Comparative Phenotypic, Genomic, and Transcriptomic Analyses of Two Contrasting Strains of the Plant Beneficial Fungus Trichoderma virens.

Trichoderma virens is a beneficial fungus that helps plants fight pathogens and abiotic stresses and thereby enhances crop yields. Unlike other Trichoderma spp., there are two well-defined strains (P and Q) of T. virens, classified by secondary metabolites profiling, primarily the biosynthesis of the nonribosomal, strong antimicrobial agents gliotoxin (Q) and gliovirin (P). We have studied the phenotypic and biocontrol properties of two well-studied representative isolates (T. virens Gv29-8 and T. virens GvW/IMI304061) that represent a Q strain and a P strain of T. virens, respectively. We refined the genome assembly of the P strain using nanopore technology, and we compared it with the Q strain. The differences between the genomes include gene expansion in the Q strain. T. virens Gv29-8 is weaker than GvW as a mycoparasite on the broad host-range plant pathogen Sclerotium rolfsii, and it is ineffective as a biocontrol agent when applied to pathogen-infested soil. T. virens Gv29-8 proved to be phytotoxic to Arabidopsis seedlings, whereas the effect of T. virens GvW was not major. Both strains colonized the surface and outer cortex layer of tomato roots, with about 40% higher colonization by T. virens Gv29-8. T. virens Gv29-8 induced the expression of a larger set of tomato genes than did T. virens GvW, although some tomato genes were uniquely induced in response to T. virens GvW. We studied the comparative transcriptome response of T. virens Gv29-8 and T. virens GvW to S. rolfsii. A larger set of genes was regulated in T. virens GvW than in T. virens Gv29-8 in the presence of the plant pathogen. IMPORTANCE 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. This study enriches our knowledge of Trichoderma-plant-pathogen interactions and identifies novel candidate genes for further research and deployment in agriculture.

Genetic Evidence in Favor of a Polyketide Origin of Acremeremophilanes, the Fungal "Sesquiterpene" Metabolites.

Eremophilanes are a large group of "sesquiterpenes" produced by plants and fungi, with more than 180 compounds being known in fungi alone. Many of these compounds are phytotoxic, antimicrobial, anticancer and immunomodulators, and hence are of great economic values. Acremeremophilanes A to O have earlier been reported in a marine isolate of Acremonium sp. We report here the presence of Acremeremophilane I, G, K, N, and O, in a plant beneficial fungus Trichoderma virens, in a strain-specific manner. We also describe a novel, P strain-specific polyketide synthase (PKS) gene cluster in T. virens. This gene cluster, designated amm cluster, is absent in the genome of a Q strain of T. virens, and in other Trichoderma spp.; instead, a near identical cluster is present in the genome of the toxic mold Stachybotrys chartarum. Using gene knockout, we provide evidence that acremeremophilanes are biosynthesized via a polyketide route, and not via the mevalonate/terpene synthesis route as believed. We propose here that the 10-carbon skeleton is a product of polyketide synthase, to which a five-carbon isoprene unit is added by a prenyl transferase (PT), a gene for which is present next to the PKS gene in the genome. Based on this evidence, we propose that at least some of the eremophilanes classified in literature as sesquiterpenes (catalyzed by terpene cyclase) are actually meroterpenes (catalyzed by PKSs and PTs), and that the core moiety is not a sesquiterpene, but a hybrid polyketide/isoprene unit. IMPORTANCE 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.

Gliotoxin, an Immunosuppressive Fungal Metabolite, Primes Plant Immunity: Evidence from Trichoderma virens-Tomato Interaction.

Beneficial interaction of members of the fungal genus Trichoderma with plant roots primes the plant immune system, promoting systemic resistance to pathogen infection. Some strains of Trichoderma virens produce gliotoxin, a fungal epidithiodioxopiperazine (ETP)-type secondary metabolite that is toxic to animal cells. It induces apoptosis, prevents NF-κB activation via the inhibition of the proteasome, and has immunosuppressive properties. Gliotoxin is known to be involved in the antagonism of rhizosphere microorganisms. To investigate whether this metabolite has a role in the interaction of Trichoderma with plant roots, we compared gliotoxin-producing and nonproducing T. virens strains. Both colonize the root surface and outer layers, but they have differential effects on root growth and architecture. The responses of tomato plants to a pathogen challenge were followed at several levels: lesion development, levels of ethylene, and reactive oxygen species. The transcriptomic signature of the shoot tissue in response to root interaction with producing and nonproducing T. virens strains was monitored. Gliotoxin producers provided stronger protection against foliar pathogens, compared to nonproducing strains. This was reflected in the transcriptomic signature, which showed the induction of defense-related genes. Two markers of plant defense response, PR1 and Pti-5, were differentially induced in response to pure gliotoxin. Gliotoxin thus acts as a microbial signal, which the plant immune system recognizes, directly or indirectly, to promote a defense response. IMPORTANCE A single fungal metabolite induces far-reaching transcriptomic reprogramming in the plant, priming immune responses and defense, in contrast to its immunosuppressive effect on animal cells. While the negative effects of gliotoxin-producing Trichoderma strains on growth may be observed only under a particular set of laboratory conditions, gliotoxin-linked molecular patterns, including the potential for limited cell death, could strongly prime plant defense, even in mature soil-grown plants in which the same Trichoderma strain promotes growth.

Gamma-induced mutants of Bacillus and Streptomyces display enhanced antagonistic activities and suppression of the root rot and wilt diseases in pulses.

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.

Dual role of a dedicated GAPDH in the biosynthesis of volatile and non-volatile metabolites- novel insights into the regulation of secondary metabolism in Trichoderma virens.

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.

Structure-function analysis reveals Trichoderma virens Tsp1 to be a novel fungal effector protein modulating plant defence.

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 Å resolutions, from two crystal forms, using Se-SAD methods. The Tsp1 adopts a ß 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.

Target highlights in CASP14: Analysis of models by structure providers.

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.

Production, stability and degradation of Trichoderma gliotoxin in growth medium, irrigation water and agricultural soil.

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.

Trichoderma virens Bys1 may competitively inhibit its own effector protein Alt a 1 to stabilize the symbiotic relationship with plant-evidence from docking and simulation studies.

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.

Deletion of the Trichoderma virens NRPS, Tex7, induces accumulation of the anti-cancer compound heptelidic acid.

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.

Expression of a heptelidic acid-insensitive recombinant GAPDH from Trichoderma virens, and its biochemical and biophysical characterization.

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.

Expression, purification, crystallization and X-ray diffraction studies of a novel root-induced secreted protein from Trichoderma virens.

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 ß-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 Šresolution and belonged to the orthorhombic space group P212121 (Rmeas = 5.4%), with unit-cell parameters a = 46.3, b = 67.0, c = 173.2 Å. The Matthews coefficient (VM) 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.

Whole Genome Sequencing Reveals Major Deletions in the Genome of M7, a Gamma Ray-Induced Mutant of Trichoderma virens That Is Repressed in Conidiation, Secondary Metabolism, and Mycoparasitism.

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.

A Novel Seed-Dressing Formulation Based on an Improved Mutant Strain of Trichoderma virens, and Its Field Evaluation.

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.

Differential expression analysis of Trichoderma virens RNA reveals a dynamic transcriptome during colonization of Zea mays roots.

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.

Regulation of conidiation and antagonistic properties of the soil-borne plant beneficial fungus Trichoderma virens by a novel proline-, glycine-, tyrosine-rich protein and a GPI-anchored cell wall protein.

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.

A dedicated glyceraldehyde-3-phosphate dehydrogenase is involved in the biosynthesis of volatile sesquiterpenes in Trichoderma virens-evidence for the role of a fungal GAPDH in secondary metabolism.

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.

Ferricrocin, the intracellular siderophore of Trichoderma virens, is involved in growth, conidiation, gliotoxin biosynthesis and induction of systemic resistance in maize.

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.

Novel Trichoderma strains isolated from tree barks as potential biocontrol agents and biofertilizers for direct seeded rice.

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.