Intra-strain crossing in trichoma harzianum via protoplast fusion to enhance chitinase productivity and biocontrol activity

ABSTRACT

Protoplasts were isolated from Trichoderma harzianum fungus using Novozyme 234 with 0.7 M KCl as osmotic stabilizer. Intra-strain T. harzianum protoplast fusion has been carried out using polyethylene glycol with STC [sorbitol, Tris - HCl, CaCl[2]] buffer. The fused protoplasts were regenerated on colloidal chitin agar selective medium. Eighteen self-fusant strains were selected to study the chitinase production and biocontrol activity. Most of the fusants exhibited fast and vigorous mycelial growth on 2% colloidal chitin agar compared to non-fusant and parent strains. High chitinase activity was measured in the culture filtrates of the self-fusant strains than the parent. Among the fusants, four [ATh1/9, ATh1/12, ATh1/14 and ATh1/17] produced maximum chitinase with a two - fold increase compared to the parent strain. Moreover, fusant AThl/7 produced 94.3% more chitinase activity than the original strain. Most self - fusant strains exhibited increased antagonistic activity against Cephalosporium acremonium, Aspergillus niger and Rhizoctonia solani than the parent strain. The crude chitinase preparations of fusants ATh1/9, ATh1/12, ATh1/14 and ATh1/17 lysed the mycelia of T. reesei, T. viride and A. niger and released the protoplasts in higher numbers than the crude chitinase preparation of parent strain. Results demonstrated the significance of the protoplast fusion approach, as a technique to develop superior hybrid strains of filamentous fungi lacking inherent sexual reproduction