Phenotypic and pathogenicity changes in Beauveria and Metarhizium isolates induced by serial subculturing and passage...

Entomopathogenic fungi are widely used as biological control agents, but repeated in vitro subculturing can lead to degenerative changes affecting their efficacy. This study investigated the impact of serial subculturing on conidial size, production, germination, thermotolerance and virulence. We used G. mellonella larvae as a model host and evaluated four fungal isolates: Beauveria bassiana (Bb88), B. pseudobassiana (Bp30), Metarhizium anisopliae (Ma129) and M. pingshaense (Mp140). Conidia were from early (Sc1), intermediate (Sc5) or late (Sc10) subcultures. Results demonstrated isolate-specific variation in conidial size with increasing number of subcultures: Bb88 exhibited no variation; Bp30 showed an increase in size with increased subculture; Mp140 and Ma129 exhibited a decrease in size with increased subculture. Conidia production declined progressively with subculturing in Bb88, Bp30 and Mp140, while conidia yield of Ma129 remained stable. In contrast, germination, thermotolerance and virulence against G. mellonella were not significantly affected across all isolates and subcultures. To determine whether degenerative traits could be restored, subcultured isolates were passaged through alternative insect hosts (Plutella xylostella, Galleria mellonella, Anthonomus eugenii and Phyllophaga sp). Host passage partially restored conidial production in Bb88 and Mp140, but not in isolate Bp30, except when recovered from P. xyllostella. While germination rates remained unchanged, host passage significantly affected conidial thermotolerance, especially in isolate Mp140. These findings highlight the necessity to monitor and manage subculturing practices and suggest that periodic host passage may sustain the biological control potential of entomopathogenic fungi.