Genetic transformation and mutagenesis of the entomopathogenic fungus Paecilomyces fumosoroseus

We have developed a DNA-mediated transformation system for the entomopathog enic fungus Paecilomyces fumosoroseus based on resistance to the herbicide glufosinate ammonium. The resistance is provided by the bar gene from Strep tomyces hygroscopicus and is under the control of the Aspergillus nidulans trpC promoter and terminator sequences. Frequencies of up to 110 transforma nts/mu g of linearized plasmid DNA were obtained aided by the addition of t he nuclease inhibitor, aurintricarboxylic acid (ATA). The transformants wer e stable for drug resistance for five consecutive vegetative transfers unde r selective and nonselective conditions. Southern analyses revealed that th e transforming DNA integrated into the P. fumosoroseus genome in single and multiple copies. Single-copy integration events were generated with higher efficiency by restriction enzyme-mediated integration (REMI), although rat es of transformation were generally not as high as those treatments contain ing ATA. Two mutant strains altered in sporulation capacity and virulence t oward the Russian wheat aphid were recovered using this approach. This tran sformation-based manipulation of P. fumosoroseus will facilitate insertiona l mutagenesis and the functional analysis of various genes.