A new method for producing hybrid strains of the entomopathogenic fungus verticillium lecanii (lecanicillium spp.) through protoplast fusion by using nitrate non-utilizing (nit) mutants

Mycotal and Vertalec are mass-produced fungal strains for insect control. Strain B-2, which was isolated in Japan, has high epiphytic ability on cucumber leaves. Protoplast fusion was performed using these strains of Verticillium lecanii to obtain new strains possessing useful characteristics as biological control agents (BCAs). We used nit mutants for visually selecting the protoplasts. Hybrid strains were subjected to molecular analysis using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) and arbitrarily primed-PCR (AP-PCR) in order to determine protoplast fusion and/or genetic recombination. We detected 126, 44, and 4 hybrid strains from the combinations of Vertalec × Mycotal, B-2 × Mycotal, and B-2 × Vertalec, respectively. Morphological characteristics of hybrid strains differed from those of their parental nit mutants. Protoplast fusion of hybrid strains was confifi rmed in genomic DNA, but not in mitochondrial DNA (mtDNA). A uniform biased tendency of the DNA banding pattern was observed depending on the combination of parental strains. The molecular analysis also revealed genetic recombination. These results showed a novel method for producing hybrid strains of the entomopathogenic fungus V. lecanii.