title: Influence of protoplast fusion between twoTrichodermaspp. on extracellular enzymes production and antagonistic activity.
abstract: Biological control plays a crucial role in grapevine pathogens disease management. The cell-wall degrading enzymes chitinase, cellulase and β-glucanase have been suggested to be essential for the mycoparasitism activity ofTrichodermaspecies against grapevine fungal pathogens. In order to develop a useful strain as a single source of these vital enzymes, it was intended to incorporate the characteristics of two parental fungicides tolerant mutants ofTrichodermabelonging to the high chitinase producing speciesT. harzianumand the high cellulase producing speciesT. viride, by fusing their protoplasts. The phylogeny of the parental strains was carried out using a sequence of the 5.8S-ITS region. The BLAST of the obtained sequence identified these isolates asT. harzianumandT. viride. Protoplasts were isolated using lysing enzymes and were fused using polyethylene glycol. The fused protoplasts have been regenerated on protoplast regeneration minimal medium supplemented with two selective fungicides. Among the 40 fast growing fusants, 17 fusants were selected based on their enhanced growth on selective media for further studies. The fusant strains were growing 60%-70% faster than the parents up to third generation. All the 17 selected fusants exhibited morphological variations. Some fusant strains displayed threefold increased chitinase enzyme activity and twofold increase in β-glucanase enzyme activity compared to the parent strains. Most fusants showed powerful antagonistic activity againstMacrophomin aphaseolina,Pythium ultimumandSclerotium rolfsiipathogens. Fusant number 15 showed the highest inhibition percentage (92.8%) againstM. phaseolinaandP. ultimum,while fusant number 9 showed the highest inhibition percentage (98.2%) against the growth ofS. rolfsii.A hyphal intertwining and degradation phenomenon was observed by scanning electron microscope. TheTrichodermaantagonistic effect against pathogenic fungal mycelia was due to the mycoparasitism effect of the extracellular enzymes.
authors.affiliation: Scientific Research Center, Biotechnology and Genetic Engineering Unit, Taif University , Taif , KSA ; Genetics Department, Faculty of Agriculture, Minufiya University , Shebin El-Kom , Egypt.
authors.forename: Mohamed M
authors.initials: MM
authors.lastname: Hassan
keywords: Trichoderma; antagonistic activity; chitinase; grapevine pathogens; protease; protoplast fusion; β-glucanase