Isolation, identification, and pathogenicity of three fungal species from silkworms infected with white muscardine disease

Abstract

White muscardine disease, caused by entomopathogenic fungi, is one of the most destructive infections in silkworms (Bombyx mori), leading to significant economic losses in sericulture. In this study, we obtained fungal isolates from diseased larvae showing characteristic white mycelial growth on the body surface. Based on colony morphology and internal transcribed spacer rDNA sequence analysis, the isolates were identified as Beauveria bassiana (KN-White), Penicillium copticola (KN-Green), and Mucor variicolumellatus (KN-Gray). Phylogenetic reconstruction using the Neighbor-Joining method (Tamura–Nei model, 1,000 bootstrap replicates) supported a close relationship between KN-White and B. bassiana ARSEF 1564, confirming its taxonomic position within the B. bassiana species complex. Pathogenicity assays, conducted by spraying fifth-instar larvae with conidial suspensions of each isolate, revealed that KN-White caused rapid mortality in a dose-dependent manner, with larval survival dropping below 20% within six days at the highest dose, whereas KN-Green and KN-Gray were non-pathogenic. Infected larvae exhibited progressive cuticle whitening and hardening, forming typical “white muscardine” cadavers. These findings demonstrate that KN-White is a virulent B. bassiana strain responsible for white muscardine disease in silkworms, whereas KN-Green and KN-Gray are saprophytic fungi associated with the rearing environment. Our results provide molecular and pathological evidence for the identification of B. bassiana as a major pathogen in sericulture and provide insights to develop enhanced fungal disease management strategies.