VIEW ARTICLE

Biochemistry and Cell Biology

Reduction of Laccase Activity and Other Hypovirulence-Associated Traits in dsRNA-Containing Strains of Diaporthe ambigua. W. A. Smit, Department of Plant Biotechnology and Pathology, Stellenbosch Institute for Fruit Technology (INFRUITEC), Private Bag X5013, Stellenbosch 7599, South Africa; B. D. Wingfield(2), and M. J. Wingfield(3). (2)(3)Department of Microbiology & Biochemistry, University of the Orange Free State, Bloemfontein 9300, South Africa. Phytopathology 86:1311-1316. Accepted for publication 9 July 1996. Copyright 1996 The American Phytopathological Society. DOI: 10.1094/Phyto-86-1311.

A single double-stranded RNA (dsRNA) segment was detected in hypovirulent but not in virulent strains of Diaporthe ambigua isolated from apple 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. Laccase and peroxidase activities were determined with 2,6-dimethoxyphenol as substrate. Oxalic acid production in virulent and hypovirulent strains was determined by ultraviolet spectrophotometric analysis of NADH. Conversion of virulent strains was achieved by pairing hypovirulent and virulent strains on dialysis membrane on the surface of Czapek-Dox agar. Pathogenicity tests were conducted on 3-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 type. dsRNA could be transmitted to strains of the same vegetative compatibility group by hyphal anastomosis. Converted strains lost virulence and showed loss of phenol oxidase activity, reduced gallic acid oxidation, diminished oxalic acid accumulation, and suppressed sporulation. From these studies, we conclude that dsRNA was transferred to virulent strains via hyphal anastomosis. This resulted in hypovirulence as tested in the field, as well as reduction of laccase activity and other hypovirulence-associated traits.