Molecular Characterization of Double-Stranded (DS) RNAS in Chalara Elegans and Biological Effects on the Fungus

Molecular characterization of cDNA clones from double-stranded (ds) RNA in Chalara elegans was accomplished. The cDNA clones derived from a 5.3 kb dsRNA present in strains of C. elegans showed nucleotide sequence homology to viruses belonging to Totiviridae. The cDNA clones derived from a 2.8 kb dsRNA in strains BK18 and WASH revealed nucleotide sequence homology to the Mitovirus group. Two cDNA clones derived from a 12 kb dsRNA in strain NC1527 revealed no significant sequence homology to any known virus group. These results suggest the occurrence of at least three different virus groups in C. elegans. The genetic relatedness among dsRNAs in C. elegans strains was determined using Northern blot and cDNA sequence analysis. The clones derived from a 2.8 kb dsRNA cross-hybridized with 2.8 kb dsRNA occurring in C. elegans strains from diverse geographic regions worldwide. However, the cDNA clones derived from either a 5.3 kb or 12 kb dsRNA cross-hybridized only with the similar-sized dsRNAs, mainly in strains originating from same geographic regions. Sequence analysis of the full-length cDNA clone from the 2.8 kb showed the presence of one large open reading frame (OW), when the mitochondrial genetic code was used. The ORF 1 contained RdRp conserved motifs present in other mitochondrial RNA viruses. This dsRNA copurified with mitochondria and named Chalara elegans 18 virus (Ce 1 8V), a new mitovirus. A latently-infected strain (BKl8C) which was obtained by high temperature incubation of 2.8 kb dsRNA-containing strain (BK18) for 2-3 months showed enhanced pathogenicity on carrots, but no differences in culture morphology or various enzymatic assays was detected. A full-length cDNA clone of the 5.3 kb dsRNA in strain CKP was also obtained. Sequence analysis revealed the presence of three ORFs, which shared some homology either to the coat protein or RdRp regions of Totiviridae. Partial cDNA clones were obtained from additional dsRNA in strain CKP which shared some homology to the RdRp regions of Totivirus, suggesting coinfection by two distinctive totivirus-like dsRNAs in strain CKP of C. elegans. Results from this study demonstrate the presence of diverse novel dsRNA elements in C. elegans with differing biological effects.