The filamentous fungus, Isaria fumosorosea, is a promising insect biological control agent. Chitinases have been implicated in targeting insect cuticle structures, with biotechnological potential in insect and fungal control. The I. fumosorosea chitinase gene, Ifchit1, was isolated and determined to encode a polypeptide of 423 amino acids (46 kDa, pI = 6.53), present as a single copy in the I. fumosorosea genome. A split marker transformation system was developed and used to construct an Ifchit1 gene knockout. The Delta Ifchit1 strain displayed minor alterations in mycelial growth on diverse media at 26 A degrees C compared to the wild type and complemented (Delta Ifchit1::Ifchit1) strains; however, colony morphology was affected, and the mutant strain had a temperature sensitive phenotype (32 A degrees C). Although sporulation was delayed for the mutant, overall conidial production was almost twice than that of wild type. Biochemical assays indicated decreased chitinase activity during growth in Czapek-Dox liquid media for the Delta Ifchit1 strain. Insect bioassays using diamondback moth, Plutella xylostella, larvae revealed decreased infectivity, i.e., increased LC (50) (threefold to fourfold) and a significantly delayed time to death, LT (50) from 3 to 6 days, for the Delta Ifchit1 strain compared to the wild type and complemented strains. These data indicate an important role for the Ifchit1 chitinase as a virulence factor in I. fumosorosea.