Gliovirin is a strong anti‐oomycete and a candidate anticancer compound. It is produced by “P” strains of the plant disease biocontrol fungus Trichoderma virens and is involved in biological control of certain plant pathogens. Even though the compound is known for more than three decades, neither the genes involved nor the biosynthetic pathway are known. We have sequenced the whole genome of a gliovirin producing strain of T. virens and discovered a novel gene cluster comprising of 22 genes. Disruption of the non‐ribosomal peptide synthetase eliminated biosynthesis of gliovirin. The gene cluster is very similar to a hitherto un‐described gene cluster of Aspergillus udagawae, a human pathogen. Our findings open‐up the possibility of strain improvement of T. virens for improved biocontrol of plant diseases through enhanced production of gliovirin. Research also can now be initiated on the role of this gene cluster in pathogenicity of the human pathogen A. udagawae.

A transcriptional comparison of wild type and a secondary metabolite deficient Trichoderma virens mutant resulted in the identification of six genes similar to those involved in secondary metabolism in other fungi, including four cytochrome P450 genes, one O-methyl transferase and one terpene cylase. Four of the genes (three cytochrome P450s and the cyclase) are located as a cluster. Transcript levels of three of the P450 genes, the O-methyl transferase and the terpene cyclase were measured. These genes are underexpressed in the mutant, which lacks the major secondary metabolites produced by this strain, viridin and viridiol. Expression levels of clones from the differential library with similarity to fungal trehalose synthase and a hydrophobin were also underexpressed in the mutant, while a heat shock protein hsp98 homolog was not. Based on the gene expression pattern and associated secondary metabolite profile, along with similarity to other secondary metabolism pathways in related fungi, we predict that the cluster is associated with the production of a terpene. The terpene could be viridin. This is the first report on cloning of secondary metabolism related genes from T. virens, and of their organization in a cluster, in this biocontrol fungus.

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.

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We dedicate this paper in the memory of Late Dr. Charles R. Howell for his immense contribution to Trichoderma virens biology, and biocontrol. He had always been generous in providing metabolite standards used in our studies.Viridin group of furano-steroidal antibiotics are known to function as anti-fungal and anti-cancer agents, in addition to their roles as radio-and chemo-sensitizers. Discovered in 1945 as a metabolite of Trichoderma virens, viridins continue to receive significant attention of several synthetic chemists and clinicians as a very strong PI3 kinase inhibitor. However, till date, researchers have not been able to discover a single gene that is involved in viridin biosynthesis. In this study, we present the complete gene cluster for the biosynthesis of viridin in T. virens, and provide genetic evidence of its involvement in viridin formation. Also, we show that the same cluster is present in a distantly related fungus Pseudogymnoascus destructans that causes bat white-nose disease, which is leading to the devastation of the bat population in North America. Our findings, thus, pave not only the way for further research on the elucidation of the complete biosynthesis pathway and possible tuning of viridin production in the plant beneficial fungus Trichoderma virens, but also are expected to trigger investigations on the role of this gene cluster in pathogenicity of the devastating bat pathogen.

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