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To produce more thermotolerable conidia of Beauveria bassiana, a well-known fungal biocontrol agent, by optimizing the medium components and culture conditions.The conidia produced on media including 0·5–6% glucose, sucrose or starch as carbon source and 50–300-μg ml−1 Cu2+, Zn2+, Mn2+ or Fe3+ as additive to Sabouraud dextrose medium at 15–30°C, pH 4–8 or KCl-adjusted water availabilities were exposed to 30-min wet heat stress at 48°C. The medium components for conidial production with greatly enhanced thermotolerance included 4% glucose as optimum or 1% starch as alternative for the carbon source and ≤50-μg ml−1 Mn2+ for the metal additive. The culture conditions were optimized as 25°C and pH 5–6. Conidial thermotolerance decreased remarkably when sucrose and Fe3+ or Cu2+ were used in the cultures, but altered slightly when 50–200-μg ml−1 Zn2+ were included.The tolerance of B. bassiana conidia to the thermal stress was significantly affected by the medium composition and culture conditions under which the conidia were produced.Proper treatment of small grains as mass production substrates for more glucose release and supplement of glucose or ≤50-μg ml−1 Mn2+ are possible means to enhancing conidial thermotolerance and field persistence for improved insect control.