3.7.55
THE IDENTITY AND POSSIBLE ORIGIN OF THE AMYLOSTEREUM SYMBIONT OF SIREX NOCTILIO IN SOUTH AFRICA

B SLIPPERS, 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
The woodwasp Sirex noctilio, a serious pest of pine in the Southern Hemisphere, carries the basidiomycete symbiont Amylostereum areolatum. This woodwasp along with its fungal symbiont was first reported from South Africa in 1994. Little is known about A. areolatum or its genetic composition in the Southern Hemisphere. The aim of this study was to confirm the identity of A. areolatum in South Africa and to study the genetic diversity of this fungus in collections from South Africa and Brazil. Many questions concerning the taxonomy and phylogeny of the genus Amylostereum are still unanswered [1]. We, therefore, also compared species of Amylostereum based on DNA sequence data.

Materials and methods
Amylostereum areolatum was isolated from the internal mycangia of female S. noctilio wasps from the Cape province of South Africa, as well as from various locations in Brazil. Additional isolates of A. areolatum (from S. noctilio, and other woodwasp species), A. chailletii, A. fereum and A. laevigatum were obtained from collections in Australasia, Europe and North America. Vegetative compatibility between heterokaryotic isolates was used to characterise the genetic diversity among A. areolatum isolates from South Africa and Brazil. Isolates from these two countries were also paired against those from Australia and New Zealand. AFLP fingerprinting was performed on a selection of isolates from South Africa and Brazil to confirm results of vegetative compatibility tests. PCR, RFLP analysis using the restriction enzyme Alu] and DNA sequencing were used to study taxonomic and phylogenetic relationships between the species of Amylostereum. Both the mitochondhal small subunit RDNA and the intergenic spacer region (IGS) of the nuclear RDNA were targeted in these analyses. Sequence data from the mt-ssu-rDNA were aligned with sequence data from other Basidiomycetes as published by Hibbett [2], and analysed using PAUP.

Results and conclusions
Isolates of A. areolatum from South Africa and Brazil represented a single vegetative compatibility group. This unusually small genetic base indicates a limited introduction of S. noctilio in both countries. South Africa and Brazil may share a common origin of this pest. Alternatively, S. noctilio may have been introduced into South Africa from Brazil, where it has been known for a longer period. The preservation of this single VCG can be explained by the vegetative spread of the fungus through its symbiosis with the woodwasp. AFLP fingerprinting, however, indicates some genetic variation within this single VCG. Isolates from South Africa and Brazil showed strong incompatibility with all isolates from Australasia. This suggests that the Australasian insect and fungus have an origin different to that in South Africa and Brazil. Mt-ssu-rDNA sequence analysis supports the separation of species as reported previously for the genus Amylostereum. Isolates from South Africa clearly group with isolates of A. areolatum from other countries. A. fereum and A. laevigatum are phylogenetically closely related to each other. These two species also appear to be more closely related to A. chailletii, than to A. areolatum. DNA sequence comparisons with other Basidiomycetes, suggest evolutionary linkage to a diverse group including Echinodontium tinctodum and Russula compacta. Preliminary IGS-RDNA sequence indicate two distinct groups within A. areolatum. This grouping is also supported by RFLP analysis.

References
1. Thomsen IM, 1996. PhD thesis. Danish Forest and Landscape Research Institute, Horsholm, Denmark.
2. Hibbett DS, Pine EM, Langer E, Langer G, Donoghue MJ 1997. Proceedings of the National Academy of Sciences, USA 94,12002-12006.