Abstract
Cotton verticillium wilt caused by Verticillium dahliae severely restricts global cotton production. Mycovirus–induced hypovirulence provides a promising strategy for sustainable disease management. In this study, V. dahliae isolates from Xinjiang were screened by virome sequencing and RT–PCR, and strain 121–11C–1 co–infected with Verticillium dahliae partitivirus 1 (VdPV1) and Verticillium dahliae magoulivirus 1 (VdMoV1) was identified. Virus–free isogenic strains were obtained via single–conidium purification, verifying that both viruses can be vertically transmitted through fungal conidia. Dual–culture assays uncovered a novel, previously unreported horizontal transmission dependency between the two viruses: VdPV1 achieves horizontal transmission only with the assistance of VdMoV1, whereas VdMoV1 can transmit independently or co–transmit with VdPV1. Biological and pathogenicity assays demonstrated that both viruses significantly inhibit mycelial growth, reduce conidial production and attenuate fungal virulence. Notably, dual infection induced a significantly stronger hypovirulent phenotype than single infection. These findings elucidate the transmission dynamics of VdPV1 and VdMoV1, expand the repertoire of hypovirulent mycoviruses in V. dahliae, and provide evidence that viral co–infection enhances fungal attenuation, offering potential applications for biological control of cotton Verticillium wilt.