Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum

Aims: To examine the ability of Agrobacterium to attach to Metarhizium anisopliae var. acridum strain CG423 under co-cultivation and to develop an Agrobacterium-mediated method of gene delivery into strain CG423, a promising agent for biological control of grasshoppers. Methods and Results: The co-cultivation of Agrobacterium tumefaciens and M. anisopliae var. acridum was analysed under scanning electron microscopy. We observed that Agrobacterium attached to and formed aggregates around Metarhizium conidia and germ tubes. We also observed the occurrence of fibril-like structures connecting neighbouring bacterial-fungal cells. The Agrobacterium-mediated transformation was applied using two binary vectors carrying a benomyl resistance gene as a selection marker. The efficiency of transformation was up to 53 transformants per 10(5) target conidia. High mitotic stability of the transformants (89-97%) was demonstrated after five successive transfers on non-selective media. Molecular analysis revealed the occurrence of high frequency of gene conversion. Conclusions: In our study, we report that A. tumefaciens strain AGL-1 attaches to and genetically transforms the entomopathogenic fungus Metarhizium anisopliae var. acridum. Significance and Impact of the Study: We report for the first time, the attachment of Agrobacterium to fungal cells opening new avenues for the study of this essential step of the T-DNA transfer process. Considering the efficiency of the transformation protocol herein described, this is a useful tool for gene disruption in M. anisopliae var. acridum.