Risks and threats due to genetic variation in Phytophthora cinnamomi for disease management in natural vegetation ecosystems

Phytophthora cinnamomi is often genetically diverse in disease fronts and diseased areas. P. cinnamomi has considerable ability to produce a wide range of pathogenic phenotypes measured from ‘plant clone by isolate by environmental factor interactions’ in each of the three pathogen clonal lineages of P. cinnamomi isolated in Australia. P. cinnamomi lineages are defined by microsatellite types. In a few disease areas, despite both mating types occurring in the same square metre of soil, or 50 g soil sample or bait plant, no evidence of sexual reproduction (genomic recombination) has been found in the field. Isolates are sexually competent in the laboratory and all lineage combinations are strongly outcrossing. Hence the substantial variation in lineages in a range of traits associated with disease development must be arising asexually. Preventing movement of any P. cinnamomi contaminated material is critical to maximising disease control and minimising risks of introducing new strains which may threaten plant communities, their diversity and the integrity of these ecosystems. Phosphite or other intended phytophthoracide usage, which does not eliminate P. cinnamomi in plant nurseries can, by camouflaging disease, increase potential risks and threats by spreading different strains in diseased materials

Optimising the conservation of genetic diversity of the last remaining population of a critically endangered shrub

An understanding of genetic diversity and the population genetic processes that impact future population viability is vital for the management and recovery of declining populations of threatened species. Styphelia longissima (Ericaceae) is a critically endangered shrub, restricted to a single fragmented population near Eneabba, 250 km north of Perth, Western Australia. For this population, we sought to characterize population genetic variation and its spatial structure, and aspects of the mating portfolio, from which strategies that optimize the conservation of this diversity are identified. A comprehensive survey was carried out and 220 adults, and 106 seedlings from 14 maternal plants, were genotyped using 13 microsatellite markers. Levels of genetic variation and its spatial structure were assessed, and mating system parameters were estimated. Paternity was assigned to the offspring of a subsection of plants, which allowed for the calculation of realized pollen dispersal. Allelic richness and levels of expected heterozygosity were higher than predicted for a small isolated population. Spatial autocorrelation analysis identified fine-scale genetic structure at a scale of 20 m, but no genetic structure was found at larger scales. Mean outcrossing rate (tm = 0.66) reflects self-compatibility and a mixed-mating system. Multiple paternity was low, where 61 % of maternal siblings shared the same sire. Realized pollen dispersal was highly restricted, with 95 % of outcrossing events occurring at 7 m or less, and a mean pollen dispersal distance of 3.8 m. Nearest-neighbour matings were common (55 % of all outcross events), and 97 % of mating events were between the three nearest-neighbours. This study has provided critical baseline data on genetic diversity, mating system and pollen dispersal for future monitoring of S. longissima. Broadly applicable conservation strategies such as implementing a genetic monitoring plan, diluting spatial genetic structure in the natural population, genetically optimizing ex situ collections and incorporating genetic knowledge into translocations will help to manage the future erosion of the high genetic variation detected

Use of RAPD-PCR to isolate a species specific DNA probe for Phytophthora cinnamomi

The products of RAPD-PCR amplification of Phytophthora cinnamomi DNA were separated by electrophoresis in agarose. Parallel Southern blots of the gels were hybridized with nick translated DNA from different species of Phytophthora. Fragments that hybridized specifically to P. cinnamomi DNA were identified. These fragments were purified and cloned into pUC18. Their specificity for P. cinnamomi was confirmed

Microsatellites in the mitochondrial genome of Phytophthora cinnamomi failed to provide highly polymorphic markers for population genetics

Microsatellites were evaluated as genetic markers for the mitochondrial genome (mtDNA) of Phytophthora cinnamomi for population studies. Two (A)(n) microsatellite loci were cloned from the mtDNA of P. cinnamomi. Amplification products from these loci showed little polymorphism among Phytophthora isolates due to their location in coding regions of mtDNA. A further three (A)(n) microsatellite loci obtained from the complete mtDNA sequence of P. infestans were also not highly polymorphic, although located in non-coding mtDNA. The presence of the (A)(n) microsatellites was not conserved in the genus Phytophthora. Unlike those of the chloroplast genome of plants, (A)(n) microsatellite loci of mtDNA do not have potential as highly polymorphic markers in Phytophthora

Three clonal lineages of Phytophthora cinnamomi in Australia revealed by microsatellites

The genetic structure of populations of Phytophthora cinnamomi, a pathogen of an enormous variety of woody plants, was investigated using microsatellites. Three intensively sampled disease sites in southwest Australia were analyzed along with a large culture collection of Austra-lian isolates and some isolates from elsewhere in the world. The mutation in the four microsatellite loci analyzed revealed spatial patterns at the disease sites that correlated with the age of the infestation. Only three clonal lineages were identified in Australian populations and these same clonal lineages were present in worldwide populations, where it is suggested that a limited number of clonal lineages have spread in most regions. No evidence for sexual reproduction between these clonal lineages in Australia has been found even though the pathogen has the opportunity. Instead, mitotic recombination is frequent within the clonal lineages. The implications of this are discusse

Continent-wide clonal lineages of Phytophthora cinnamomi show frequent mitotic recombination

Genetic studies of Phytophthora cinnamomi using isozymes have revealed low levels of diversity suggesting, though not proving, clonality in a large proportion of worldwide populations (1). In Australia, only three isozyme types (representing both mating types) are found with no evidence for sexual recombination (2). Using microsatellite markers, we have shown that these isozyme types are clonal lineages of P. cinnamomi and that these same lineages are found elsewhere in the world (3). Our study used 647 isolates from three intensively and hierarchically sampled P. cinnamomi disease fronts located in south-west Australia. In addition 133 isolates from an Australia-wide culture collection and 27 isolates from elsewhere in the world were analysed with four microsatellite markers. One disease front contained all three clonal lineages within close proximity in soil and plant tissue but no sexual recombinant isolates were found, even with very intensive sampling. However, within these clonal lineages we frequently found evidence for mitotic recombination (mitotic crossing over). This mechanism for producing genetic variation may explain phenotypic variation known to occur within the identified clonal lineages

Non-mendelian inheritance revealed in a genetic analysis of sexual progeny of Phytophthora cinnamomi with microsatellite markers

We report the development of four microsatellite loci into genetic markers for the diploid oomycete plant pathogen Phytophthora cinnamomi and that (AC)n and (AG)n microsatellites are significantly less frequent than in plant and mammal genomes. A minisatellite motif 14 bp long was also discovered. The four microsatellite loci were used to analyze sexual progeny from four separate crosses of P. cinnamomi. A large proportion of non-Mendelian inheritance was observed across all loci in all four crosses, including inheritance of more than two alleles at a locus and noninheritance of alleles from either parent at a locus. The aberrant inheritance is best explained by nondisjunction at meiosis in both the A1 parent and the A2 trisomic parents, resulting in aneuploid progeny. Two loci on the putative trisomic chromosome showed linkage and no loci were linked to mating type. One aneuploid offspring was shown to have lost alleles at two loci following subculture over 4 years, indicating that aneuploid progeny may not be mitotically stable

Genetic diversity of Phytophthora cinnamomi in disease fronts of natural vegetation

Phytophthora cinnamomi, a global plant pathogen, causes devastating damage to many natural ecosystems in southern Australia due to the low resistance of the dominant vegetation to this introduced pathogen. It is a serious threat to ecosystem biodiversity particularly in southwest Australia. The number of introductions of P. cinnamomi to southern Australia is unknown, although three isozyme types and both A I and A2 mating types have been found. We aimed to survey the genetic structure of P. cinnamomi and assess the potential for sexual reproduction in disease fronts of southwestern Australia. We hierarchically sampled three P. cinnamomi disease fronts, widely spaced in southwestern Australia, which were 600-900 m long with natural vegetation dominated by Banksia spp. P. cinnamomi was isolated from bark of recently dead susceptible plants and adjacent soil samples. The mating types of isolates were determined and genetic analysis performed using five microsatellite loci. The isolates of P. cinnamomi we recovered grouped into three clonal types as indicated by their multilocus microsatellite genotypes, two of A2 and one of Al mating type. One A2 clonal type was common to all three disease fronts, and one disease front had all three clonal types. At this disease front no isolates with recombinant genotypes were found even though Al and A2 mating types were found in close proximity, i.e. the potential for sexual reproduction exists but we have found no evidence for it. Founder effects can explain the genetic diversity of P. cinnamomi in the disease fronts we examined

Phenotypic variation in a clonal lineage of two Phytophthora cinnamomi populations from Western Australia

Seventy-three isolates of Phytophthora cinnamomi were collected from diseased Eucalyptus marginata (jarrah) and Corymbia calophylla (marri) trees in two forest communities in the southwest of Western Australia. Both populations of P. cinnamomi were examined for phenotypic and genotypic variation. Microsatellite DNA analysis showed that all isolates were of the same clonal lineage. We show, for the first time for P. cinnamomi, that morphological and pathogenic variation between populations of the clonal lineage are very broad and continuous. The phenotypes examined included growth rates and colony morphology on potato dextrose agar at different temperatures, sporangial and gametangial morphology, ability to form lesions in detached jarrah and marri stems, and ability to cause deaths of clonal jarrah plants in a glasshouse trial. Phenotype variation was derived asexually. All phenotypes investigated varied independently from one another. Cluster analysis of 24 morphological and pathogenicity phenotypes identified two main clusters of isolates corresponding to each population. The ability to cause deaths in both populations ranged from killing all plants within 59 d to plants being symptomless 182 d after inoculation

Patterns and drivers of structure, diversity, and composition in species‐rich shrublands restored after mining

Long‐term studies of vegetation recovery following post‐mining restoration in low‐productivity, high‐stress areas are limited, but essential for understanding underlying ecological processes and evaluating management practices. This study's goal was to describe temporal patterns of recovery (up to 37 years) in vegetation structure, floristic diversity, and composition following post‐mining restoration at two sites, and identify potential drivers of restoration outcomes, in the nutrient‐poor, seasonally dry, species‐rich, fire‐prone kwongan vegetation of southwest Western Australia. Vegetation development is progressing and restoration measures are within range of native reference values, but there is large variation in both patterns observed and restoration outcomes. Several patterns described share similarities with post‐fire recovery of kwongan, and post‐disturbance recovery of other low‐productivity, high‐stress, fire‐prone systems. However, differences in some patterns between sites indicate differences in the underlying mechanisms of recovery. Many management and environmental variables emerge as significant drivers of restoration outcomes but age, fire, and the planting of seedlings account for the largest amount of variation. Adaptive management at both sites appears to be facilitating improved restoration outcomes over time. Using a combination of space‐for‐time, plot level, and linear mixed effects modeling perspectives to examine patterns provides greater insights into restoration recovery than a chronosequence perspective alone. This study will inform restoration practices and outcomes not only in kwongan but in other comparable systems also