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

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

UV-and-EMS-induced mutations affecting synthesis of alkaloids and lipase in penicillium roquefortii

Mutants of a Penicillium roquefortii strain were obtained by ultraviolet irradiation [UV] and ethylmethansulfonate [EMS]. Based on alkaloid production, the mutant strains could be divided into four groups: [1] unable to synthesize alkaloids, [2] with a non functional chain of clavin formation, [3] with roquefortine and 3, 12-dihydroroquefortine that were not found in the alkaloid fraction and [4] with new compounds, that were not shown in the wild type strain. These data indicate the presence of not less than three different pathways of alkaloids formation in Penicillium roquefortii. On the other hand, all selected mutants produced more lipase than the wild type strain, when using olive oil as a substrate; when cotton seed oil was used many mutant strains produced less lipase activity than the parent strain