1.11.47
PHYLOGENETIC RELATIONSHIPS BETWEEN DOUBLE-STRANDED RNA VIRUSES INFECTING SOUTH AFRICAN ISOLATES OF SPHAEROPSIS SAPINEA

NA VAN DER MERWE, O PREISIG, ET STEENKAMP, BD WINGFIELD and MJ WINGFIELD

Tree Pathology Co-operative Programme, Forestry and Agricultural Biotechnology Institute (FABI), Department of Genetics, University of Pretoria, Pretoria 0001, South Africa

Background and objectives
Sphaeropsis sapinea is an important fungal pathogen of pine trees in South Africa. The fungus causes stress-related diseases such as extensive die-back after hail damage. In healthy trees, it apparently exists in cones in the absence of symptoms, which develop after the onset of stress. Hypovirulence, associated with dsRNA viruses, is of interest for the potential biological control of S. sapinea. dsRNA has recently been described in this pathogen, although very little is known regarding its effect (Steenkamp ET, Wingfield BD, Swart WJ, Wingfield MJ, unpublished data). Two S. sapinea double-stranded RNA viruses, known as SsRV1 and SsRV2, are simultaneously present in the same fungal isolate, namely CMW4254 (see co-presentation: O Preisig et al.). Both of these viruses belong to the family Totiviridae and show identical genome size and organization. They are, however, only 36-38% similar at the amino acid sequence level. The objective of this study was to analyse the diversity of SsRVs in a collection of South African isolates of S. sapinea, containing dsRNA. This analysis was based on the level of sequence similarity shown to SsRV1 and SsRV2 by other SsRVs.

Materials and methods
A rapid screening method was employed to identify dsRNA-containing S. sapinea isolates. This was followed by extraction and purification of the dsRNA. Reverse transcription PCR with two specific primer sets, OLI13/OL114 and OLI13/OLI15, respectively, was used to amplify a region in the RNA-dependent RNA polymerase gene of dsRNA viruses from three S. sapinea isolates. The isolates used were CMW4231, CMW4236, and CMW4254, all of which have different geographic origins in South Africa. Only isolate CMW4254 was known to contain SsRV1 and SsRV2. The RT-PCR products were cloned, sequenced, and translated into amino acid sequences. These sequences were aligned with each other, as well as with previously published sequences from other Totiviruses. Aligned sequences were then used to generate a phylogenetic tree.

Results and conclusions
Since primer set OLI 1 3/OLI 1 4 is specific for SsRV1, and OLI 1 3/OLI 1 5 is specific for SsRV2, we could determine that isolates CMW4241 and CMW4236 both contained SsRV1. The SsRV2 virus could not be detected in either one of the isolates using PCR with specific primers. The phylogenetic analysis from the aligned sequences revealed that SsRV1 from isolate CMW4254, the CMW4236 virus, and the CMW4231 virus grouped closely together. Surprisingly, SsRV2 was more distantly related to the SsRV1-viruses than the Helminthosporium victoriae 190S 121 virus.

This observation implies that the South African SsRVs do not necessarily share a common origin. Further investigations, using additional infected isolates, are required to resolve this question further.