1.11.45
TWO DISTINCT dsRNA VIRUSES (FAMILY TOTIVIRIDAE) CO-INFECTING THE PINE PATHOGEN SPHAEROPSIS SAPINEA

O PREISIG, BD WINGFIELD and MJ WINGFIELD

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

Background and objectives
Mycoviruses can play an important role in the biological control of fungal pathogens. A well known example is the hypovirus that mediates hypovirulence in the chestnut blight pathogen Cryphonectria parasitica. While most mycoviruses are members of the family Totiviridae, the C. parasitica hypovirus resides in its own family known as the Hypoviridae. Totiviruses are dsRNA viruses with a monopartite genome of 4.5 to 6 kbp and are known to persistently infect fungi (e.g. Saccharomyces cerevisiae) and protozoa (e.g. Leishmania braziliensis). The effect of their presence is variable and can result in no symptoms or a lytic reaction.

In South Africa, the introduced filamentous fungus Sphaeropsis sapinea is the most important pathogen of pine trees. The fungus is a common latent pathogen in pine cones. After onset of stress (e.g. hail damage or drought) the fungus is able to invade trees causing severe die-back. Therefore, any means to reduce the impact of this pathogen is of considerable interest to forestry in South Africa. Due to the emerging possibilities of utilising mycoviruses for biological control, we have an interest in viruses of S. sapinea. Recently, 4 kbp dsRNA fragments were isolated from South African isolates of S. sapinea (Steenkamp ET, Wingfield BD, Swart WJ, Wingfield MJ, unpublished data). In order to investigate a possible role for these mycoviruses in biological control, we have begun to characterise them intensively.

Materials and methods
The nature of the potential virus infecting S. sapinea isolate CMW 4254 was determined by the following experiments: Firstly, cell extract was applied to a virus purification protocol and then examined using a TEM. Secondly, the nucleotide sequence of the S. sapinea RNA virus (SSRV) genome was established by random cDNA cloning and RT-PCR with sequence derived primers.

Results and conciusions
Virus-like particles of an isometric shape and a size of ca 35 nm in diameter were found in the cell contents of the dsRNA-containing isolate. These features were similar to those found for particles of Totiviruses. Surprisingly, our sequence data revealed that the S. sapinea isolate used in these studies is co-infected with two distinctly related Totiviruses which we have named SsRV1 and SsRV2. In both cases, the genome consists of 5.2 kb dsRNA and two overlapping ORFs are present encoding a coat protein (ORF1) and a RNA-dependent RNA polymerase (ORF2). At the amino acid level, the respective gene products of the two entities of SSRV share only 36 to 38 % identity. Interestingly the gene products of the most closely related dsRNA virus, the Helminthosporium victoriae 19OSV (HvlgOSV) [1], show nearly the same or even higher degree of homology to the gene products of SsRVs. HvlgOSV is associated with a lytic disease of H. victoriae, but we have not, as yet, seen any evidence of an equivalent phenomenon in S. sapinea. This fascinating situation clearly deserves further in-depth study. We will now construct a full-length cDNA clone of each of the SsRVs which will enable us to study the virus-host interaction in greater detail.

References
1. Huang S, Ghabdal SA 1996. Proc. Natl. Acad. Sci. USA 93, 12541-12546.