Temperature extremes are an important adverse factor limiting the effectiveness of microbial pest control agents. They reduce virulence and persistence in the plant root-colonizing insect pathogen Metarhizium robertsii. Small heat shock proteins have been shown to confer thermotolerance in many organisms. In this study, we report on the cloning and characterization of a small heat shock protein gene hsp25 from M. robertsii. hsp25 expression was upregulated when the fungus was grown at extreme temperatures (4, 35, and 42 A degrees C) or in the presence of oxidative or osmotic agents. Expression of hsp25 in Escherichia coli increased bacterial thermotolerance confirming that hsp25 encodes a functional heat shock protein. Overexpressing hsp25 in M. robertsii increased fungal growth under heat stress either in nutrient-rich medium or on locust wings and enhanced the tolerance of heat shock-treated conidia to osmotic stress. In addition, overexpression of hsp25 increased the persistence of M. robertsii in rhizospheric soils in outdoor microcosms, though it did not affect survival in bulk soil, indicating that M. robertsii's survival in soil is dependent on interactions with plant roots.