MOLECULAR CHARACTERIZATION OF CONIOTHYRIUM ZULUENSE ISOLATES FROM SOUTH AFRICA AND THAILAND LM VAN ZYL 1, MJ WINGFIELD 1, TA COUTINHO 1 BD WINGFIELD 1, and K PONGPANICH 2 1 Tree Pathology Co-operative Programme, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa. 2 Forest Research Office, Royal Forest Department, Chatuchak, Bangkok 10900, Thailand Background and objectives Coniothyrium canker caused by Coniothyrium zuluense is a serious stem canker disease of Eucalyptus spp. in subtropical parts of South Africa [1]. This disease was first noted in 1988 in an isolated stand in Kwazulu-Natal on a single clone of Eucalyptus grandis , with outstanding sylvicuftural properties. It has subsequently become widespread in that area and occurs not only on a wide range of E. grandis clones, but also on hybrids of this and other species. The earliest signs of infection are small, discrete, necrotic lesions on the young green bark. These lesions coalesce to form large necrotic patches on the stems from which copius amounts of red/brown kino exude. A Coniothyrium sp. associated with similar symptoms on E. camaldulensis was observed for the first time in Thailand in 1998. Isolates of C. zuluense are morphologically variable and also vary markedly in their virulence. It has thus been suggested that more than one species might be associated with Coniothyrium . The aim of this study was to compare South African isolates of C. zuluense at the molecular level and also to consider whether the Coniothyrium sp. from Thailand is the same as the South African fungus Materials and methods Isolates used in this study included a range of morphological forms of C. zuluense from South Africa as well as isolates of the Coniothyrium sp. from Thailand. C. ovatum from Eucalyptus spp., C. palmarum , C. fuckelii and Massarina corni
  • were included for comparative purposes. These comparisons were based on partial rRNA sequence data generated by sequencing the ITS region of the rRNA operon and the 5.8S gene. DNA was isolated and the ITSI and 1TS4, as well as the 5.8Sgene of the ribosomal RNA operon were amplified using the polymerase chain reaction (PCR). An automated DNA sequencing system was used to sequence the PCR products. Sequence data were manually aligned and phylogenetic comparisons were made using PAUP (Phylogenetic Analysis Using Parsimony). A bootstrap analysis was performed to assess the confidence intervals of the branch points. Results and conclusions All Coniothyrium isolates associated with stem cankers on Eucalyptus grouped together in a single major clade. This clade was clearly distant from isolates of other Coniothyrium spp. included for comparison purposes. Isolates representing C. zuluense and the Coniothyrium sp. from Thailand formed two distinct subgroups in the major clade. Although these fungi from South Africa and Thailand could be distinguished from each other, we believe that the latter isolates represent C. zuluense . This would thus be a first record of C. zuluense outside South Africa. The data also suggest that C. zuluense represents a single species despite the considerable variation in morphology and virulence of isolates. References 1. Wingfield MJ, Coutinho TA, Crous PW, 1997. Mycopathologia 138,139-147.