MOLECULAR CHARACTERIZATION OF SPHAEROPSIS SAPINEA ISOLATES FROM SOUTH AFRICA, MEXICO AND INDONESIA J DE WET , MJ WINGFIELD TA COUTINHO and BD WINGFIELD Tree Pathology Co-operative Programme, Forestry and Agricultural Biotechnology Institute (FABI), Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South Africa Background and objectives Sphaeropsis sapinea is a well-known and common pathogen of Pinus spp. in many parts of the world. Generally it is known as an opportunist that infects trees after the onset of predisposition, which can include adverse environmental conditions or severe wounding such as through hail damage. Symptoms of infection by S. sapinea indude die-back, whorl cankers, root disease and blue stain. In South Africa, this is the most important pathogen of pines were it infects and kills large numbers of trees after hail damage [1]. Recently, it has also been shown that S. sapinea commonly inhabits cone tissue of healthy trees and rapidly colonizes the vascular tissue after hail. S. sapinea is recognized to have two distinct morphotypes. The A morphotype has fluffy white to grey-green mycelia and the B morphotype has white to black mycelia suppressed to the agar. The obiective of this study was to characterize the morphotypes of South African, Indonesian and Mexican isolates of S. sapinea Materials and methods For RAPD analyses, 21 S. sapinea isolates were studied of which six United States isolates of known morphotype were used for comparison. The RAPD reaction consisted of a low as well as a high stringency cycle with primers D52 and D53, previously used to distinguish between the S. sapinea morphotypes [2]. The RAPD data obtained were scored and analyzed using PAUP to generate a dendrogram. For sequence analyses, the region between the large subunit and small subunit of the DNA operon of seven S. sapinea isolates from different parts of the world were sequenced. Sequencing was performed on purified amplification products using primers ITS 1, ITS4, CS2 and CS3. The ITS sequences of Botryosphaeria dothidea and Lasiodiplodia threobromae were used as outgroups. Sequence data were analyzed using Sequence Navigator and PAUP was used to generate a dendrogram. Results and discussion Both RAPD data and comparison of ITS sequences were successfully used to characterize the test fungi as belonging to either the A, B or an unknown morphotype. Using RAPDs, Indonesian isolates were found to represent three distinct groups including the A and B type and one apparently distinct form. Prior to this study, the B morphotype was known only from the North Central United States. The discovery of additional isolates of this type from Indonesia is of considerable interest. We have confidence in these results as the control isolates gave consistent and expected results. Using this technique, all South African and Mexican isolates grouped with the A morphotype. This is consistent with the notion that the A type is predominant in the world [1]. Based on rDNA sequence data, it was also possible to distinguish between the A and B morphotypes of S. sapinea. This approach will, therefore, be useful in further studies to distinguish morphotypes in S. sapinea. Selected Indonesian, Mexican and South African isolates showed a high degree of relatedness to the more common A type. Data also indicate a high degree of sequence homology between the A and B morphotypes. It is, therefore, unlikely that these morphotypes might represent distinct species. References 1. SwartWJ &Wingfield MJ, 1991. Plant Disease 75,761-766. 2. Smith DR & Stanosz GR, 1995. Phytopathology 85~ 699-704.