A novel hexa-segmented dsRNA mycovirus confers hypovirulence in the phytopathogenic fungus Diaporthe pseudophoenicicola. Environmental microbiology A novel hexa-segmented double-stranded RNA (dsRNA) mycovirus was isolated and characterized from the filamentous phytopathogenic fungus Diaporthe pseudophoenicicola and was named Diaporthe pseudophoenicicola chrysovirus 1 (DpCV1). The full-length cDNAs of dsRNA1-6 were 3335, 3030, 3039, 2980, 963, and 780 bp, respectively. Sequence analysis indicated the presence of nine open reading frames (ORFs) in the DpCV1 genome. ORF1 in dsRNA1 putatively encoded the RNA-dependent RNA polymerase (RdRp) and ORF3 in dsRNA2 encoded a capsid protein (CP). The seven remaining ORFs, ORF2 in dsRNA2, ORF4 in dsRNA3, ORF6, seven in dsRNA4, ORF8 in dsRNA5, and ORF9 in dsRNA6, encoded proteins with unknown functions. Phylogenetic analysis revealed that DpCV1 is closely related to members of the cluster I group within the family Chrysoviridae but formed a separate clade. Importantly, all the six segments of DpCV1 were cured successfully through single spore isolation to obtain the isogenic virus-free strains. DpCV1 can confer hypovirulence to the fungal host of Diaporthe pseudophoenicicola. Compared with the virus-free strain, WC02 harbouring the DpCV1 is more sensitive to fungicide prochloraz. Furthermore, the cell wall of DpCV1 infected strain was loose and enlarged. This is the first report of a hexa-segmented tentative chrysovirus in D. pseudophoenicicola. 10.1111/1462-2920.15963