Genetic enhancement of Trichoderma asperellum biocontrol potentials and carbendazim tolerance for chickpea dry root rot disease management

Abstract

Advances in biocontrol potentials and fungicide resistance are highly desirable for Trichoderma. Thus, it is profitable to use mutagenic agents to develop superior strains with enhanced biocontrol properties and fungicide tolerance in Trichoderma. This study investigates the N-methyl-n-nitro-N-nitrosoguanidine (NTG) (100 mg/L) induced mutants of Trichoderma asperellum. Six NTG (3 each from 1st & 2nd round) induced mutants were developed and evaluated their biocontrol activities and carbendazim tolerance. Among the mutant N2-3, N2-1, N1 and N2-2 gave the best antagonistic and volatile metabolite activities on inhibition of chickpea F. oxysporum f. sp. ciceri, B. cinerea and R. bataticola mycelium under in vitro condition. Mutant N2-2 (5626.40 μg/ml) showed the highest EC50 value against carbendazim followed by N2-3 (206.36 μg/ml) and N2-1 (16.41 μg/ml); and succeeded to sporulate even at 2000 μg/ml of carbendazim. The biocontrol activity of N2-2 and N2 with half-dose of carbendazim was evaluated on chickpea dry root rot under controlled environment. Disease reduction and progress of the dry root rot was extremely low in T7 (N2-2 + with half-dose of carbendazim) treatment. Further, carbendazim resistant mutants demonstrated mutation in tub2 gene of β-tubulin family which was suggested through the 37 and 183 residue changes in the superimposed protein structures encoded by tub2 gene in N2 and N2-2 with WT respectively. This study conclusively implies that the enhanced carbendazim tolerance in N2-2 mutant did not affect the mycoparasitism and plant growth activity of Trichoderma. These mutants were as good as the wild-type with respect to all inherent attributes.

Fig 1. Carbendazim tolerance in N-methyl-n-nitro-N-nitrosoguanidine irradiated Trichoderma mutants.

Mutants N2-1, N2-2, N2-3 is compared with N2 mutant at carbendazim 250, 500, 750 and 1000 μg/ml in PDA plates, and observation are recorded when the control plates cover the whole plate.

Fig 2. Carbendazim tolerance and dry mycelial productions in N-methyl-n-nitro-N-nitrosoguanidine irradiated Trichoderma mutants.

(a) The 1^st round NTG induced N1, N2, N3 mutants carbendazim tolerance at 0–120 μg/ml carbendazim amended PDA; (b) 2^st round NTG induced N2-1, N2-2, N2-3 mutants carbendazim tolerance at 0–1500 μg/ml carbendazim amended PDA; (c) dry mycelium production ability of N2-2, N2 and WT in 0–2000 μg/ml carbendazim amended potato dextrose broth.

Fig 3. Integrated management of chickpea dry root rot disease under glasshouse condition.

(a) Overview of the experiments at 44 days after sowing; (b) complete dry rot infected plants in T₁ (untreated) treatment; (c) partially dry rot infected plants in dry root rot in T₇ (N2-2+0.5 RD carbendazim) treatment at 60 days after sowing in JG 62 cultivar.

Fig 4. Ribbon representations of Trichoderma N2, N2-2 and WT strain tub2 protein.

Deduced amino acids structural superimposition between N2 and N2-2 with WT of tub2 gene differ by 37 and 183 mutant residues. In both the images—yellow (mutant protein), cyan (WT protein) and magenta (mutant residues).