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

Abstract

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.

Keywords: Trichoderma; chickpea; farmers’ fields; formulation; lentil; mutant; tamarind seeds.

FIGURE 1

Colony morphology of wild-type and mutant strain of Trichoderma virens, after growth on potato dextrose agar for 5 days.

FIGURE 2

Inhibition of Pythium aphanidermatum by culture filtrate of WT and G2.

FIGURE 3

Confrontation assay between Sclerotium rolfsii and Trichoderma virens mutant/wild-type strain.

FIGURE 4

Parasitism of sclerotia of Sclerotium rolfsii by wild type and mutant of Trichoderma virens. (A) Sclerotia colonized after 5 days of co-incubation of Trichoderma and sclerotia of S. rolfsii. (B) Growth of S. rolfsii from sclerotia after 3 days of incubation on benomyl-amended PDA medium.

FIGURE 5

HPLC analysis of culture filtrate of WT and G2.

FIGURE 6

Genes upregulated in T. virens mutant G2, over wild type, when grown on PDA for 3 days. Hypo: hypothetical/unknown proteins; SSCP: small, cysteine-rich secreted proteins; OXIDO: oxidoreductases; GH: glycosyl hydrolases; SEC. META.: gene predicted to be involved in secondary metabolism (includes PKS, P450, NRPS-like, OMT-B, and MFS transporters); HYD: hydrophobins; PEP: peptidases.

FIGURE 7

Growth of Trichoderma virens wild type and mutant on tamarind seeds, 7 days after inoculation.

FIGURE 8

Formulated product of Trichoderma virens wild type and mutant.

FIGURE 9

Colony-forming units of Trichoderma virens on PDA-rose bengal medium.

FIGURE 10

Seeds treated with wild type and G2 formulations.

FIGURE 11

Pot assay for biocontrol of Sclerotium rolfsii in chickpea treated with wild type or mutant, in the presence and absence of the pathogen. Treatments with same letters (case-sensitive) are not significantly different (P < 0.05).

FIGURE 12

Demonstration trial on control of collar rot of chickpea in experimental field at Raipur (2015–2016).

FIGURE 13

Yield data of farmers’ field trials taken at 30 farmers’ fields in Nadia district of West Bengal (2017–2018).

FIGURE 14

Yield data of farmers’ field trials taken at 30 farmers’ fields in Nadia district of West Bengal (2018–2019).