PurposeTo establish an efficient transformation system of the nematopathogenic fungus Purpureocillium lilacinum and obtain its insertional mutagenesis.

MethodsThe benomyl resistance gene beta-tubulin being as the selective marker, Agrobacterium tumefaciens-mediated transformation technique was developed to screen different pathogenicity mutants in P. lilacinum. PCR amplification and Southern hybridization were used to verify the transformation events, and Southern blotting of beta-tubulin gene and cloning of transforming DNA (T-DNA) flanking sequences were used to determine insert number and site of T-DNA in the fungal genome, respectively.

ResultsA reliable transformation method was established for P. lilacinum. Specifically, pre-germinating spores of P. lilacinum used at co-cultivated period was a prerequisite．P. lilacinum germinating spores co-cultivated with A. tumefaciens EHA105 at 25 ℃ for 48 h achieved the highest transformation efficiency, which was 1 200-3 200 transformants per 10⁶ spores, and the ratio of positive resistant transformants was 96%. The transformants were cultivated up to 5 generations on beta-tubulin-containing medium and confirmed by PCR and those genetic traits remained stable. Southern hybridization showed that 83.3% of the transformants were single copy insertions of T-DNA, and 16 mutants with virulence variants were screened from 20 transformants.

Conclusion This study successfully constructed an efficient genetic transformation system mediated by A. tumefaciens with beta-tubulin gene as a selective marker, and obtained an insertion mutant with pathogenicity variation, which was P. lilacinum. It provides insights into studying gene function, pathogenic mechanism and breeding excellent strains.