REDUCTION OF LACCASE ACTIVITY AND OTHER HYPOVIRULENCE-ASSOCIATED TRAITS IN DSRNA-CONTAINING STRAINS OF DIAPORTHE AMBIGUA

A single double-stranded RNA (dsRNA) segment was detected in hypovirul ent but not in virulent strains of Diaporthe ambigua isolated from app le rootstocks in South Africa. To test for phenol oxidase activity and gallic acid oxidation (Bavendamm's tests), the strains were grown on malt extract agar containing tannic and gallic acid, respectively. Lac case and peroxidase activities were determined with 2,6-dimethoxypheno l as substrate. Oxalic acid production in virulent and hypovirulent st rains was determined by ultraviolet spectrophotometric analysis of NAD H. Conversion of virulent strains was achieved by pairing hypovirulent and virulent strains on dialysis membrane on the surface of Czapek-Do x agar. Pathogenicity tests were conducted on S-year-old M793 and M25 apple rootstock cultivars. In both Bavendamm's tests, virulent strains produced a strong color reaction, whereas hypovirulent strains showed weak or no activity. The enzyme responsible for the color reaction on Bavendamm's medium was identified as phenol oxidase of the laccase ty pe. dsRNA could be transmitted to strains of the same vegetative compa tibility group by hyphal anastomosis. Converted strains lost virulence and showed loss of phenol oxidase activity, reduced gallic acid oxida tion, diminished oxalic acid accumulation, and suppressed sporulation. From these studies, we conclude that dsRNA was transferred to virulen t strains via hyphal anastomosis. This resulted in hypovirulence as te sted in the field, as well as reduction of laccase activity and other hypovirulence-associated traits.