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Trichoderma virens populations that were earlier classified into two strains (P and Q) based on secondary metabolites profiling are also phenotypically and genetically distinct, with the latter being ineffective in controlling the devastating, broad host range plant pathogen Sclerotium rolfsii . The two strains also provoke distinct as well as overlapping transcriptional responses to the presence of the plant and the pathogen.

Trichoderma spp. are widely used as plant disease biocontrol agents in agriculture. Mycoparasitism, which is an ancestral trait of Trichoderma, is one of the most important mechanisms of reducing the pathogen inocula. Mycoparasitism is a complex physiological process that should be viewed in the broad perspective of microbial competition, and involves the production of enzymes and secondary metabolites. Trichoderma spp. have traditionally been viewed as necrotrophic mycoparasites; however, there are evidences that, at least in some instances, they behave as hemibiotrophs, causing minor damage to the host cell wall and having an intracellular existence in the host cell for a significant period. In this review, we cover different aspects of Trichoderma as mycoparasites, ranging from evolution to genomics and interactions with “non-target” fungi.

This study aims to increase Bacillus and Streptomyces antagonistic activity against the root rot and wilt diseases of pulses caused by Macrophomina phaseolina and Fusarium oxysporum f. sp. udum, respectively. To increase antagonistic action, Bacillus subtilis BRBac4, Bacillus siamensis BRBac21, and Streptomyces cavourensis BRAcB10 were subjected to random mutagenesis using varying doses of gamma irradiation (0.5–3.0 kGy). Following the irradiation, 250 bacterial colonies were chosen at random for each antagonistic strain and their effects against pathogens were evaluated in a plate assay. The ERIC, BOX, and random amplified polymorphic studies demonstrated a clear distinction between mutant and wild-type strains. When mutants were compared to wild-type strains, they showed improved plant growth-promoting characteristics and hydrolytic enzyme activity. The disease suppression potential of the selected mutants, B. subtilis BRBac4-M6, B. siamensisi BRBac21-M10, and S. cavourensis BRAcB10-M2, was tested in green gram, black gram, and red gram. The combined inoculation of B. siamensis BRBac21-M10 and S. cavourensis BRAcB10-M2 reduced the incidence of root rot and wilt disease. The same treatment also increased the activity of the defensive enzymes peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase. These findings suggested that gamma-induced mutation can be exploited effectively to improve the biocontrol characteristics of Bacillus and Streptomyces. Following the field testing, a combined bio-formulation of these two bacteria may be utilised to address wilt and root-rot pathogens in pulses.

Trichoderma virens colonizes roots and develops a symbiotic relationship with plants where the fungal partner derives nutrients from plants and offers defence, in return. Tsp1, a small secreted cysteine-rich protein, was earlier found to be upregulated in co-cultivation of T. virens with maize roots. Tsp1 is well conserved in Ascomycota division of fungi, but none of its homologs have been studied yet. We have expressed and purified recombinant Tsp1, and resolved its structure to 1.25 A resolutions, from two crystal forms, using Se-SAD methods. The Tsp1 adopts a β barrel fold and forms dimer in structure as well as in solution form. DALI based structure analysis revealed the structure similarity with two known fungal effector proteins: Alt a1 and PevD1. Structure and evolutionary analysis suggested that Tsp1 belongs to a novel effector protein family. Tsp1 acted as an inducer of salicylic acid mediated susceptibility in plants, rendering maize plants more susceptible to a necrotrophic pathogen Cochliobolus heterostrophus, as observed using plant defence assay and RT-qPCR analysis.

The article contradicts the established fact that acremeremophilane metabolites produced by fungi are sesquiterpenes; instead, our findings suggest that at least some of these well-studied metabolites are of polyketide origin. Acremeremophilane metabolites are of medicinal significance, and the present findings have implications for the metabolic engineering of these metabolites and also their overproduction in microbial cell factories.

Small secreted cysteine-rich proteins (SSCPs) from fungi play an important role in fungi–host interactions. The plant-beneficial fungi Trichoderma spp. are in use worldwide as biocontrol agents and protect the host plant from soil-borne as well as foliar pathogens. Recently, a novel SSCP, Tsp1, has been identified in the secreted protein pool of T. virens and is overinduced upon its interaction with the roots of the maize plant. The protein was observed to be well conserved in the Ascomycota division of fungi, and its homologs are present in many plant-pathogenic fungi such as Fusarium oxysporum and Magnaporthe oryzae. However, none of these homologs have yet been characterized. Recombinant Tsp1 protein has been expressed and purified using an Escherichia coli expression system. The protein, with four conserved cysteines, forms a dimer in solution as observed by size-exclusion chromatography. The dimerization, however, does not involve disulfide bonds. Circular-dichroism data suggested that the protein has a β-strand-rich secondary structure that matched well with the secondary structure predicted using bioinformatics methods. The protein was crystallized using sodium malonate as a precipitant. The crystals diffracted X-rays to 1.7 Å resolution and belonged to the orthorhombic space group P212121 (R meas = 5.4%), with unit-cell parameters a = 46.3, b = 67.0, c = 173.2 Å. The Matthews coefficient (V M) of the crystal is 2.32 Å3 Da−1, which corresponds to nearly 47% solvent content with four subunits of Tsp1 protein in the asymmetric unit. This is the first report of the structural study of any homolog of the novel Tsp1 protein. These structural studies will help in understanding the classification and function of the protein.

This study aims to increase

Gliotoxin produced by Trichoderma virens is inhibitory against various phytopathogenic fungi and bacteria. However, its stability in soil-ecosystem has not yet been well-defined. This study aimed to decipher its persistence and behaviour in growth media, irrigation water and soil ecosystems. Gliotoxin production was noticed at logarithmic growth phase and converted into bis-thiomethyl gliotoxin at late stationary growth phase of T. virens in acidic growth medium. But, no gliotoxin production was observed in neutral and alkaline growth medium. Gliotoxin was stable for several days in acidic water but degraded in alkaline water. Degradation of gliotoxin was more in unsterile soil than sterile soil and also that was higher under wet soil than dry soil. Degradation of gliotoxin was hastened by alkaline pH in wet soil but not in dry soil. Under unsterile soil conditions, high soil moisture increased the degradation of gliotoxin and the degradation of gliotoxin occurred quickly in alkaline soil (in 5 days) compared to acidic soil (in 10 days). Under sterile soil conditions, high soil moisture also enhanced the degradation of gliotoxin but level of degradation was less compared to unsterile conditions. Thus, gliotoxin stability is influenced mainly by the soil wetness, soil microbial community and pH conditions.

The filamentous fungi Trichoderma spp. are widely used for plant growth promotion and disease control. They form stable symbiosis-like relationship with roots. Unlike plant pathogens and mycorrhizae, the molecular events leading to the development of this association is not well understood. Pathogens deploy effector proteins to suppress or evade plant defence. Indirect evidences suggest that Trichoderma spp. can also deploy effector-like proteins to suppress plant defence favouring colonization of roots. Here, using computer simulation, we provide evidence that Trichoderma virens may deploy analogues of host defence proteins to “neutralize” its own effector protein to minimize damage to host tissues, as one of the mechanisms to achieve a stable symbiotic relationship with plants. We provide evidence that T. virens Bys1 protein has a structure similar to plant PR5/thaumatin-like protein and can bind Alt a 1 with a very high affinity, which might lead to the inactivation of its own effector protein. We have, for the first time, predicted a fungal protein that is a competitive inhibitor of a fungal effector protein deployed by many pathogenic fungi to suppress plant defence, and this protein/gene can potentially be used to enhance plant defence through transgenic or other approaches. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02652-8.

Trichoderma virens produces viridin/viridiol, heptelidic (koningic) acid, several volatile sesquiterpenes and gliotoxin (Q strains) or gliovirin (P strains). We earlier reported that deletion of the terpene cyclase vir4 and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH, designated as vGPD) associated with the "vir" cluster abrogated the biosynthesis of several volatile sesquiterpene metabolites. Here we show that, the deletion of this GAPDH also impairs the biosynthesis of heptelidic acid (a non-volatile sesquiterpene), viridin (steroid) and gliovirin (non-ribosomal peptide), indicating regulation of non-volatile metabolite biosynthesis by this GAPDH that is associated with a secondary metabolism gene cluster. To gain further insights into the details of this novel form of regulation, we identified the terpene cyclase gene responsible for heptelidic acid biosynthesis (hereafter designated as has1) and prove that the expression of this gene is regulated by vGPD. Interestingly, deletion of has1 impaired biosynthesis of heptelidic acid (HA), viridin and gliovirin, but not of volatile sesquiterpenes. Deletion of the vir cluster associated terpene cyclase gene (vir4), located next to the vGPD gene, did not impair biosynthesis of HA, viridin or gliovirin. We thus unveil a novel circuitry of regulation of secondary metabolism where an HA-tolerant GAPDH isoform (vGPD) regulates HA biosynthesis through the transcriptional regulation of the HA-synthase gene (which is not part of the "vir" cluster). Interestingly, impairment of HA biosynthesis leads to the down-regulation of biosynthesis of other non-volatile secondary metabolites, but not of volatile secondary metabolites. We thus provide evidence that the "vir" cluster associated, HA-tolerant GAPDH in T. virens participates in the biosynthesis of volatile sesquiterpenes as a biosynthetic enzyme, and regulates the production of non-volatile metabolites via regulation of HA biosynthesis. The orthologue of the "vir" cluster in Aspergillus oryzae was earlier reported to synthesize HA by another group. Our study thus proves that the same gene cluster can code for unrelated metabolites in different species.

International audience; The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.

Trichoderma virens is a commercial biofungicide used in agriculture. We have earlier isolated a mutant of T. virens using gamma ray-induced mutagenesis. This mutant, designated as M7, is defective in morphogenesis, secondary metabolism, and mycoparasitism. The mutant does not produce conidia, and the colony is hydrophilic. M7 cannot utilize cellulose and chitin as a sole carbon source and is unable to parasitize the plant pathogens Rhizoctonia solani and Pythium aphanidermatum in confrontation assay. Several volatile (germacrenes, beta-caryophyllene, alloaromadendrene, gamma-muurolene) and non-volatile (viridin, viridiol, gliovirin, heptelidic acid) metabolites are not detected in M7. In transcriptome analysis, many genes related to secondary metabolism, carbohydrate metabolism, hydrophobicity, and transportation, among others, were found to be downregulated in the mutant. Using whole genome sequencing, we identified five deletions in the mutant genome, totaling about 250 kb (encompassing 71 predicted ORFs), which was confirmed by PCR. This study provides novel insight into genetics of morphogenesis, secondary metabolism, and mycoparasitism and eventually could lead to the identification of novel regulators of beneficial traits in plant beneficial fungi Trichoderma spp. We also suggest that this mutant can be developed as a microbial cell factory for the production of secondary metabolites and proteins.

The anticancer antibiotic heptelidic acid is a sesquiterpene lactone produced by the beneficial plant fungus Trichoderma virens. This species has been separated into two strains, referred to as P and Q, based on its biosynthesis of secondary metabolites; notably, only P-strains were reported to produce heptelidic acid. While characterizing a Q-strain of T. virens containing a directed mutation in the non-ribosomal peptide synthetase encoding gene Tex7, the appearance of an unknown compound in anomalously large quantities was visualized by TLC. Using a combination of HPLC, LC-MS/MS, and NMR spectroscopy, this compound was identified as heptelidic acid. This discovery alters the strain classification structure of T. virens. Additionally, the Tex7 mutants inhibited growth of maize seedlings, while retaining the ability to induce systemic resistance against the foliar fungal pathogen, Cochliobolus heterostrophus.

Fungal siderophores are known to be involved in iron acquisition and storage, as well as pathogenicity of mammals and plants. As avirulent plant symbionts, Trichoderma spp. colonize roots and induce resistance responses both locally and systemically. To study the role of intracellular siderophore(s) in Trichoderma-plant interactions, we have obtained mutants in a non-ribosomal peptide synthetase, TvTex10, that was predicted to be involved in intracellular siderophore(s) biosynthesis. This gene has a detectable basal level of expression and is also upregulated under iron-deplete conditions. This is unlike two other siderophore-encoding genes, which are tightly regulated by iron. Disruption of tex10 gene using homologous recombination resulted in mutants with enhanced growth rate, reduced conidiation and hyper-sensitivity to oxidative stress as compared to wildtype strain. The mutants also produced reduced levels of gliotoxin and dimethyl gliotoxin but have enhanced ability to colonize maize seedling roots. The mutants were also impaired in induction of induced systemic resistance (ISR) in maize against the foliar pathogen Cochliobolus heterostrophus.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyses the sixth step of glycolysis, and is also known to perform other (moonlighting) activities in animal cells. We have earlier identified an additional GAPDH gene in Trichoderma virens genome. This gene is consistently associated with the vir cluster responsible for biosynthesis of a range of volatile sesquiterpenes in Trichoderma virens. This gene is also associated with an orthologous gene cluster in Aspergillus spp. Both glycolytic GAPDH and the vir cluster-associated GAPDH show more than 80% similarity with essentially conserved NAD+ cofactor- and substrate-binding sites. However, a conserved indel is consistently present only in GAPDH associated with the vir cluster, both in T. virens and Aspergillus spp. Using gene knockout, we demonstrate here that the vir cluster-associated GAPDH is involved in biosynthesis of volatile sesquiterpenes in T. virens. We thus, for the first time, elucidate the non-glycolytic role of a GAPDH in a fungal system, and also prove for the first time that a GAPDH, a primary metabolism protein, is involved in secondary metabolism.

Trichoderma species are opportunistic fungi residing primarily in soil, tree bark and on wild mushrooms. Trichoderma is capable of killing other fungi and penetrating plant roots, and is commonly used as both a biofungicide and inducer of plant defence against pathogens. These fungi also exert other beneficial effects on plants including growth promotion and tolerance to abiotic stresses, primarily mediated by their intimate interactions with roots. In root–microbe interactions (both beneficial and harmful), fungal secreted proteins play a crucial role in establishing contact with the roots, fungal attachment, root penetration and triggering of plant responses. In Trichoderma–root interactions, the sucrose present in root exudates has been demonstrated to be important in fungal attraction. Attachment to roots is mediated by hydrophobin-like proteins, and secreted swollenins and plant cell wall degrading enzymes facilitate internalization of the fungal hyphae. During the early stage of penetration, suppression of plant defence is vital to successful initial root colonisation; this is mediated by small soluble cysteine-rich secreted proteins (effector-like proteins). Up to this stage, Trichoderma's behaviour is similar to that of a plant pathogen invading root structures. However, subsequent events like oxidative bursts, the synthesis of salicylic acid by the plants, and secretion of elicitor-like proteins by Trichoderma spp. differentiate this fungus from pathogens. These processes induce immunity in plants that help counter subsequent invasion by plant pathogens and insects. In this review, we present an inventory of soluble secreted proteins from Trichoderma that might play an active role in beneficial Trichoderma–plant interactions, and review the function of such proteins where known.

Various plasma membrane compartments in fungal cell wall are involved in a wide range of cellular functions. Eisosome protein Pil1 ( p hosphorylation is i nhibited by l ong chain bases) in yeast cells are known to be involved genetic pathway that controls the cellular responses to heat stress. The PIL gene superfamily is well conserved among different organisms. In this study, a full-length ThPIL1 gene that encodes for a 357 amino acid protein was cloned from Trichoderma harzianum T35 strain. A comparative analysis of ThPIL1 gene sequence with other PIL1 genes of different organisms and Trichoderma spp. shows degree of sequence identity ranging from 78 to 95%. Southern blot analysis showed that there existed only one copy of the ThPIL1 gene of in genome of T. harzianum T35, but alternative splicing of ThPIL1 generates two transcript isoforms, ThPIL1 -α and β. Spliced transcript ThPIL1 -β retains an unspliced first intron that carries a premature termination codon. The expression characteristics of the both isoforms of ThPIL1 gene in the mycelium grown at different temperatures were investigated using quantitative real-time PCR (qRT-PCR). The results showed that they are differentially expressed in mycelia exposed to different temperatures, the major product being ThPIL1 -α. The transcripts of ThPIL1 -β accumulated markedly when the Trichoderma mycelium was subjected to heat (40 °C and 45 °C). The expression level of the ThPIL1 -α was highest in treatment when mycelium was grown at 45 °C for 2 h, and was approximately 4-fold higher than at 45 °C for 1 h. The highest expression level of ThPIL1 -β was detected at the same temperature, and very negligible expression was found at lower temperature. These results provide new insight into roles in the expression regulation of fungal eisosome genes and suggest that ThPIL1 gene may play a role in growth of T. harzianum T35 under the heat stress response to environmental stresses.

Using gamma-ray-induced mutagenesis, we have developed a mutant (named G2) of Trichoderma virens that produced two- to three-fold excesses of secondary metabolites, including viridin, viridiol, and some yet-to-be identified compounds. Consequently, this mutant had improved antibiosis against the oomycete test pathogen Pythium aphanidermatum. A transcriptome analysis of the mutant vis-à-vis the wild-type strain showed upregulation of several secondary-metabolism-related genes. In addition, many genes predicted to be involved in mycoparasitism and plant interactions were also upregulated. We used tamarind seeds as a mass multiplication medium in solid-state fermentation and, using talcum powder as a carrier, developed a novel seed dressing formulation. A comparative evaluation of the wild type and the mutant in greenhouse under high disease pressure (using the test pathogen Sclerotium rolfsii) revealed superiority of the mutant over wild type in protecting chickpea (Cicer arietinum) seeds and seedlings from infection. We then undertook extensive field evaluation (replicated micro-plot trials, on-farm demonstration trials, and large-scale trials in farmers’ fields) of our mutant-based formulation (named TrichoBARC) for management of collar rot (S. rolfsii) in chickpea and lentil (Lens culinaris) over multiple locations in India. In certain experiments, other available formulations were included for comparison. This formulation consistently, over multiple locations and years, improved seed germination, reduced seedling mortality, and improved plant growth and yield. We also noticed growth promotion, improved pod bearing, and early flowering (7–10 days) in TrichoBARC-treated chickpea and lentil plants under field conditions. In toxicological studies in animal models, this formulation exhibited no toxicity to mammals, birds, or fish.

Background Trichoderma spp. are majorly composed of plant-beneficial symbionts widely used in agriculture as bio-control agents. Studying the mechanisms behind Trichoderma-derived plant benefits has yielded tangible bio-industrial products. To better take advantage of this fungal-plant symbiosis it is necessary to obtain detailed knowledge of which genes Trichoderma utilizes during interaction with its plant host. In this study, we explored the transcriptional activity undergone by T. virens during two phases of symbiosis with maize; recognition of roots and after ingress into the root cortex. Results We present a model of T. virens – maize interaction wherein T. virens experiences global repression of transcription upon recognition of maize roots and then induces expression of a broad spectrum of genes during colonization of maize roots. The genes expressed indicate that, during colonization of maize roots, T. virens modulates biosynthesis of phytohormone-like compounds, secretes a plant-environment specific array of cell wall degrading enzymes and secondary metabolites, remodels both actin-based and cell membrane structures, and shifts metabolic activity. We also highlight transcription factors and signal transduction genes important in future research seeking to unravel the molecular mechanisms of T. virens activity in maize roots. Conclusions T. virens displays distinctly different transcriptional profiles between recognizing the presence of maize roots and active colonization of these roots. A though understanding of these processes will allow development of T. virens as a bio-control agent. Further, the publication of these datasets will target future research endeavors specifically to genes of interest when considering T. virens – maize symbiosis. Electronic supplementary material The online version of this article (10.1186/s12864-019-5651-z) contains supplementary material, which is available to authorized users.

Trichoderma species are filamentous ascomycetous fungi that have wide biotechnological applications in industry as well as agriculture. Having nearly 300 species, this genus represents one of the most diverse groups of fungi. Secondary metabolites are useful natural products having widespread applications in agriculture and medicine. Trichoderma species are prolific producers of secondary metabolites (natural products) with proven role in disease suppression. Genes for biosynthesis of these metabolites are often present as gene clusters, and one such cluster may be responsible for synthesis of a range of metabolites and intermediates. Depending on the chemical nature, these metabolites could be grouped as non-ribosomal peptides, polyketides, terpenes, steroids, etc. Three species of Trichoderma (T. virens, T. atroviride, and T. reesei) are well studied from genomics point of view, and this article focuses mainly on these three species. We discuss here the level of diversity with respect to secondary metabolite biosynthesis machinery at the genus, species, and strain level with genetic evidence where available. The article highlights the untapped potential of Trichoderma spp. as a source of a variety of secondary metabolites with potential applications in agriculture and medicine.

Translationally controlled tumour proteins (TCTPs) are omnipresent in eukaryotes and play important physiological roles. This protein is identified as an allergen in the fungi Alternaria alternata and Cladosporium herbarum, and is involved in maintaining a balance between sexual and asexual differentiation in Aspergillus nidulans. MoTCTP regulates growth and conidiation in the plant pathogen Magnaporthe oryzae. We present here the functions of a TCTP orthologue (Tcp1) in the plant beneficial fungus Trichoderma virens. T. virens Tcp1 shares 42.94% and 39.88% sequence similarity with the evolutionarily distant human TCTP and maize TCTP, respectively. Based on prediction models, the secondary structure elements (α-helices and β-sheets) were found to be very well conserved barring a few insertions/deletions in the loop region. Using homologous recombination, we obtained three independent deletion mutants in this gene and a comparison of phenotypes with the wild type strain revealed that this protein has multiple functions in T. virens. Tcp1 knockout mutants showed slow radial growth and dry weight production. Δtcp1 mutants also lost the ability to overgrow the plant pathogenic fungi Sclerotium rolfsii and Rhizoctonia solani, but retained this property against the oomycete Pythium aphanidermatum reflecting selective loss of antagonistic ability. The mutants also lost the ability to colonize and kill the sclerotia of S. rolfsii.

Trichoderma spp. are plant beneficial fungi that colonize roots, but unlike plant pathogens, impart defence. Recently, however, some small secreted cysteine-rich proteins have been identified in Trichoderma secretome, deletion of which improves induced defence. The mechanism of such “unusual” behaviour of a plant-beneficial fungus is not understood. In the present study, through in silico analysis, we attempted to explore if such proteins indeed suppress defence by interacting with plant proteins, much like pathogens, taking Alt a 1 protein of T. virens and maize PR5, a thaumatin-like defence protein as predicted interacting partners (with cue from some plant-pathogen interactions). Using computational analysis, we test the hypothesis that Trichoderma Alt a 1 inactivates maize PR5 to suppress plant defence in order to be able to colonize roots. The tertiary structures of both Alt a 1 and PR5 were modelled through homology as well as de novo modelling, followed by model validation through different quality check tools. Prior to protein-protein docking, MD (molecular dynamics) optimisation and refinement of computational models were carried-out. Docking results indicated that Alt a 1 exhibits strong binding affinity with PR5 suggesting that Alt a 1 and PR5 might interact much like in a plant-pathogen interactions resulting in suppression of plant defence to facilitate colonization.

Gliotoxin (GT) is the most important epidithiodioxopiperazine (ETP)-type fungal toxin. GT was originally isolated from Trichoderma species as an antibiotic substance involved in biological control of plant pathogenic fungi. A few isolates of GT-producing Trichoderma virens are commercially marketed for biological control and widely used in agriculture. Furthermore, GT is long known as an immunosuppressive agent and also reported to have anti-tumour properties. However, recent publications suggest that GT is a virulence determinant of the human pathogen Aspergillus fumigatus. This compound is thus important on several counts - it has medicinal properties, is a pathogenicity determinant, is a potential diagnostic marker and is important in biological crop protection. The present article addresses this paradox and the ecological role of GT. We discuss the function of GT as defence molecule, the role in aspergillosis and suggest solutions for safe application of Trichoderma-based biofungicides.

Trichoderma virens genome harbors two isoforms of GAPDH, one (gGPD) involved in glycolysis and the other one (vGPD) in secondary metabolism. vGPD is expressed as part of the “vir” cluster responsible for the biosynthesis of volatile sesquiterpenes. The secondary metabolism-associated GAPDH is tolerant to the anti-cancer metabolite heptelidic acid (HA), produced by T. virens. Characterizing the HA-tolerant form of GAPDH, thus has implications in cancer therapy. In order to get insight into the mechanism of HA-tolerance of vGPD, we have purified recombinant form of this protein. The protein displays biochemical and biophysical characteristics analogous to the gGPD isoform. It exists as a tetramer with Tm of about 56.5 °C, and displays phosphorylation enzyme activity with Km and Kcat of 0.38 mM and 2.55 sec−1, respectively. The protein weakly binds to the sequence upstream of the vir4 gene that codes for the core enzyme (a terpene cyclase) of the “vir” cluster. The EMSA analysis indicates that vGPD may not act as a transcription factor driving the “vir” cluster, at least not by directly binding to the promoter region. We also succeeded in obtaining small crystals of this protein. We have constructed structural models of vGPD and gGPD of T. virens. In silico constrained docking analysis reveals weaker binding of heptelidic acid in vGPD, compared to gGPD protein.

Trichoderma virens is a plant beneficial fungus that interacts intimately with roots and boosts plant immunity against invading pathogens. Some Trichoderma species have been used to control vascular wilt pathogen Fusarium oxysporum f. sp. cubense by enhancing host plant defence. In order to understand the molecular mechanisms of such interactions of Trichoderma with banana roots, this study analyses the secretome of T. virens induced by tissue culture raised banana cv. Rasthali roots, using a gel-free proteome approach. Tissue cultured banana roots were co-incubated with germinated conidia of T. virens for four days. The filtrate was harvested and proteins were identified using mass spectrometry (trypsin digestion and LC-MS/MS analysis). A total of 77 proteins were identified to be upregulated in T. virens in contact with banana roots, compared with T. virens incubated alone. Among these, 11 and 6 belonged to the glycoside hydrolases and small cysteine-rich secreted proteins (SSCPs), respectively. These two groups of proteins are known to be involved in plant-Trichoderma interactions, facilitating penetration, colonization, and inducing defense responses. Contrary to this, 27 proteins were downregulated in contact with banana roots and 32 did not show any significant changes. This is the first study on proteome analysis of Trichoderma in contact with banana roots leading to the identification of potential elicitor proteins that could be relevant to disease management in banana. A novel cerato-platanin family protein and a Nep-1 like protein were found to be upregulated in contact with banana roots. Since such proteins are known to be involved in plant-microbe interactions, these are being studied for their role in Trichoderma-banana plant interactions especially in induced defence.

Trichoderma spp. are widely used as commercial biofungicides, and most commercial formulations are conidia based. Identification of genes that regulate conidiation would thus be of help in genetic reprogramming of these species to optimize sporulation. In this study, we constructed an SSH (suppression subtractive hybridization) library from RNA samples of the wild type strain and a non-conidiating mutant, M7, grown under constant illumination for 2 days. We identified several genes that are underexpressed in the mutant. Some of these genes are related to secondary metabolism, and a few could be associated with conidiation. Genes coding for the following proteins, among others, were identified: O-methyl transferase, ATPase, alpha/beta-hydrolase, WD repeat containing protein, dehydrogenase, thioesterase, translationally controlled tumour protein, and a proline–glycine–tyrosine-rich protein (PGYRP) that has been annotated in T.reesei as a signalling protein. Two of these genes, encoding Pgy1, a novel PGYRP, and Ecm33, a GPI-anchored cell wall protein, were further studied in detail by generation of deletion mutants. We demonstrate here that both these genes not only regulate radial growth and conidiation in Trichoderma virens, but are also involved in antagonism against soil-borne wide host range plant pathogens. Furthermore, deletion of ecm33 affected hydrophobicity and cell wall integrity.

This study is the first time report of utilization of Trichoderma spp. isolated from different tree bark from Odisha state of India for rice crop health management and higher productivity. Six isolates of Trichoderma spp. were identified based on the morphological characteristics and species determination was performed by molecular assays. One of the isolated strains determined as Trichoderma erinaceum outperformed others. Trichoderma erinaceum controlled three soil borne plant pathogens i.e. Rhizoctonia solani, Sclerotium rolfsii and Sclerotium oryzae effectively under controlled condition and R. solani and Helminthosporium oryzae under filed condition. Seed treatments with the formulated isolates improved the germination rate of rice and enhanced vigour. These parameters along with higher chlorophyll content could be related to higher yield observed in two rice varieties; Karuna and Sahabhagidhan. Among the six isolates tested, Trichoderma erinaceum treatment recorded highest yield. Significantly higher expression of some stress related enzymes was observed in Trichoderma treated plants which helped in better crop growth both under biotic and abiotic stresses. These isolates helped both the varieties to accumulate more nutrients. This study proves that Trichoderma erinaceum obtained from tree bark may be incorporated in integrated rice crop management both as biocontrol agent and biofertilizer.

Coevolution of plants with microbes has resulted in beneficial, harmful, or neutral host-parasite interactions. With the domestication of crop plants and intensive plant breeding, not only the plant genetic base narrowed down, but also it neglected the positive role of associated microbes, especially those dwelling the rhizosphere. The green revolution not only helped to feed the hungry population, but also brought along the concept of high-input agriculture, especially the extensive use of chemical fertilizers and pesticides. It was soon realized that in order to sustain the productivity, there is a need for more emphasis on biological-based inputs. Many plant beneficial microbes have been identified and are being used for enhancing plant growth and productivity through supply of macro- and micronutrients and suppression of pests and pathogens. The rhizobia, mycorrhizae, Bacillus thuringiensis, and Trichoderma spp. are classical examples of exploitation of beneficial microbes in today's agriculture. This chapter provides an overview of microbe-based agriculture—its genesis, status, and prospects.

Background Cytochrome P450s form an important group of enzymes involved in xenobiotics degradation and metabolism, both primary and secondary. These enzymes are also useful in industry as biotechnological tools for bioconversion and a few are reported to be involved in pathogenicity. Trichoderma spp. are widely used in industry and agriculture and are known for their biosynthetic potential of a large number of secondary metabolites. For realising the full biosynthetic potential of an organism, it is important to do a genome-wide annotation and cataloguing of these enzymes. Results Here, we have studied the genomes of seven species (T. asperellum, T. atroviride, T. citrinoviride, T. longibrachiatum, T. reesei , T. harzianum and T. virens) and identified a total of 477 cytochrome P450s. We present here the classification, evolution and structure as well as predicted function of these proteins. This study would pave the way for functional characterization of these groups of enzymes and will also help in realization of their full economic potential. Conclusion Our CYPome annotation and evolutionary studies of the seven Trichoderma species now provides opportunities for exploration of research-driven strategies to select Trichoderma species for various applications especially in relation to secondary metabolism and degradation of environmental pollutants. Electronic supplementary material The online version of this article (10.1186/s40694-018-0056-3) contains supplementary material, which is available to authorized users.

When this paper was first published the artwork for Figures 5 and 6 were switched in error. They are correctly printed below:[Figure presented] Fig. 5 – Sequence alignments of ceratoplatanin and Sm1/Epl1 orthologs. The sequences used to generate the alignment are: six Trichoderma sequences (Tr, T. reesei XP_006970000.1; Ta_Epl1, T. atroviride XP_013937770; Tasp_Epl1, T. asperellum CAL80753.1; Tv_Sm1, T. virens XP_013959806.1; Tvir_Epl1 T. viride CAL80756; Th_Snod-Prot1, T. harzianum KKP05841.1), ceratoplatanin itself (Ceratocystis platani AGQ22226.1), and predicted Sm1/Epl1 orthologs from other Sordariomycetes (Fo_SnodProt1, Fusarium oxysporum XP_018240137; Nc_CCG-14, Neurospora crassa XP_958708.1; Mo_SnodProt1, Magnaporthe oryzae XP_003710181.1; Ct_Epl1, Colletotrichum tofieldiae KZL71046.1; Ci_Epl1, Colletotrichum incanum KZL84107.1). The alignment was generated by MUSCLE at www.phylogeny. fr and plotted by EMBO MView online tool at http://www.ebi.ac.uk/Tools/msa/mview/; colour coding is by identity and amino acid chemical properties.[Figure presented] Fig. 6 – Similarity of some Trichoderma secreted proteins to expansins. Domain analyses of: a typical plant expansin (Zea mays NP_001288510.1 expansin-B1 precursor), T. reesei swollenin (CAB92328.1), ceratoplatanin (Ceratocystis fimbriata, AGQ22233.1), T. virens Sm1 (XP_013959806.1), and T. virens MRSP1 (XP_013952052.1). The maps are shown in descending order with respect to their expansin-like properties, from a plant expansin down to MRSP1, whose only hint of similarity to expansins is the double j/b-barrel (DPBB_1) fold (expansin Domain 1, grey). Prediction of domain structures: for expansin B1 and swollenin, Prosite (http://prosite.expasy.org/); for ceratoplatanin and Sm1, Pfam (CP domain is apparently not in the Prosite database); for MRSP1, coordinates generated by JGI's automated pipeline were taken from the protein page (Prosite and Pfam scans did not detect the DPBB_1 fold; which was originally detected by a combination of other methods (see Horwitz et al. (2013)). Combined graphics were generated by the Prosite domain drawing tool (via ExPasy).

The biocontrol agent, Trichoderma virens, has the ability to protect plants from pathogens by eliciting plant defense responses, involvement in mycoparasitism, or secreting antagonistic secondary metabolites. SM1, an elicitor of induced systemic resistance (ISR), was found to have three paralogs within the T. virens genome. The paralog sm2 is highly expressed in the presence of plant roots. Gene deletion mutants of sm2 were generated and the mutants were found to overproduce SM1. The ability to elicit ISR in maize against Colletotrichum graminicola was not compromised for the mutants compared to that of wild type isolate. However, the deletion strains had a significantly lowered ability to colonize maize roots. This appears to be the first report on the involvement of an effector-like protein in colonization of roots by Trichoderma.

Using a gene disruption strategy, we generated mutants in the gliP locus of the plant-beneficial fungus Trichoderma virens that were no longer capable of producing gliotoxin. Phenotypic assays demonstrated that the gliP-disrupted mutants grew faster, were more sensitive to oxidative stress and exhibited a sparse colony edge compared with the WT strain. In a plate confrontation assay, the mutants deficient in gliotoxin production were ineffective as mycoparasites against the oomycete, Pythium ultimum, and the necrotrophic fungal pathogen, Sclerotinia sclerotiorum, but retained mycoparasitic ability against Rhizoctonia solani. Biocontrol assays in soil showed that the mutants were incapable of protecting cotton seedlings from attack by P. ultimum, against which the WT strain was highly effective. The mutants, however, were as effective as the WT strain in protecting cotton seedlings against R. solani. Loss of gliotoxin production also resulted in a reduced ability of the mutants to attack the sclerotia of S. sclerotiorum compared with the WT. The addition of exogenous gliotoxin to the sclerotia colonized by the mutants partially restored their degradative abilities. Interestingly, as in Aspergillus fumigatus, an opportunistic human pathogen, gliotoxin was found to be involved in pathogenicity of T. virens against larvae of the wax moth, Galleria mellonella. The loss of gliotoxin production in T. virens was restored by complementation with the gliP gene from A. fumigatus. We have, thus, demonstrated that the putative gliP cluster of T. virens is responsible for the biosynthesis of gliotoxin, and gliotoxin is involved in mycoparasitism and biocontrol properties of this plant-beneficial fungus Fil: Vargas, Walter Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentina Fil: Mukherjee, Prasun K.. Bhabha Atomic Research Centre; India Fil: Laughlin, David. Texas A&M University; Estados Unidos Fil: Wiest, Aric. University Of Missouri; Estados Unidos Fil: Moran Diez, Maria E.. Texas A&M University; Estados Unidos Fil: Kenerley, Charles M.. Texas A; Estados Unidos

Trichoderma species are widely used in agriculture and industry as biopesticides and sources of enzymes, respectively. These fungi reproduce asexually by production of conidia and chlamydospores and in wild habitats by ascospores. Trichoderma species are efficient mycoparasites and prolific producers of secondary metabolites, some of which have clinical importance. However, the ecological or biological significance of this metabolite diversity is sorely lagging behind the chemical significance. Many strains produce elicitors and induce resistance in plants through colonization of roots. Seven species have now been sequenced. Comparison of a primarily saprophytic species with two mycoparasitic species has provided striking contrasts and has established that mycoparasitism is an ancestral trait of this genus. Among the interesting outcomes of genome comparison is the discovery of a vast repertoire of secondary metabolism pathways and of numerous small cysteine-rich secreted proteins. Genomics has also facilitated investigation of sexual crossing in Trichoderma reesei, suggesting the possibility of strain improvement through hybridization.

A putative terpene cyclase vir4, which is a member of a secondary metabolite cluster, has been deleted in Trichoderma virens to determine its function. The deletion mutants were compared for volatile production with the wild-type as well as two other Trichoderma spp. This gene cluster was originally predicted to function in the synthesis of viridin and viridiol. However, the experimental evidence demonstrates that this gene cluster is involved in the synthesis of volatile terpene compounds. The entire vir4-containing gene cluster is absent in two other species of Trichoderma, T. atroviride and T. reesei. Neither of these two species synthesizes volatile terpenes associated with this cluster in T. virens. We have thus identified a novel class of volatile fungal sesquiterpenes as well as the gene cluster involved in their biosynthesis.

Trichoderma spp. are a rich source of secondary metabolites (SMs). The recent publication of the genome sequences of three Trichoderma spp. has revealed a vast repertoire of genes putatively involved in the biosynthesis of SMs, such as non-ribosomal peptides, polyketides, terpenoids and pyrones. Interestingly, the genomes of the mycoparasitic species Trichoderma virens and Trichoderma atroviride are enriched in secondary metabolism-related genes compared with the biomass-degrading Trichoderma reesei: 18 and 18 polyketide synthases compared with 11; 28 and 16 non-ribosomal peptide synthetases compared with 10, respectively. All three species produce a special class of non-ribosomally synthesized peptides known as peptaibols, containing non-proteinogenic amino acids (particularly α-aminoisobutyric acid). In common with other filamentous ascomycetes, Trichoderma spp. may require siderophores (also produced by non-ribosomal peptide synthetases) to grow in iron-poor conditions and to compete with their hosts for available iron. Two generalizations can be made about fungal SM genes: they are often found in clusters, and many are not expressed under standard laboratory conditions. This has made it difficult to identify the compounds. Trichoderma, in particular, interacts with other microbes in the soil and with plant roots in the rhizosphere. A detailed metabolomic-genomic study would eventually unravel the roles of many of these SMs in natural ecosystems. Novel genetic tools developed recently, combined with biological understanding of the function of SMs as toxins or signals, should lead to 'awakening' of these 'silent' clusters. Knowledge of the SM repertoire should precede application of Trichoderma strains for biocontrol: some metabolites could be toxic to plants and their consumers, and thus should be avoided. Others could be beneficial, antagonizing pathogens or inducing resistance in crop plants.