Keywords

1 Introduction

Trichoderma spp. are among the most diverse group of fungi with nearly 300 defined species (Robbertse et al. 2017). These fungi are widely used in industry as source of enzymes and in agriculture as biofungicides and plant growth promoters (Mukherjee et al. 2013a, b). Several formulation products for agricultural usage are available worldwide, and in India alone, a few hundred products are there in the market (Singh et al. 2017). Trichoderma species are strong mycoparasites and can kill other fungi in contact (Druzhinina et al. 2011). These fungi are prolific producers of secondary metabolites/natural products which can be broadly classified as high molecular weight (peptaibols) and low molecular weight (non-ribosomal peptides, polyketides, terpenes, steroids, etc.). There are more than 1000 peptaibols reported from Trichoderma, and similarly, more than 1000 small molecular weight compounds are known to be produced by this single genus (Zeilinger et al. 2016). Secondary metabolites play an important role in Trichoderma-fungus and Trichoderma-plant interactions (Mukherjee et al. 2012a). These compounds may be used to weaken the prey fungus before mycoparasitic attack generally mediated by hydrolytic enzymes like chitinases, beta-glucanases, and proteases. Some of these compounds exhibit strong antimicrobial properties (antibiosis), while others may be involved in inducing resistance response against plant pathogens (Viterbo et al. 2007; Mukherjee et al. 2012b). Trichoderma spp. also produce phytohormones that promote plant growth (Contreras-Cornejo et al. 2009). In short, secondary metabolites are important components of the package of benefits that Trichoderma spp. provide to plants. In this article, we discuss about the diversity of secondary metabolites that these fungi produce and, where information is available, provide genetic evidence for the origin of such vast chemo-diversity in these beneficial filamentous fungi. We focus on three species (T. atroviride, T. virens, and T. reesei) as these have been well characterized subsequent to the publication of the whole genome sequences.

2 Secondary Metabolites of Trichoderma spp. and Their Biosynthesis

Trichoderma species produce a plethora of secondary metabolites belonging to various classes like non-ribosomal peptides, polyketides, terpenes, etc. Table 17.1 presents some examples of secondary metabolites produced by the three well-characterized species of Trichoderma.

Table 17.1 Some examples of secondary metabolites produced by T. reesei, T. atroviride, and T. virens
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2.1 Non-ribosomal Peptides (NRPs)

The non-ribosomal peptides contain both proteinogenic and non-proteinogenic amino acids and may exist in linear or cyclic form. They are synthesized by multi-modular non-ribosomal peptide synthetases. Each module includes adenylation, peptidyl carrier, and condensation domains. The important NRPs produced by Trichoderma spp. are peptaibols, epipolythiodioxopiperazines (ETPs), and siderophores.

2.1.1 Peptaibols

Peptaibols are short peptides containing α-aminoisobutyric acid (Aib) and a C-terminal alcohol. These are the most prominent NRPs produced by Trichoderma species. Peptaibols are reported to have antimicrobial property and cytotoxic activity, and these can induce systemic resistance in plants. The antibiotic property of peptaibols is majorly due to amphipathic nature of peptaibols which allow concentration-dependent membrane permeabilizing activity (Bortolus et al. 2013). Peptaibols are synthesized by peptaibol synthetases (NRPSs) consisting of different modules. Seven-, 14-, and 18–20-module peptaibol synthetases are present in Trichoderma genomes (Mukherjee et al. 2012b). The first peptaibol synthetase enzyme (Tex1) has been reported in Trichoderma virens (Wiest et al. 2002). Tex1 is an 18-module peptaibol synthetase and produces 18-residue trichovirin II type peptaibols. T. virens also possess 14-module peptaibol synthetase enzyme which produces two classes of peptaibol (the 11- and 14-residue peptaibols) [Mukherjee et al. 2011]. T. atroviride is reported to produce 19-residue atroviridins which are produced by a 19-module peptaibol synthetase. Like T. virens, T. atroviride and T. reesei also produce 11- and 14-residue peptaibols catalyzed by a 14-module peptaibol synthetase (Degenkolb et al. 2012).

2.1.2 Epipolythiodioxopiperazines (ETPs)

Epipolythiodioxopiperazines (ETPs) are secondary metabolites with a characteristic cyclic peptide-derived diketopiperazine ring. Gliotoxin and gliovirin are members of the ETP class of peptides. Gliotoxin is produced by “Q” strains of T. virens, whereas gliovirin is produced by “P” strains of T. virens (Howell et al. 1993). Gliotoxin has antimicrobial activity and fungistatic property. In Aspergillus fumigatus which is a human pathogen, the gliotoxin acts as a virulence factor (Scharf et al. 2016). GliP is the NRPS dioxopiperazine synthetase enzyme involved in the biosynthesis of gliotoxin. The gliotoxin biosynthetic gene cluster in A. fumigatus consists of 12 genes inclusive of GliP gene, while in T. virens, the gliotoxin gene cluster consists of only eight genes (Fig. 17.1). In T. virens, the deletion of part of the gliP gene confirmed the role of it in gliotoxin production (Vargas et al. 2014). The gliotoxin biosynthetic gene cluster with only six genes was also identified in T. reesei, albeit this species is not reported to produce gliotoxin (Mukherjee et al. 2012a). The T. virens genome also contains a putative SirP gene cluster which is associated with the production of phytotoxin sirodesmin PL in the phytopathogen Leptosphaeria maculans. However, the product of SirP gene cluster is not known yet in Trichoderma. We have recently discovered the glv gene cluster responsible for gliovirin biosynthesis in “P” strains of T. virens (Sherkhane et al. 2017).

Fig. 17.1
figure 1

The gliotoxin biosynthesis cluster of Trichoderma virens and Aspergillus fumigatus – P, non-ribosomal peptide synthetase (NRPS); G, glutathione-S-transferase; J, dipeptidase; N, N-methyl transferase; F and C, cytochrome P450; M, O-methyl transferase; I, C-S-bond lyase; K, gamma-glutamate cyclotransferase. (Adapted from Zeilinger et al. 2016)

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2.1.3 Siderophores

Siderophores are secondary metabolites that can bind, transport, and store iron. Siderophore-mediated iron acquisition is important for microbial competition, biocontrol, and in interactions with plants and other microbes. There are two types of siderophores, intracellular and extracellular. Ferricrocin is an intracellular siderophore and is reported to protect cells from oxidation-induced stress. The product of NPS6 is an extracellular siderophore that acts as virulence factor in Cochliobolus heterostrophus as well as protects fungus from oxidative stress. In Trichoderma spp., three different NRPSs involved in siderophore biosynthesis have been identified. Intracellular ferricrocin-associated gene cluster has been found in all the three Trichoderma species, but the role for the gene for ferricrocin biosynthesis has been established only in T. virens (Mukherjee et al. 2018). NPS6 and SidD are the NRPSs reported to be involved in extracellular siderophore biosynthesis. A gene cluster with NPS6 as a core enzyme is found in all the three Trichoderma species, but the function is confirmed only in T. virens with gene deletion experiment (Mukherjee et al. 2013a). Another gene cluster with SidD as a core enzyme is found only in T. virens and T. reesei (Mukherjee et al. 2012b)

2.2 Polyketides

Polyketides are secondary metabolites, many of which are having antimicrobial and anti-cancer property. Some polyketides are important for competition for substrate and for interaction with other organisms. Polyketides are synthesized by the polyketide synthases, which are complex enzymes with ketoacyl synthase, an acyl transferase, and a phosphopantetheine attachment site domain. Few studies have been published on the biosynthesis and functional role of polyketides in Trichoderma species, although the genomes of Trichoderma species are rich in PKS-encoding genes. Orthologues of PKS genes associated with the conidial pigment biosynthesis cluster have been identified in all the three Trichoderma species (Baker et al. 2012). Additional 20 putative PKS gene clusters have also been reported in these three species (Bansal and Mukherjee 2016). The role of PKS genes in green pigmentation of conidia, teleomorphic structure, conidial cell wall stability, and antagonistic abilities has been confirmed in T. reesei by deletion of pks4 gene which is an orthologue of pigment forming PKS in Fusarium spp.

2.3 PK/NRPs

Several PKS-NRPS hybrid enzymes are present in Trichoderma genomes. Functional study provided evidence for the role of one of the PKS-NRPS hybrid enzymes (Tex13) in inducing the defense-related pal gene in maize seedlings. The metabolite produced by Tex13 cluster is still not known (Mukherjee et al. 2012b).

2.4 Terpenoids

Terpenoids represent a diverse class of secondary metabolites produced by almost all the organisms including fungi. They are composed of five-carbon isoprene units (C5H8) producing hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, sesterterpenes, triterpenes, tetraterpenes, or polyterpenes. Terpenoid biosynthetic gene clusters have terpene cyclase as the core enzyme. Only a few terpene cyclases may be responsible for production of diverse form of terpenoids. Trichoderma species are reported to produce all forms of terpenoids like volatile terpenoids, diterpenes, sesquiterpenes, and triterpenes (Zeilinger et al. 2016).

In most organisms, the mevalonate pathway is responsible for the formation of isoprene units (Zeilinger et al. 2016). Hydroxy-methylglutaryl-CoA reductase (HMGR) encoded by hmgR gene is the first enzyme in the mevalonate pathway involved in conversion of hydroxy-methylglutaryl-CoA into mevalonate. Deletion of hmgR gene in Trichoderma harzianum showed reduction in antifungal activity against Rhizoctonia solani and Fusarium oxysporum and decrease in ergosterol levels. Ergosterol encoding gene (erg1) silencing also showed decrease in the ergosterol level in T. harzianum, whereas overexpression of erg1 gene increases the antifungal activity of T. harzianum (Cardoza et al. 2006, 2014). The genome analysis of T. reesei, T. atroviride, and T. virens revealed that T. virens (11) has the highest number of terpene cyclases followed by T. atroviride (7) and T. reesei (6), but the terpene cyclases associated with the biosynthetic gene cluster are six in T. virens, three in T. atroviride, and two in T. reesei (Bansal and Mukherjee 2016). The first terpenoid biosynthetic gene cluster was identified in T. virens using suppression subtractive hybridization technique (Mukherjee et al. 2006). The cluster was initially predicted to be associated with viridin production but later found to be responsible for biosynthesis of volatile sesquiterpenes (Crutcher et al. 2013). Deletion of terpene cyclase present in the cluster abolished the production of all the volatile sesquiterpene compounds. This cluster named as vir cluster was found to be present in T. virens and in few Aspergillus species but not in other species of Trichoderma. The reason for existence of the vir cluster in distantly related Trichoderma and Aspergillus species could be explained by horizontal gene transfer. Another terpene cyclase Tri5 is responsible for the production of a phytotoxic agent, trichodermin, in Trichoderma brevicompactum. Overexpression of Tri5 enhanced the production of trichodermin in T. brevicompactum (Tijerino et al. 2011a, b).

2.5 Steroids

Viridin is a triterpene steroidal metabolite produced by both “P” and “Q” strains of T. virens. It has antifungal and anticancer properties. The reduced form of viridin is known as viridiol. Viridiol has herbicidal properties (Jones and Hancock 1987). Both viridin and viridiol are produced abundantly by T. virens, and the vdn cluster for viridin biosynthesis has recently been discovered (Fig. 17.2). Interestingly, an orthologous gene cluster is also present in the bat white nose fungus Pseudogymnoascus destructans (Bansal et al. 2018).

Fig. 17.2
figure 2

The viridin-biosynthesis gene cluster of Trichoderma virens and its orthologue in Pseudogymnoascus destructans. (Adapted from Bansal et al. 2018). For details, please refer to Bansal et al. (2018)

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2.6 6-Pentyl Pyrone (6-PP)

6-PP belongs to the volatile class of secondary metabolites. The characteristic “coconut aroma” produced by some Trichoderma species is due to 6-PP metabolite production. 6-PP has antifungal and plant growth-promoting property (Vinale et al. 2008). There is not much information available on the biosynthetic pathway for the 6-PP production, but a lipoxygenase enzyme has been predicted to be involved in the 6-PP biosynthesis as it is present exclusively in T. atroviride, but not in other Trichoderma species which are devoid of 6-PP production like T. virens and T. reesei (Kubicek et al. 2011).

3 Secondary Metabolism Genes and Gene Clusters: Diversity at the Genus Level

In order to have a comparative assessment of why T. atroviride and T. virens are strong mycoparasites compared to T. reesei, a comparative genomics analysis was performed, and it was found that Trichoderma atroviride and Trichoderma virens, the two aggressive mycoparasites, harbor more genes for hydrolytic enzymes like chitinases and glucanases (Kubicek et al. 2011). Interestingly, however, these two genomes also are richer in secondary metabolite biosynthesis genes (Table 17.2). Many of the secondary metabolite biosynthetic genes form gene clusters inclusive of core enzymes such as non-ribosomal peptide synthetases (NRPSs), polyketide synthases (PKSs), terpene synthases/cyclases, and others. These gene clusters harbor other genes as well like cytochrome P450s, oxidoreductases, methyl transferases, genes for transporters, and transcription factors (Bansal and Mukherjee 2016).

Table 17.2 Secondary metabolism related core genes in the genomes of T. reesei, T. atroviride, and T. virens
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4 Intra-species Diversity in Secondary Metabolites

Trichoderma virens produces a plethora of secondary metabolites, some of which may be strain specific (Howell et al. 1993). Gliotoxin and gliovirin are the best examples showing intra-species diversity of secondary metabolites in T. virens. As discussed above, there are two strains of T. virens, P and Q. “Q” strains of T. virens produce gliotoxin, while “P” strains produce gliovirin (Fig. 17.3). Genome of T. virens “Q” strain Gv29-8 had already been sequenced (Kubicek et al. 2011), and by mining this genome, the gliotoxin biosynthesis gene cluster could easily be identified as information on gliotoxin gene cluster in Aspergillus fumigatus was already known (Mukherjee et al. 2012a). However, the gliovirin gene cluster remained elusive until the whole genome of a “P” strain (IMI 304061, a strain isolated from India) was sequenced by us. By comparative genome analysis, we were able to identify the whole cluster responsible for biosynthesis of gliovirin; its biosynthetic role was confirmed by gene knockout and LC-MS/MS analysis (Sherkhane et al. 2017). Gliotoxin gene cluster is absent in “P” strain. Interestingly, an orthologue of the gliovirin cluster is present in a distantly related fungus Aspergillus udagawae (Fig. 17.4).

Fig. 17.3
figure 3

HPLC analysis of filtrates of T. virens strains. Note the presence of gliotoxin in “Q” strains (T-152, MTCC 2977, MTCC 2884, NRC 8) and its absence in “P” strains

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Fig. 17.4
figure 4

The gliovirin gene cluster of T. virens and its orthologous cluster in A. udagawae. (Adapted from Sherkhane et al. 2017). Please refer to Sherkhane et al. (2017) for details

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5 One Gene Cluster: Many Metabolites

The diversity in secondary metabolites is also brought about by single gene cluster. In T. virens, the vir cluster has been reported to produce 22 volatile secondary metabolites. These include both monoterpenes and sesquiterpenes. The volatile compounds produced by the vir cluster have important roles. For example, beta-caryophyllene has anti-inflammatory and antimicrobial property, and germacrene D has antioxidant and antibacterial property. Interestingly, a glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was found to be present in the vir cluster in both Trichoderma and Aspergillus species (Mukherjee et al. 2006). This GAPDH is isomer of the GAPDH involved in glycolysis in the genome of Trichoderma and Aspergillus species. Deletion of GAPDH in the vir cluster confirms its role in volatile sesquiterpene compound biosynthesis (Fig. 17.5) [Pachauri et al. 2018].

Fig. 17.5
figure 5

(a) The vir cluster responsible for production of volatile sesquiterpene compounds in Trichoderma virens. (b) GC-MS profile of wild-type T. virens and vir8 knockout mutant. No volatile sesquiterpene compounds are produced by vir8 knockout mutant. (Adapted from Pachauri et al. 2018)

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6 One Gene: Many Metabolites

Deletion of tex2 encoding a 14-module non-ribosomal peptide synthetase (NRPS) resulted in abolition of two types of peptaibols, 14-residue peptaibols and 11-residue peptaibols. A total of 88 peptaibols were reported to be produced by Tex2 (53 14-residue peptaibols and 35 11-residue peptaibols). Module skipping and degeneracy gave rise to such diversity (Fig. 17.6).

Fig. 17.6
figure 6

(a) Modular organization of peptaibol synthetase Tex2. (b) sequence alignments of 11- and 14-residue peptaibols produced by T. virens. Sequences are given in standard single-letter code (Ac, acetyl-, U, Aib, Vx, Val/Iva, Lx, Leu/Ile, and ol represents the C-terminal amino-alcohol). The proposed module order is represented by numbers above each box. (Adapted from Mukherjee et al. 2011)

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7 Regulation of Secondary Metabolism

Secondary metabolite diversity may also be regulated by environmental conditions including biotic and abiotic stresses. Though several gene clusters are present in a genome, only a few are expressed under standard laboratory cultivation conditions. Inducing the expression of such gene clusters by using biological or genetic tools gives rise to chemo-diversity (Brakhage and Schroeckh 2011). Many secondary metabolite biosynthetic gene clusters possess putative transcription factors. Thctf1 is a transcription factor reported to be associated with the production of 6-PP in T. harzianum. Deletion of Thctf1 in T. harzianum resulted in decrease in the expression of two secondary metabolites derived from 6-PP and altered antimicrobial activity (Rubio et al. 2009). The secondary metabolites produced by Trichoderma species are reported to be influenced by other microorganisms, pH signaling, and the velvet-complex proteins. The interaction of T. atroviride, T. virens, and T. reesei with Rhizoctonia solani was studied using transcriptome analysis, and this analysis highlighted important genes influenced by the presence of R. solani. Interaction of T. atroviride and T. reesei with R. solani showed upregulation of two PKSs (Atanasova et al. 2013; Kubicek et al. 2011). In T. atroviride, the presence of R. solani also upregulates a 6-PP biosynthesis-related lipoxygenase gene (Kubiceket al 2011). In T. virens, the presence of R. solani upregulates expression of all the genes associated with the gliotoxin biosynthetic gene cluster (Atanasova et al. 2013). In another report, the presence of mycotoxin fusaric acid (FA) produced by Fusarium reduced the production of 6-PP and increased the 1-octen-3-ol biosynthesis. But in certain Trichoderma strains, the presence of fusaric acid does not alter the volatile profile; instead, 6-PP production inhibits FA production (Stoppacher et al. 2010). Change in the pH of the environment causes induction of a pH regulator PacC which further regulates expression of many genes in the fungus. For example, the deletion of a PacC orthologue in T. virens alters the expression of genes associated with secondary metabolite biosynthesis and iron transport, and the mutant also had decreased biocontrol activity (Mukherjee et al. 2012a). The velvet complex is best studied in Aspergillus nidulans and includes a methyltransferase LaeA and the two velvet proteins VeA and VelB. The velvet complex is responsible for coupling light response to the regulation of sexual development and secondary metabolite biosynthesis. Vel1 is an orthologue of veA in T. virens, and deletion of vel1 gene in T. virens ceased the production of gliotoxin and downregulated many secondary metabolism-related genes (Mukherjee and Kenerley 2010). The T. reesei LaeA orthologue Lae1 is required for the expression of lignocellulose-degrading enzymes, and this enzyme is also found to be regulated epigenetically (Karimi-Aghcheh et al. 2013b). Lae1 gene deletion in T. atroviride resulted in the reduced expression of PKS-encoding genes and 6-PP-related lipoxygenase gene (Karimi-Aghcheh et al. 2013a). The mutant also displayed decreased mycoparasitic activity and reduction in the production of antifungal water-soluble metabolites and VOCs. The adenylyl cyclase-inhibiting Gα subunit is encoded by tga1 gene, and the adenylyl cyclase-stimulating Gα subunit is encoded by tga3 gene in T. atroviride. Deletion of tga1 gene decreased the production of 6-PP, but the peptaibol production increased (Reithner et al. 2005), whereas deletion of tga3 gene completely abolished the production of peptaibols, and its production was found to be regulated by two blue light regulators BLR1 and BLR2 in T. atroviride (Komon-Zelazowska et al. 2007). In T. virens, the role of tac1 gene encoding adenylate cyclase has been identified (Mukherjee et al. 2007). MAPK-dependent signaling pathway was also shown to be involved in secondary metabolite biosynthesis and regulation. MAPK-encoding gene tmk1 deletion in T. atroviride increased the production of peptaibols and 6-PP and also increased the antifungal activity (Reithner et al. 2007). But there was no change in secondary metabolites biosynthesis in T. virens in tmk1 deletion mutants (Mendoza-Mendoza et al. 2003).

8 Conclusions

Trichoderma spp. are genetically diverse group of fungi which produce a plethora of secondary metabolites with known and unknown functions and applications. Tremendous amount of variability in secondary metabolite biosynthesis exists even within the same species. The genomes of these fungi are rich in secondary metabolism-related gene clusters, many of which are silent under standard laboratory culture conditions. With new genetic tools available, it is possible to induce the expression of such “silent” clusters which will add to the metabolic diversity of these fungi. Since many secondary metabolites are bioactive, it’s possible to discover novel molecules from these “biocontrol fungi” that might actually find direct applications in agriculture and medical science.