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Phomopsis viticola is easily transformed with hph and Bml sup (r) genes Vitis vinifera L.
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Journal Article
Phomopsis viticola is easily transformed with hph and Bml sup (r) genes Vitis vinifera L.
2003
Overview
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 with plasmids carrying either the bacterial hph gene, conferring resistance to hygromycin B (pAN7-1, pOHT, pOHT-AMA1), or the Bml sup (r) gene from Neurospora crassa, causing resistance to benzimidazole fungicides (pBT6). Up to more than 300 transformants per microng of plasmid DNA were obtained with the hph marker gene. The highest effectiveness was obtained with pOHT, whereas pBT6 yielded around 25 transformants per microng 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
[Phomopsis viticola (Sacc.) Sacc. e' il fungo fitopatogeno agente di una malattia grave della vite denominata escoriosi. Protoplasmi derivanti dal micelio di P. viticola sono stati trasformati con plasmidi portanti sia il gene batterico hph, che conferisce la resistenza all'igromicina B (pAN7-1, pOHT, pOHT-AMA1), sia il gene Bml sup (r) di Neurospora crassa, che causa la resistenza ai fungicidi benzimidazolici (pBT6). Con il gene marcatore hph sono stati ottenuti fino a piu' di 300 trasformanti per microng di DNA plasmidiale. La maggiore efficienza e' stata ottenuta con pOHT, mentre pBT6 produceva circa 25 trasformanti per microng di DNA plasmidiale. L'analisi Southern blot ha evidenziato il verificarsi di eventi di integrazione multipla di tutti i plasmidi sperimentati nel genoma del fungo. Esperimenti di co-trasformazione con pOHT e pBT6 hanno avuto successo e circa il 70% dei trasformanti e' risultato resistente sia all'igromicina B, sia al benomyl. Sono state provate la tecnica dell'Instant Gene Bank e la mutagenesi attraverso l'integrazione mediata da enzimi di restrizione (REMI). Come riportato per altri funghi fitopatogeni, la tecnica REMI si e' dimostrata un metodo efficace per ottenere ceppi mutanti con variazione dei caratteri fenotipici]
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