Characterization of Double-Stranded RNA and Fungal Virus From Diaporthe Phaseolorum Var. Caulivora.

Abstract

Fifty-two isolates of Diaporthe phaseolorum var. caulivora originating from eight different states of the United States were collected and grown on potato dextrose broth for 2-3 wk. Double-stranded RNA was extracted by two cycles of cellulose (CF-11) chromatography and electrophoresed on 6% polyacrylamide gel. Among all isolates tested, 28 (54%) contained at least one molecule of dsRNA. Molecular sizes of dsRNA ranged from 0.4 to 4.5 Kb, and seven different sizes of dsRNA were detected. The 4.5 Kb dsRNA did not have sequence homology with smaller dsRNAs (2.1, 1.8, and 1.4 Kb), suggesting that the smaller dsRNAs are not deletion products of the 4.5 Kb dsRNA. The dsRNAs were located in the cytoplasm of the fungi. The dsRNA of D. p. caulivora was apparently not associated with pathogenicity, toxin production, growth rate, or phenol oxidase activity. Isometric virus particles about 30-40 nm in diameter were detected from a virulent isolate. The viral genome was composed of one double-stranded RNA molecule with a molecular size of 4.5 Kb. The molecular size of viral dsRNA was the same as that extracted from mycelia. Three polypeptides (93 Kd, 90 Kd, and 88 Kd) were associated with the virus particles. Polyclonal antibodies generated to virus particles reacted to all three viral polypeptides. These data suggest that this virus, designated as DpcV, is a possible member of the Totoviridae. To compare southern and northern isolates of this pathogen, fourteen selected isolates representing six southern states (AR, FL, GA, LA, MS, and TN) and two northern states (IO and OH) were compared for colony morphology, mycelial growth at 22 and 30 C, type of perithecia formation, phenol oxidase activity, pathogenicity, and phenol-soluble polypeptides by two-dimensional electrophoresis. Southern isolates showed more variable colony morphology, mycelial growth at both temperatures, phenol oxidase activity, type of perithecia formation, and level of pathogenicity to soybean cultivar Bedford than northern isolates. Greater genetic diversity was observed among southern isolates than among northern isolates in polypeptide analysis. However, polypeptide analysis by two-dimensional electrophoresis indicated a strong genetic relationship between the two populations, suggesting that the fungi causing southern and northern stem canker are the same fungus.