Detection and characterisation of pr1 virulent gene deficiencies in the insect pathogenic fungus Metarhizium anisopliae.

In an initial attempt to characterise single-spore isolates from Metarhizium anisopliae wild-type strain V275, three genetically identical spontaneous pr1A- and pr1B-deficient mutants were detected. These mutants were identified by Nested-PCR amplification and verified by Southern hybridisation. Sequencing and comparison of the highly conserved sequences of 28S rDNA domains 9-11 confirmed that the mutant isolates were Metarhizium anisopliae, but each had both morphological and genetic differences from the wild-type parent. RAPD analyses indicated that the overall genetic similarity between wild-type strain and the mutant was less than 70%. Enzyme assays proved that the mutant isolates had significantly lower Pr1 and elastase activities. Bioassays showed reduced lethal activity of the mutants (20%) on Tenebrio molitor, but lethal activities were similar to those of the wild-type parent on the larvae of Galleria mellonella. This paper shows for the first time that a stable mutant strain lacking the most important pr1 genes is still able to infect its respective hosts. Our observations suggest that it is not uncommon for Metarhizium anisopliae to lose its virulence genes during maintenance on artificial medium.