Molecular and biological characterization of a mitovirus in Chalara elegans (Thielaviopsis basicola)

A 2.8-kb double-stranded RNA (dsRNA) element in strain BK18 of Chalara elegans originally isolated from cotton soil in California was characterized by obtaining a full-length cDNA sequence (2,896 nucleotides in length) from a series of overlapping clones. Sequence analysis revealed the presence of one large open reading frame (ORF I) using the mitochondrial genetic code, with 20 to 34% amino acid identity to the ORF I of other previously reported fungal mitochondrial RNA viruses. The ORF I encoded a putative protein of 705 amino acids and contained the conserved motif characteristic of RNA-dependent RNA polymerases. Purification of mitochondria from strain BK18 confirmed the co-localization of this dsRNA, and northern blot hybridization with a strand-specific probe revealed the (+) single-stranded nature. This Chalara elegans mitovirus (CeMV) is designated as a new member of the genus Mitovirus of the family Narnaviridae. Using dsRNA-specific primers, the ORF I region (positions 427 to 2544) was obtained from an additional 2.8-kb dsRNA element in strain HA2 originating from carrot roots in the Netherlands. Both ORFs had 98% homology at the nucleotide and amino acid levels. CeMV was also found to be present in five additional strains of C. elegans from different geographic locations worldwide, and a 97 to 100% nucleotide sequence identity was observed within a 300-bp region of ORF I in these strains. To determine the biological effects of CeMV on C. elegans, attempts to cure strain BK18 of the dsRNA were made. Sequential transfers of mycelium at 35 to 37 degrees C yielded a colony which lacked the 2.8-kb dsRNA when visualized on agarose gels and also in northern blot hybridization analysis. However, reverse transcription-polymerase chain reaction with specific primer sets revealed a band, indicating that dsRNA replication had been significantly repressed (latent). The wild type and latently infected strains were compared for colony morphology, growth rate, melanin production, various enzymatic assays (polyphenoloxidase, laccase, tyrosinase, and esterase), and virulence on carrot roots. Colony morphology on V8 agar was comparable between the two strains, while growth rate, melanin production, and virulence were enhanced in the latently infected strain. There were no detectable differences in enzymatic activity. Transmission electron microscopy of hyphae of the wild type and latently infected strains revealed differences in the number and size of the mitochondria, which were enhanced in the latently infected strain. Our results show that CeMV is a new member of the genus Mitovirus with some disruptive effects on its fungal host and is present in C. elegans strains from different locations worldwide.