Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens

Abstract

A simple, highly efficient, and reliable Agrobacterium tumefaciens-mediated transformation method was developed for the insect pathogenic fungus Metarhizium anisopliae. Expression of the green fluorescent protein gene, egfp, and the benomyl resistance gene, benA3, were used as markers in transformed M. anisopliae. Transformation efficiencies were dependent on the strain of A. tumefaciens used. With strain AGL-1, 17.0 ± 1.4 transformants per plate could be obtained using conidial concentrations of 106 conidia/mL and a 2 day co-cultivation in the presence of 200 µmol/L acetosyringone. On the other hand, transformations using strain LBA4404 were unsuccessful. Ten transformants were tested by Southern analysis and found to contain a single copy T-DNA. Twenty transformants were subcultured for five generations on nonselective media, and 95% of the transformants were mitotically stable. Agrobacterium tumefaciens-mediated transformation of M. anisopliae can serve as a useful tool to investigate genes involved in insect pathogenicity.Key words: entomopathogenic fungi, Metarhizium anisopliae, Agrobacterium tumefaciens, genetic transformation.