Molecular Characterization Of A DsRNA Totirivus Infecting The Sclerotial Parasite Coniothyrium Minitans

Coniothyrium minitans, an important sclerotial mycoparasite of Sclerotinia sclerotiorum with worldwide distribution, has great potential for biological control of plant diseases caused by S. sclerotiorum and other Sclerotinia spp. The complete nucleotide sequence, 4975bp, of the double-stranded RNA (dsRNA) mycovirus infecting the sclerotial parasite Coniothyrium minitans (CmRV) was determined. Sequence analysis revealed the occurrence of two overlapping open reading frames (ORFs): the 5' -proximal large ORF (ORF1; nucleotide positions 62-2389) encodes a putative coat protein (CP) with a predicted molecular mass of 80344 Da, and the 3'-proximal ORF (ORF2, nucleotide positions 2386-4875) encodes a putative RNA dependent RNA Polymerase (RdRp) with a predicted molecular mass of 82551 Da. The tetranucleotide AUGA at nucleotide positions 2386-2389 includes the predicted start codon of ORF2, which overlaps with the stop codon for ORF1. Based on genome organization and sequence analysis encoded proteins, the virus infecting C. minitans strain Chy-1, designated C. minitans RNA virus (CmRV), belongs to the family Totiviridae. Pairwise sequence comparisons of the deduced amino acid sequences encoded by CmRV as well as phylogentic analysis indicated that it is more closely related to the totiviruses that infect filamentous fungi than to those infecting protozoa, yeast and smut fungi.To test the stability of CmRV in C. minitans, 100 single-conidium-isolation cultures were obtained and observed for development of colony morphology, all of the 100 cultures had a similar colony morphology compared to the original strain Chy-1, among them 32 cultures randomly selected were proved to carry dsRNA genome of CmRV. Spores of Chy-1 were buried into field soil for two months, and 8 cultures were recovered from soil with sclerotial baiting. All the 8 cultures had the same phenotype of Chy-1, and viral dsRNA was extracted from all the 8 cultures. Northern blottings showed that all the viral dsRNA of the 8 cultures were the viral genome of CmRV. These results suggested that CmRV in C. minitans was stable.Twenty-eight C. minitans strains (including 5 from United Kingdom and 3 UV-treated Chy-1) were used to check the dsRNA of mycovirus, among them 12 strains were proved infected by mycovirus. Northern blotting analysis revealed that the viral dsRNA in all these ten strains could be hybridized with the probe made from a cDNA clone derived from CmRV dsRNA, suggesting that those viruses might be in the same group (species). It inferred that virus in C. minitans might be very common.