Crop Plants

12.3.1 Anthraquinones

Metabolites of this class are the most common metabolites of Trichoderma. Slater et al. (1967) reported chrysophanol, pachybasin, and emodin from T. viride. Similar compounds were observed by Donnelly and Sheridan (1986) in T. polysporum grown with Basidiomycetes fungi. Extraction of chrysophanol and emodin was done from T. aureoviride in 1990. Combined cultures of Trichoderma and Fusarium solani produces trichodermaol. Anthraquinones are majorly involved in the pigmentation’s acetyl and O-methyl derivatives of pachybasin, chrysophanol, and emodin and has been found to show a decrease in the mycelial growth of strains of F. annosus. Emodin possesses monoamine and tyrosine kinase inhibition activity. Trichodermaol exhibits antibacterial activity, and 50 μg/mL conc. of trichodermaol is found to be toxic to Bacillus subtilis and Streptococcus aureus (Adachi et al. 1983). Chrysophanol has been found to possess antifungal activity against Candida albicans and Aspergillus fumigatus at 25–250 μg/mL concentration (Agarwal et al. 2000).

12.3.2 Daucanes

This belongs to the class of sesquiterpenes and is also known as carotenes. During a long analysis of secondary metabolites produced by Trichoderma species, T. virens were found to produce a novel carotene-type metabolite having antifungal activity against yeast and dermatophytes (Watanabe et al. 1990). An oleic acid ester L-735,334 was isolated from the T. virens. These compounds were found to exhibit the growth of etiolated wheat coleoptiles (Macias et al. 2000).

12.3.3 Simple Pyrones

The pyrone 6-pentyl-2H-pyran-2-one is the representative metabolite of this series. This compound is responsible for the aromatic fragrance associated with the fungus. This compound was first identified by Collins and Halim in 1972. This compound has been found to be present in T. viride, T. harzianum (Claydon et al. 1987), and T. koningii (Simon et al. 1988). This metabolite is found to exhibit antagonism against Rhizoctonia solani and Fusarium oxysporum f. sp. lycopersici (Scarselletti and Faull 1994). This compound has been found to significantly reduce the growth of B. cinerea. From T. harzianum, four analogues of pyrone have been isolated exhibiting phytopathogenic action: Penicillium spp., Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans (Claydon et al. 1987; Parker et al. 1997). In 1995 Hill et al. patented the hydro derivatives massoilactone and delta-decanolactone for their ability to control Botrytis or Phytophthora. These compounds inhibit the growth of Aspergillus niger, Candida albicans, and Staphylococcus aureus (Kishimoto et al. 2005). From T. viride viridepyronone has been isolated which has been found to show antagonistic activity in Sclerotium rolfsii at 196 μg/mL of concentration (Evidente et al. 2003).

12.3.4 Koninginins

This belongs to pyranes; they are found to be present in some species of Trichoderma. The culture broth of Trichoderma koningii showed the presence of koninginins (Cutler et al. 1989, 1991a). Koninginin has been found to show an inhibitory effect on the growth of Rhizoctonia solani, Phytophthora cinnamomi, Pythium middletonii, Fusarium oxysporum, and Bipolaris sorokiniana.

12.3.5 Trichodermides

Trichodermides have been isolated from the cultures of T. virens. These trichodermides showed inhibitory action toward human colon carcinoma, and the inhibitor concentration was 0.32 μg/mL and it was found to have a low cytotoxic effect against P388, A-549, and HL-60 cancer lines (Garo et al. 2003; Liu et al. 2005).

12.3.6 Viridans

These compounds possess an unusual furan ring which is fused with c-4 and c-6 carbons of the steroid framework (Hanson 1995). These were first described in 1945 (Brian and McGowan 1945). T. koningii, T. virens, and T. viride have been found to possess this metabolite. Trichoderma viridans have been found to possess the inhibitory action against the spore germination of Botrytis allii, Colletotrichum lini, and Fusarium caeruleum (MIC of 0.003–0.006 lg/mL), Penicillium expansum, Aspergillus niger, and Stachybotrys atra (6 lg/mL) (Brian and McGowan 1945; Ghisalberti 2002). The viridiol obtained from the T. viride and Gliocladium species has been found to possess phytotoxic and antifungal properties.

12.3.7 Viridiofungins

The structural element of this metabolite is citric acid. T. viride produces these metabolites from solid-state fermentation (Harris et al. 1993; Mandala et al. 1997). Viridiofungins are antifungal compounds and have been found to possess inhibitory action toward Aspergillus species (Harris et al. 1993).

12.4 Nitrogen Heterocyclic Compounds

Harzianopyridone is the chief representative of this class of metabolite and has been found to show antifungal activity against Rhizoctonia solani (Dickinson et al. 1989), Gaeumannomyces graminis var. tritici, and Pythium ultimum (Vinale et al. 2006). Harzianic acid also belongs to this category and was obtained from T. harzianum (Sawa et al. 1994).

12.4.1 Derivatives of Trichodenones and Cyclopentenone

5-Hydroxy-3-methoxy-5-vinylcyclopent-2-en-1-one was isolated from the cultures of T. album in 1977 (Strunz et al. 1977).

Harziphilone and fleephilone were isolated from the culture filtrate of T. harzianum, Rhizoctonia solani, Pythium ultimum, and Gaeumannomyces graminis var. tritici (Vinale et al. 2006).

12.4.2 Harzialactones and Derivatives

From T. harzianum harzialactones and their derivatives have been isolated. Harzianoilde is a secondary metabolite with butenolide ring that has been identified. These compounds have been found to show inhibitory action toward Gaeumannomyces graminis var. tritici, Rhizoctonia solani, and Pythium ultimum (Vinale et al. 2006). Vertinolide is a different series of metabolites isolated from the T. longibrachiatum. Sparapano and Evident (1995) reported the biological activity of these compounds.

12.4.3 Trichothecenes

Trichoderma genus is the predominant producer of these metabolites (Grove 1988, 1993, 1996). Trichothecenes can be divided into four categories. Type A has a functional group other than keto at C-8 position, type B has a keto group at C-8 position, and type C and D have a second epoxide ring at c-7,8 or C-9,10 positions and a macrocylic ring was present between C-4 and C-5 with ester linkages. Trichodermin belongs to the category of trichothecenes and has been isolated from T. viride.

12.4.4 Isocyano Metabolites

These metabolites have a characteristic five-membered ring. Forty years ago the first report on cyclopentene in Trichoderma was published (Pyke and Dietz 1966; Meyer 1966). Isonitrile trichoviridin was first isolated by Tamura et al. form T. koningii.

12.4.5 Setin-like Metabolites

Various species of Trichoderma secrete setin-like metabolites which are phytopathogenic against Fusarium species.

12.4.6 Bisorbicillinoids

Bisorbicillinoids are a family of natural products which have many biological activities. Trichodermerol has been isolated from T. longibrachiatum.

12.4.7 Diketopiperazines

Trichoderma harzianum, T. hamatum, and T. koningii have been found to produce diketopiperazines. Gliotoxin was the first member of this category to be identified. There are various species of Trichoderma like T. harzianum, T. hamatum, T. virens, and T. koningii which produce this compound. These metabolites have been found to play an inhibitory effect against Rhizoctonia solani and Pythium ultimum (Howell and Stipanovic 1983).

12.4.8 Ergosterol Derivatives

In Trichoderma sterol production was first detected by Kamal et al. (1971) from the fermentation broth of T. pseudokoningii. Ergosterol is the most common sterol, and in 1975 this has been isolated from the T. hamatum (Hussain et al. 1975). Ergokinin, a class of sterol, has been isolated from T. koningii. Ergokinin has been patented for its use in the inhibition of yeast and fungal mycelia (Reichenbach et al. 1990). Ergokinin is effective against Candida and Aspergillus but is ineffective against Cryptococcus, Fusarium, and Saccharomyces (Vicente et al. 2001).

12.4.9 Peptabiols

This is a large family of natural products. Trichoderma species are the main producers of this metabolite class. From T. viride, first compound, named alamethicin, of this class was isolated. The use of alamethicin, produced by Trichoderma viride, induces a defense response in Phaseolus lunatus and Arabidopsis thaliana.

12.4.10 Cyclonerodiol Derivatives

Cyclonerodiol was first reported from T. koningii (Cutler et al. 1991b; Huang et al. 1995) and from T. harzianum (Ghisalberti and Rowland 1993). Metabolites of this class have been found to have antimicrobial activity against various pathogens.

12.4.11 Statins

It is a diverse group of metabolites which have the ability to inhibit HMG-CoA reductase activity. Compactin belongs to this class and has been isolated from T. longibrachiatum and T. pseudokoningii (Fig. 12.2).

Fig. 12.2
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Structure of secondary metabolites present in Trichoderma

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12.5 Secondary Metabolites Present in Trichoderma Isolates Associated with Pulse Rhizosphere

ICAR-IIPR 160 isolates of Trichoderma are present which have been characterized through ITS and TEF (Fig. 12.3). The mycoparasitic activity of all the isolates has been checked through dual culture. To check the production of secondary metabolites, inverse plate technique was performed (Fig. 12.4). Out of 160 isolates metabolite profiling of five isolates was done (Table 12.2), and it was observed that metabolites related to phytopathogenic activity and plant growth promotion activity were present in all the isolates (Fig. 12.5).

Fig. 12.3
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Diversity of Trichoderma isolates from pulses rhizosphere

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Fig. 12.4
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Inhibition of phytopathogen mycelial growth by the secondary metabolites produced by Trichoderma spp. (a) Inverse plate technique and (b) binary culture technique

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Table 12.2 Metabolite profiling of potential ICAR-IIPR Trichoderma isolates
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Fig. 12.5
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(I): Chitinase and lipase enzyme production by Trichoderma species on their specific medium. (II): (a) Phosphate and (b) siderophore enzyme production by Trichoderma

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12.5.1 Lytic Enzyme

Apart from secondary metabolites, Trichoderma species can produce lytic enzymes (Fig. 12.5).

12.5.2 Proteases

Trichoderma spp. are well-known producers of proteases. Proteolytic activity of Trichoderma viride is aimed to be involved in the biocontrol activity against Sclerotium rolfsii. T. harzianum produces serine proteases which are involved in the mycoparasitism against plant pathogens (Geremia et al. 1993). Proteases are reported to degrade cell walls, membranes, and proteins released by the lysis of pathogen (Goldman et al. 1994).

12.5.3 B1,3-Glucanases

β1,3-glucanases lyse the host cell wall and lead to the leakage of protoplasmic contents (Cherif and Benhamou 1990; Elad et al. 1983; Tronsmo et al. 1993). T. harzianum has been reported to produce N-acetylglucosaminidase, endochitinase, chitobiosidase, and endo β-1,3-glucanases which play a significant role against R. solani.

12.5.4 Chitinase

Chitinases are the linear polymer of β-1,4-N-acetyl glucosamine, the second most abundant polysaccharide present in nature (Deshpande 1986; Nicol 1991). The chitinolytic enzymes of Trichoderma are very effective against pathogens (Harman et al. 1993).

12.6 Future Line of Research

Isolation of metabolic compounds which affect plant metabolism may help in dealing with the problems related to living microorganisms. The use of Trichoderma metabolites for inhibiting pathogen growth and inducing plant growth is a topic of research at present. These metabolites can be produced on a large scale and can be separated from the fungal biomass and formulated for foliar spray.

12.7 Conclusion

Due to the growing interest in the biological control properties of Trichoderma, several bioactive metabolites have been isolated and identified which inhibit the growth of phytopathogens. Trichoderma species produce different volatile and non-volatile metabolites which inhibit the growth of phytopathogens. Among Trichoderma gliovirin, gliotoxin, viridin, pyrones, peptabiols, and others are the main metabolites that are extensively secreted by Trichoderma (Vey et al. 2001). Apart from secondary metabolites, Trichoderma species also secrete ethylene oxide, hydrogen cyanide, alcohol, aldehyde, and ketones which also play an important role in biocontrol activity. Peptabiols are synthesized non-ribosomally and are antimicrobial peptides that have antifungal and antibacterial properties and play an important role in antagonism (Landreau et al. 2002). At the present time around 309 metabolites have been sequenced out of which 180 are from Trichoderma. Further research is needed to study the toxicity and mechanisms of action. Trichoderma species are well-known biocontrol agents that are used efficiently for crop production. Trichoderma species are well known for their capacity to generate antibiotic substances which inhibit phytopathogens. In addition, Trichoderma species secrete several plant growth-promoting metabolites which significantly increase the plant growth and impart tolerance to abiotic stress. The ecological influence of Trichoderma metabolites should be studied for managing the secure and sustainable use of Trichoderma metabolites. For the green era of economy, use of Trichoderma species should be promoted so that we can save the environment and human health from the harmful effects of pesticides.

Metabolomics and expressomics are the techniques that should be used for the identification of molecular bioactive which are involved in the interaction among plants, microbes, and pathogens. Novel techniques developed should be used to identify Trichoderma strains that can produce beneficial metabolites (antibiotics, plant growth promoters, or inducers) (Mukherjee et al. 2012). The application of secondary metabolites to promote crop yield is an innovative and interesting approach, but further studies are needed to study the fate of metabolites applied.