B-glucosidase from trichoderma harzianum T12 as green fungicide against Macrophomina phaseolina in soybean (Glycine Max L.)

Abstract

Macrophomina phaseolina (Tassi) Goid remains the prevailing causal agent of charcoal rot disease that can significantly suppress yields of a variety of crops. Its wide host range and survivability under arid conditions as well as the ineffectiveness of fungicides have spurred scientific endeavors in search of alternative avenues to control this phytopathogen. The present study is aimed to provide empirical evidence on the efficacy of β-glucosidase from Trichoderma harzianum T12 as a biological control agent against M. phaseolina. In-vitro pathogenicity tests on 60 isolates of M. phaseolina and 30 isolates of T. harzianum, collected from different areas of the Mazandaran province in Iran revealed the isolates, M2 of M. phaseolina and the T12 of T. harzianum were the most virulent and effective in inhibiting growth of M. phaseolina, respectively. The present study showed that biochemical and phylogenetic analyses and BIOLOG results confirmed the fungal antagonists and phytopathogen were T. harzianum (Rifai) and M. phaseolina (Tassi) Goid, respectively. Purified extracellular β-glucosidase of T. harzianum inhibited the growth of M. phaseolina as seen from the large halo zones, indicating its possible application as a green fungicide against M. phaseolina. The β-glucosidase had an optimum pH (7) and temperature 45˚C, respectively, remarkably stable up to 240 min with a half-live of t1/2 = 210 min at 40 ˚C to 60 ˚C. Zn2+, Mn2+, and Tween 80 enhanced its activity while was substantially inhibited by Fe3+. Enzyme activity was the highest when wheat bran and (NH4)2SO4 were used as carbon and nitrogen sources respectively. The kinetic parameters for β-glucosidase T12, Km, Vmax and kcat were estimated as 0.79 mM, 8.45 mM min-1 mg-1 protein and 10.69 s-1, respectively, to give a turnover number of 10.69 s-1. Optimization by the Box-Behnken Design (BBD) based on: temperature, carbon sources, inoculum size and pH (7), exhibited the highest β-glucosidase activity (1260 U/mL) at 45˚C, pH 7, using a carbon source 10 % (w/v) and inoculum size of 5 % (w/v). The BBD optimization for the application of the β-glucosidase formulation from T. harzianum to control infestation of M. phaseolina M2 was carried out on soybean plants grown under a greenhouse condition. Under an optimized condition, the lowest plant disease index (PDI) of 4.32% (R2 = 0.9676) was attained using 10 mM Zn2+, Tween 80 at 2 % (w/v) an enzyme concentration at 15 mg/L and an irrigation frequency of 2 times/week. A comparative study showed the developed formulation gave the lowest PDI (4.14 %) (p < 0.05) followed by the antagonist T. harzianum Rifai (26.13 %) and the commercial fungicide, Carbendazim (32.45 %). The assessments cost revealed that the enzyme formulation only costs at USD34/acre as compared to Carbendazim at USD240/acre. Hence, the findings affirmed that the novel use of crude β-glucosidase from the growth supernatant of T. harzianum was efficient in combating charcoal rot disease. Since the enzyme formulation was substantially cheaper and its application combines the practicality of an in-situ spraying for rapid control of M. phaseolina infestation, the technique proposed here was prospectively feasible to control such disease in crops.