An overview of Australia’s Phytophthora species assemblage in natural ecosystems recovered from a survey in Victoria

Although Phytophthora species cause serious diseases worldwide, until recently the main focus on disease in natural ecosystems in southern Australia has been on the distribution and impact of P. cinnamomi. However, new Phytophthora pathogens have emerged from natural ecosystems, and there is a need to better understand the diversity and distribution of these species in our natural forests, woodlands and heathlands. From a survey along a 70 km pipeline easement in Victoria, Phytophthora species were isolated from 249 rhizosphere samples and 25 bait bags deployed in 21 stream, river, or wetland locations. Of the 186 Phytophthora isolates recovered, 130 were identified to species based on ITS sequence data. Ninety-five isolates corresponded to 13 described Phytophthora species while additionally 35 isolates were identified as Clade 6 hybrids. Phytophthora cinnamomi was the most common species isolated (31 %), followed by P. elongata (6 %), both species were only recovered from soil. Samples from sites with the highest soil moisture at the time of sampling had the highest yield of isolates. Consistent with other studies throughout the world, Clade 6 species and their hybrids dominated water samples, although many of these species were also recovered less frequently from soil samples. Many of the species recovered in this study have not previously been reported from eastern Australia, reinforcing that Phytophthora species are widespread, abundant and diverse in natural ecosystems. We have probably been underestimating Phytophthora diversity in Australia

Pathways to false positive diagnoses using molecular genetic detection methods; Phytophthora cinnamomi a case study

Phytophthora cinnamomi is one of the world's most invasive plant pathogens affecting ornamental plants, horticultural crops and natural ecosystems. Accurate diagnosis is very important to determine the presence or absence of this pathogen in diseased and asymptomatic plants. In previous studies, P. cinnamomi species-specific primers were designed and tested using various polymerase chain reaction (PCR) techniques including conventional PCR, nested PCR and quantitative real-time PCR. In all cases, the primers were stated to be highly specific and sensitive to P. cinnamomi. However, few of these studies tested their primers against closely related Phytophthora species (Phytophthora clade 7). In this study, we tested these purported P. cinnamomi-specific primer sets against 11 other species from clade 7 and determined their specificity; of the eight tested primer sets only three were specific to P. cinnamomi. This study demonstrated the importance of testing primers against closely related species within the same clade, and not just other species within the same genus. The findings of this study are relevant to all species-specific microbial diagnosis

Multiple new Phytophthora species from ITS Clade 6 associated with natural ecosystems in Australia: evolutionary and ecological implications

During surveys of dying vegetation in natural ecosystems and associated waterways in Australia many new taxa have been identified from Phytophthora ITS Clade 6. For representative isolates, the region spanning the internal transcribed spacer region of the ribosomal DNA, the nuclear gene encoding heat shock protein 90 and the mitochondrial cox1 gene were PCR amplified and sequenced. Based on phylogenetic analysis and morphological and physiological comparison, four species and one informally designated taxon have been described; Phytophthora gibbosa, P. gregata, P. litoralis, P. thermophila and P. taxon paludosa. Phytophthora gibbosa, P. gregata and P. taxon paludosa form a new cluster and share a common ancestor; they are homothallic and generally associated with dying vegetation in swampy or water-logged areas. Phytophthora thermophila and P. litoralis are sister species to each other and more distantly to P. gonapodyides. Both new species are common in waterways and cause scattered mortality within native vegetation. They are self-sterile and appear well adapted for survival in an aquatic environment and inundated soils, filling the niche occupied by P. gonapodyides and P. taxon salixsoil in the northern hemisphere. Currently the origin of these new taxa, their pathogenicity and their role in natural ecosystems are unknown. Following the precautionary principle, they should be regarded as a potential threat to native ecosystems and managed to minimise their further spread

Current and projected global distribution of Phytophthora cinnamomi, one of the world's worst plant pathogens

Globally, Phytophthora cinnamomi is listed as one of the 100 worst invasive alien species and active management is required to reduce impact and prevent spread in both horticulture and natural ecosystems. Conversely, there are regions thought to be suitable for the pathogen where no disease is observed. We developed a climex model for the global distribution of P. cinnamomi based on the pathogen's response to temperature and moisture and by incorporating extensive empirical evidence on the presence and absence of the pathogen. The climex model captured areas of climatic suitability where P. cinnamomi occurs that is congruent with all available records. The model was validated by the collection of soil samples from asymptomatic vegetation in areas projected to be suitable by the model for which there were few records. DNA was extracted, and the presence or absence of P. cinnamomi was determined by high-throughput sequencing (HTS). While not detected using traditional isolation methods, HTS detected P. cinnamomi at higher elevations in eastern Australia and central Tasmania as projected by the climex model. Further support for the climex model was obtained using the large data set from south-west Australia where the proportion of positive records in an area is related to the Ecoclimatic Index value for the same area. We provide for the first time a comprehensive global map of the current P. cinnamomi distribution, an improved climex model of the distribution, and a projection to 2080 of the distribution with predicted climate change. This information provides the basis for more detailed regional-scale modelling and supports risk assessment for governments to plan management of this important soil-borne plant pathogen

Distribution and diversity of Phytophthora across Australia

The introduction and subsequent impact of Phytophthora cinnamomi within native vegetation is one of the major conservation issues for biodiversity in Australia. Recently, many new Phytophthora species have been described from Australia's native ecosystems; however, their distribution, origin, and potential impact remain unknown. Historical bias in Phytophthora detection has been towards sites showing symptoms of disease, and traditional isolation methods show variable effectiveness of detecting different Phytophthora species. However, we now have at our disposal new techniques based on the sampling of environmental DNA and metabarcoding through the use of high-throughput sequencing. Here, we report on the diversity and distribution of Phytophthora in Australia using metabarcoding of 640 soil samples and we compare the diversity detected using this technique with that available in curated databases. Phytophthora was detected in 65% of sites, and phylogenetic analysis revealed 68 distinct Phytophthora phylotypes. Of these, 21 were identified as potentially unique taxa and 25 were new detections in natural areas and/or new introductions to Australia. There are 66Phytophthora taxa listed in Australian databases, 43 of which were also detected in this metabarcoding study. This study revealed high Phytophthora richness within native vegetation and the additional records provide a valuable baseline resource for future studies. Many of the Phytophthora species now uncovered in Australia's native ecosystems are newly described and until more is known we need to be cautious with regard to the spread and conservation management of these new species in Australia's unique ecosystems

Control of Phytophthora cinnamomi with phosphite: some recent developments in application methods

Phytophthora cinnamomi has a world-wide distribution, causes disease in a very wide range of plants and is responsible for the destruction of certain plant communities in Europe and Australia. P. cinnamomi was probably introduced into Australia in the nineteenth century and is now established in south-western Australia and Tasmania, and throughout eastern Australia, from South Australia to the wet tropics. P. cinnamomi is listed as a Key Threatening Process under the Commonwealth Environment Protection and Biodiversity Conservation Act 1999

Factors affecting the establishment of selected edible ectomycorrhizal fungi of Pinus in South-Western Australia

Establishment of a new industry in plantations of Pinus spp. in south-western Australia, based on edible ectomycorrhizal fungi, will be dependant on the ability of new fungal introductions to survive and proliferate under limitations imposed by biotic and abiotic factors. This thesis investigated factors that would influence establishment of two most valuable fungi, Matsutake (Tricholoma matsutake) and the Pine Mushroom {T. magnivelare). Factors that were investigated included, extant ectomycorrhizal fungi, plant host - fungus compatibility, climate and soils. A survey of ectomycorrhizal (ECM) fimgi associated with introduced Pinus spp. in south-western Australia increased the number of identified species to 15-16 species, from seven previously reported in the literature. Most species form a sub-set of species that have also been introduced to Eastern Australia, and are early colonizers of pines, forming ECM associations with pine seedlings, but are also found with plantationgrown pine trees older than sixty years. Four species of basidiomycetes with resupinate fruitbodies, are new records for Australia, and probably for the Southern Flemisphere. One resupinate fungus (Amphinema byssoides) has been introduced. In contrast, three Tomentella spp., that are part of a suite of at least 25 tomentelloid fungi found in native forests and woodlands in south-western Australia, appear to have made a transition from native to introduced Pinus hosts. Species within other genera of resupinate fungi, that are known to form ECM associations with pines in the Northern Hemisphere, were also identified but not confirmed as ECM fungi of pines in Western Australia. rDNA sequence data strongly suggested that some previous records of ECM fungi have been mis-identified. In two field experiments, Thelephoraceae {Thelephora terrestris and Tomentella stuposa) formed 50-60% of ectomycorrhizas but sporocarps of both Ilmgi were found infrequently or not at all in surveys. The apparent below-ground dominance by representatives of the Thelephoraceae, as ECM fungi of pines in south-western Australia, is similar to some results from natural conifer forests in Europe and North America. Sporocarp production, by naturally occurring pine ECM fungi, was measured over a season in experimental plots of five year-old P. radiata trees, that comprised trees planted with a standard treatment and trees planted for agro-forestry, with additional P fertilization and a cover crop of the legume Trifolium subterraneum. Sporocarp production in agro-forestry plots was estimated to be at least ca. 73 kg'1 ha'1 yr'1 d.w., that was significantly greater than that of the standard treatment, and up to an order of magnitude greater than productivity in natural conifer forests. There were also changes in phenology of sporocarp production by different species of fungi between treatments. Comparison of climate between selected locations within the ranges of T. matsutake and T. magnivelare, and locations within south-western Australia strongly suggests that T. magnivelare, and T. nauseosum, syn. T. matsutake from the Western Mediterranean, are more suited to the climate of south-western Australia than Asian T. matsutake. Soils within south-western Australia that are potentially suitable for Matsutake, that are derived from similar parent materials and with similar physical and chemical properties, occupy an undefined proportion of about 3250 km'2 of soil sub-systems within the area that may be climatically suitable. In vitro synthesis experiments between isolates of Tricholoma matsutake and T.magnivelare and host plants, Pinus dens [flora, P. radiata and P. pinaster, showed that internal structures comparable with natural Pinus densiflora-T. matsutake ECM were formed between P. radiata and T. matsutake. Macro- and micro-morphological features of infected short roots suggested that P. radiata and P. pinaster are suitable hosts for both T. matsutake and T. magnivelare. In contrast to in vitro synthesis experiments, ECM associations failed to form between T. matsutake and mature P. radiata trees after inoculation with two forms of vegetative mycelium, and ECM also failed to form between T. matsutake and Pinus spp. inoculated with two forms of vegetative mycelium under more controlled conditions in a glasshouse experiment

Molecular testing uncovers new Phytophthora taxa from natural ecosystems in Western Australia

Verification of mapping of the extent of Phytophthora dieback disease, based on shadowless colour aerial photography, involves the routine testing of soil and root samples collected from beneath dying, Phytophthora-sensitive native plant “indicator species” for the presence of the pathogen. In addition to P. cinnamomi, other isolates have been recovered on selective agar following the baiting of soil, or the direct plating of plant tissue, during these operations. These have been identified, using morphological characters, as P. citricola, P. megasperma, P. cryptogea, P. drechsleri, P. nicotianae, and P. boehmeriae

Containment and eradication of Phytophthora cinnamomi in native vegetation in South-Western Australia and Tasmania

The aim of our experiments was to develop protocols that can be used to contain and eradicate spot infestations of P. cinnamomi that, if untreated, are likely to threaten extensive areas of native vegetation or areas of high conservation value. Treatment regimes were guided by two assumptions: 1) within the selected sites, transmission of the pathogen is by root-to-root contact, and 2) the pathogen is a weakly competitive saprotroph. In Western Australia (WA), treatment and control plots were set-up along an active disease front within scrub-heath vegetation dominated by Banksia spp. Treatments, applied sequentially and in combination, included: 1) destruction of the largest plants within disease free vegetation forward of the disease front; 2) destruction of all plants to create a ‘dead zone’; 3) installation of physical root barriers and subsurface irrigation for the application of fungicide/s; 4) surface applications of fungicides selective against oomycetes (triadiazole and metalaxyl-M), and 5) surface injection and deep (± 1 m) treatments with Metham-sodium. In a separate experiment in Tasmania (TAS), combined treatments including vegetation removal, Ridomil and Metham-sodium and root barriers, or Ridomil and root barriers alone, were applied to experimental plots within active disease centres in Eucalyptus-Banksia woodland. In the WA experiment, P. cinnamomi was not recovered (by soil baiting) from plots after treatment with Ridomil and metham-sodium. In the TAS experiment, similar results were achieved with combined treatments (vegetation removal + Ridomil + metham sodium) but in plots treated with Ridomil alone, recoveries of P. cinnamomi increased after initially showing a significant reduction in recoveries

The diversity of ectomycorrhizal fungi associated with introduced Pinus spp. in the Southern Hemisphere, with particular reference to Western Australia

Although pines have been established in plantations in Western Australia for over 100 years, knowledge of the ectomycorrhizal fungal flora is incomplete, or lies in unpublished reports. A survey of ectomycorrhizal fungi associated with Pinus spp. was conducted throughout south-western Australia. Compared with other regions in the Southern Hemisphere where pines have been introduced, the ectomycorrhizal flora of pines in Western Australia is particularly depauperate, with only nine species of fungi identified from sporocarps and a further two taxa identified from mycorrhizas. Species identified from sporocarps (Hebeloma crustuliniforme, Lactarius deliciosus, Paxillus involutus, Rhizopogon luteolus, R. roseolus, R. vulgaris, Suillus luteus, S. granulatus, Thelephora terrestris) and Cenococcum geophilum are a subset of a larger pine mycorrhizal flora found in eastern Australia, and 8 of the 10 identified species are common to all regions in the Southern Hemisphere where pines have been introduced. These fungi are typically associated with trees, including pines, in the Northern Hemisphere and, apart from Cenococcum geophilum and T. terrestris, are not associated with indigenous vegetation in Western Australia. The mycorrhizal flora colonising roots in a plantation of Pinus radiata D. Don was also investigated, and compared with species identified as present by above-ground sporocarp production. Potential reasons for the limited ectomycorrhizal flora of pines in Western Australia are discussed