Isolation and characterization of conditional lethal mutants of Beauveria bassiana

Abstract

Temperature-sensitive mutants of Beauveria bassiana GK2016 were isolated and characterized. Heat-sensitive mutants that grew at 20 °C but not at 30 °C were generated using mutagenesis with ultraviolet light and several rounds of selection. After 2160 colonies from a heat-sensitive mutant enriched population were screened, 11 heat-sensitive strains were isolated for further study. Five mutant strains, HS1, HS2, HS6, HS9, and HS11, were stable and closely resembled the wild-type strain with respect to morphology, growth rate, and enzyme synthesis at 20 °C. Characterization of macromolecular synthesis at 30 °C using a radiolabelled precursor uptake assay indicated that three mutants, HS6, HS9, and HS11, had reduced levels of DNA, RNA, and protein synthesis at the nonpermissive temperature. Mutants affected in cell division and cell wall synthesis were characterized by microscopy. At 30 °C, mutant HS6 was defective in cellular compartmentalization and formed long, branched, aseptate mycelia that fragmented easily. Mutant HS1 was defective in cell wall biosynthesis and at 30 °C ceased to maintain cell wall integrity and lysed. Mutants HS2 and HS9 possessed temperature-sensitive lesions that could not be specified. None of the mutants were directly affected in either nuclear division or mitosis as evidenced by the accumulation of Giemsa-stainable mitotic nuclei at 30 °C. Bioassays conducted with grasshoppers showed the efficacy of the mutant strains, with the exception of HS11, to be comparable to that of the parent strain, at 20 °C. Conversely, at 32 °C the mutant strains were uninfective whereas the parental strain GK2016 was infective. It is our intention to use these strains to examine fundamental aspects of entomopathology by dissecting fungal growth and development in vitro and extending these observations to pathogenesis in insects.Key words: Beauveria bassiana, temperature-sensitive, mutants, characterization, infection.