In April 2006, grapevine plants with typical symptoms of yellows (GY) were observed in some South African vineyards. The affected plants showed premature yellowing or reddening and downward rolling of leaves. In some cases, these symptoms were associated with extensive lack of cane lignification that was undistinguishable from yellows symptoms reported in grapevine in the major viticultural areas of the world. Nucleic acids were extracted separately from 0.1 g of fresh leaf midribs and cane phloem scrapes from three symptomatic and three asymptomatic grapevine plants, cv. Shiraz, and from three symptomatic plants, cv. Cabernet, collected from three different locations using Qiagen (Milan, Italy) DNAeasy Plant Mini Kit. A nested polymerase chain reaction (PCR) assay was employed for phytoplasma detection with 2.5 μl of the extracted DNA. Direct and nested PCR assays were performed with P1/P7 (2) and R16F2/R2 (1) universal primer pairs, respectively, obtaining the expected products only from phloem scrapes of the symptomatic plant samples cv. Shiraz. Restriction fragment length polymorphism (RFLP) analyses of R16F2/R2 amplicons with TruI and Tsp509I restriction enzymes, discriminating among phytoplasma ribosomal group and subgroups, showed profiles corresponding to those of “Candidatus Phytoplasma aurantifolia” (ribosomal subgroup 16SrII-B) in all three positive samples. A Stolbur phytoplasma profile (ribosomal subgroup 16SrXII-A) was also observed in one of those samples, indicating the presence of mixed phytoplasma infection (1). Sequencing of the obtained amplicons confirmed the RFLP phytoplasma identification; in particular 16SrXII-A could be the same phytoplasma associated with the ‘Bois Noir’ disease reported in grapevine; the 1601-bp sequence of 16SrII-B phytoplasma showed 98% similarity to U15442, i.e., to the phytoplasma associated with lime witches'-broom disease in Oman (“Ca. P. aurantifolia”) confirming RFLP results. To our knowledge, this is the first report of phytoplasmas in grapevine in South Africa.
References: (1) I.-M. Lee et al. Phytopathology 85:728, 1995. (2) B. Schneider et al. Pages 369--380 in: Molecular and Diagnostic Procedures in Mycoplasmology Vol. I. Academic Press Inc., 1995.