PHOMOPSIS VITICOLA IS EASILY TRANSFORMED WITH hph AND Bmlr GENES

SUMMARY Phomopsis viticola (Sacc.) Sacc. is the phytopathogenic fungus causing a severe disease of grapevine known as Phomopsis cane and leaf spot. Protoplasts from mycelium of P. viticola were successfully transformed either with plasmids carrying the bacterial hph gene, conferring resistance to hygromycin B (pAN7-1, pOHT, pOHT-AMA1), or the Bml r gene from Neurospora crassa, causing resistance to benzimidazole fungicides (pBT6). Up to more than 300 transformants per μg of plasmid DNA were obtained with the hph marker gene. The highest effectiveness was obtained with pOHT, whereas pBT6 yielded around 25 transformants per μg of plasmid DNA. Southern blot analysis showed the occurrence of multiple integration events in the fungal genome of all tested plasmids. Experiments of co-transformation with pOHT and pBT6 were successful and about 70% of transformants were resistant to both hygromycin B and benomyl. The “Instant Gene Bank” technique and mutagenesis through Restriction Enzyme Mediated Integration (REMI) were attempted. As reported for other phytopathogenic fungi, the REMI technique proved to be a powerful method for obtaining mutant strains with variation in phenotypic traits.

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