GENETIC DIVERSITY OF PHYTOPHTHORA CINNAMOMI IN DISEASE FRONTS OF NATURAL VEGETATION
MP DOBROWOLSKI1, IC TOMMERUP2, BL SHEARER3 and PA OĠBRIEN1
1School of Biological Sciences and Biotechnology, Murdoch University, Perth, WA, 6150, Australia; 2CSIRO Forestry and Forest Products, Perth, WA, 6014, Australia; 3Science and Information Division, Department of Conservation and Land Management, Como, WA, 6152, Australia
Background and objectives
Phytophthora cinnamomi is a global plant pathogen causing root rot in a wide range of hosts. It causes devastating damage to many natural ecosystems in southern Australia due to the low resistance of the dominant vegetation to this introduced pathogen. The number of introductions of P. cinnamomi to southern Australia is not known, although three isozyme types and both A1 and A2 mating types have been found . Although both mating types co-occur in New South Wales no new genotypes arising through sexual reproduction were detected by isozyme analysis . We aimed to survey the genetic structure of P. cinnamomi and assess the potential for sexual reproduction in disease fronts of southwestern Australia, using recently developed microsatellite markers for P. cinnamomi . Microsatellites are molecular markers well suited for population analysis and, compared to isozymes, allow a finer resolution of genotypes.
Materials and methods
Three P. cinnamomi disease fronts widely spaced in southwestern Australia (approximate locations: front 1, 31ĦS 116ĦE; front 2, 33ĦS 116ĦE; front 3, 35ĦS 118ĦE) were chosen for hierarchical sampling. The fronts were 600-900 m long with natural vegetation dominated by Banksia spp: B. attenuata, B. laricina and B. menziesii in front 1, B. attenuata and B. ilicifolia in front 2, and B. attenuata, B. coccinea, B. ilicifolia and B. nutans in front 3. Bark samples and adjacent soil samples were collected from recently dead susceptible plants, mainly Banksia spp., and P. cinnamomi was isolated from these by plating on selective agar and baiting in water for zoospores, respectively. Up to 100 samples were taken at each disease front. Mating types of all isolates were determined by pairing with a known A1 or A2 test isolate on V8 agar, followed by microscopic examination for oospore production. Genetic analysis was performed using five microsatellite loci: amplification of each locus with specific primers using the polymerase chain reaction (PCR) and separation of products by polyacrylamide gel electrophoresis with ethidium bromide staining.
Results and conclusions
High recoveries of P. cinnamomi were obtained from soil samples of all three disease fronts (66-98%) with slightly fewer recoveries from bark samples. No association was apparent between recovery rate and host species. From fronts 1 and 2 only the A2 mating type was recovered, and from the third front both mating types were found within 1 m proximity in the soil. At this disease front 12% of the pairs of isolates, from host bark and adjacent soil sample, did not have matching mating types. The co-occurence of both mating types at this disease front allows the potential for new genotypes to arise through sexual reproduction. Genetic analysis of these isolates with microsatellite markers will be presented to test this hypothesis.