Characterization of a new mitovirus OMV1c in a Canadian isolate of the Dutch Elm Disease pathogen Ophiostoma novo-ulmi 93-1224
Date
2012-04-30
Authors
Kassatenko, Irina
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Abstract
The fungal pathogen Ophiostoma novo-ulmi is the causal agent of Dutch elm disease (DED) and has been responsible for the catastrophic decline of elms in North America and Europe. Double-stranded RNA (dsRNA) viruses are common to all fungal classes and although these viruses do not always cause disease symptoms, the presence of certain dsRNA viruses have been associated with reduced virulence (hypovirulence) in O. novo-ulmi. A new mitovirus was found in a Canadian isolate of O. novo-ulmi (93-1224) and has been named Ophiostoma mitovirus 1c (OMV1c). The positive strand of the dsRNA of OMV1c was 3,003 nucleotides in length and when the mitochondrial codon usage pattern was employed (mitochondria use UGA to encode tryptophan rather than as a chain terminator), a single large open reading frame (ORF) was found. This ORF had the potential to encode a protein of 784 amino acids, and revealed a high degree of nucleotide identity to genes encoding RNA-dependent RNA polymerase (RdRp) in other mitoviruses. The putative RdRp region of the newly characterized virus had the highest sequence similarity to Ophiostoma mitovirus 1b. The 5’- terminal sequence of the positive strand could potentially be folded into a double-stranded stem-loop structure with a free energy of 16.6 kcal/mol. Attempts to cure the O. novo-ulmi isolate 93-1224 of virus were unsuccessful. Screening of the re-cultured isolates for the presence of OMV1c revealed that it was still present in the fungus despite repeated hyphal tip transfer, a method known to cure cytoplasmic but not mitochondrial viruses. Based on the genome size, phylogenetic analysis, and the observation that infected isolates could not be cured, it was surmised that the virus was a member of the genus Mitovirus (family Narnaviridae). To assess the distribution of the virus in O. novo-ulmi at the disease front in Winnipeg, a small sample of thirteen isolates were screened for the presence of the new mitovirus. All proved to be negative for OMV1c, which indicated this dsRNA virus was rare and that isolate 93-1224 was the only isolate identified to date infected with OMV1c.
It was also discovered that the isolate O. novo-ulmi 93-1224 potentially harboured more than one virus. Electron microscopy of fractionated cells revealed the presence of two flexuous rod-shaped particles that may represent additional novel viruses.
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Keywords
mitovirus, sequencing virus genome, Ophiostoma