ABSTRACT Rosellinia necatrix mycoreovirus 3 (W370) (RnMYRV-3/W370, described as RnMYRV-3 in this paper), a member of the newly established genus Mycoreovirus within the family Reoviridae, is the hypovirulence factor of the white root rot fungus, Rosellinia necatrix. Two virus-free fungal isolates (W37 and W97) that were somatically incompatible with the virus-harboring field isolate (W370) were transfected with purified RnMYRV-3 particles. Virus infection was confirmed by electrophoresis and northern hybridization of viral double-stranded RNA. RnMYRV-3 was transmissible from transfected strains to their respective, virus-free counterparts via hyphal anastomosis. Virus-transfected strains produced smaller lesions on apple fruits than did their virus-free counterparts. Virus-cured strains were indistinguishable from wild-type strains in culture morphology and displayed approximately the same virulence level on apples. Virus-transfected strains had "mosaic" colony portions consisting of thin, fast-growing and dense, slow-growing mycelia, and grew more slowly as a whole than their virus-free, parental strains. The level of virus accumulation varied among virus-transfected subcultures and within its single colonies. Virus-transfected strains were occasionally cured, as was W370. Such a phenomenon may be ascribed to uneven viral distribution in single colonies and the difficulty in viral transmission to virus-free hyphae.