Bill Dunstan

Damping-off oomycete and fungal plant pathogens reduce the germination, emergence, and survival of seedlings. In agricultural systems, this poses a significant problem as it may reduce productivity; in contrast, the impact is more subtle in natural ecosystems and may be essential for maintaining the structure and diversity of plant communities. The oomycete genus Phytophthora is frequently detected in natural plant communities causing disease in mature plants, but it is rarely assessed as a damping-off pathogen. A pathogenicity experiment was established with five oomycete treatments, three putatively native Phytophthora species, the invasive P. cinnamomi, and Pythium irregulare, to determine the damping-off host range in selected plant species from a hyper-diverse Mediterranean-type ecosystem. Additionally, plant and seed traits were analysed to determine if they were correlated with susceptibility to pre-emergent damping-off. Phytophthora arenaria was the only putatively native generalist damping-off pathogen, causing 12 of 19 plant species to experience significantly reduced seedling emergence or survival relative to the control treatment. Pythium irregulare and the introduced P. cinnamomi caused significant damping-off for 12 and 13 plant species, respectively. Plant species that were slowest to germinate and emerge were the most susceptible to pre-emergence damping-off caused by P. arenaria. These results suggest native and invasive Phytophthora may substantially influence the structure and diversity of natural plant communities through damping-off. While mature plant species are the most common focus in Phytophthora research, our study indicates damping-off should be considered to determine the full impact of these soil-borne plant pathogens.

DNA and RNA detected in soil using molecular techniques may originate from a living or dead organism. It is therefore of interest to know how long the DNA and RNA from a decaying organism can persist in soil, and how environmental conditions such as soil temperature, moisture, and microbial populations impact on the survival time. This study determined the difference between the persistence of Phytophthora cinnamomi mRNA and DNA in different soil types. DNA and RNA were extracted from P. cinnamomi and 10 ng/250 mg of soil was applied to five different soil types that were either air‐dried or maintained at 70% field capacity. The persistence of DNA at 20°C was tested after intervals of 0, 3, 7, 14, 90, 241, and 378 days, and for RNA at 0, 1, 3, and 7 days using qPCR and RT‐qPCR techniques, respectively. Persistence was longer in dry than moist soil, P. cinnamomi DNA could be readily detected in dry soil conditions for up to 90 days and was found at extremely low levels at 241 and 378 days. RNA was detected only on day 1, except for dry river sand, and moist sandy loam in which it persisted for 3 days; it was not detected after seven days. These results confirm that RNA degrades very quickly, making it a valuable tool for determining the presence of viable Phytophthora in soil. In contrast, DNA can be remarkably stable in some environments, and positive results could be obtained even after the death of the organism for a year or more prior to the test. For diagnostics, the use of an RNA‐based test avoids the possibility of such false positive results. In the context of the research project, this study is relevant to determining how long viable Phytophthora remains in soil after the eradication protocols have been instigated. In a broader context, the persistence of DNA is relevant to any study using environmental DNA for diagnostics or for metabarcoding when undertaking community ecology or microbiome studies. These results are relevant for studies using detection of P. cinnamomi nucleic acids in soils for purposes of diagnostics, ecological research, or projects on eradication.

Severe tree decline in natural ecosystems around the world has driven Phytophthora research, resulting in a better understanding of the diversity and association of Phytophthora species with different host plants. Improved molecular techniques have contributed significantly to that understanding. The devastating impact of Phytophthora dieback in native vegetation in the southwest of Western Australia (SWWA) has motivated a committed research effort to understand the survival and spread of this pathogen. Hot, dry summers characterise the climate of this ecosystem, and are supposedly unfavourable for a moisture-loving pathogen to survive, spread and thrive. However, Phytophthora cinnamomi can survive within the roots of native plant species allowing for its persistence. Random plant sampling, with metabarcoding from root samples, revealed the presence of at least 23 Phytophthora species on 18 of the 20 plant species growing on mining stockpiles of the Huntly mine site (Alcoa of Australia Limited) in SWWA. Phytophthora cinnamomi was detected on 16 of the 20 plant species. This finding supports the idea that native plant species have a significant role in the survival and spread of P. cinnamomi in the environment. The presence of other Phytophthora species challenges the assumption that P. cinnamomi is the main cause of Phytophthora dieback; the disease may be complex, involving several Phytophthora species. These unexpected detections indicate that plants in the natural forest can serve as the reservoir of inoculum of not only P. cinnamomi but also for other Phytophthora species.

While eradication from haul roads was achieved, more work is required to eradicate P. cinnamomi from stockpiles and bunds. We can now implement different management strategies to the construction of bunds and stockpiles to facilitate eradication. Infestation by Phytophthora cinnamomi results in large financial and management constraints to environmental managers. This pathogen was considered impossible to eradicate until recent success with treatments including host removal, herbicide and fungicide application, soil fumigation and physical root barriers. We investigated the most benign of these treatments; keeping the area devoid of living host material. In a Western Australian mine site within a Mediterranean climate, haul roads, stockpiles and roadside bunds had P. cinnamomi colonised Pinus stem plugs buried at multiple depths. Over time, we examined the effects of soil moisture and temperature in different soil conditions and types to compare the recovery of the pathogen. Results: Within 12 months, the pathogen could not be recovered from the haul roads. In the stockpiles, depth produced significantly different results. In 3 of the 4 sites, the pathogen was not recovered at 10 cm after 20 months. By 12 months, at 50 cm, there was an 80% reduction in recovery, but only one stockpile had no recovery from 50 cm, which occurred by 36 months. Bunds were up to 1.75 m high and had variable results for plugs buried at 30 cm, influenced by height, the types of soils and shading. One of the smallest bunds was the only bund where the pathogen was not recoverable (by 22 months). This study provides strong support for using a fallow period to reduce or eliminate P. cinnamomi inoculum.

The genus Phytophthora contains species that are major pathogens worldwide, affecting a multitude of plant species across agriculture, horticulture, forestry, and natural ecosystems. Here, we concentrate on those species that are dispersed through soil and water, attacking the roots of the plants, causing them to rot and die. The intention of this study was to compare the soil baiting protocol developed by the Centre for Phytophthora Science and Management (CPSM) with two other baiting methods used in Australia. The aim was to demonstrate the effectiveness of each protocol for soil baiting Phytophthora species in different substrates. Three experiments were conducted: the first to test the sensitivity of each method to detect Phytophthora cinnamomi, the second to test the effect of substrate type (sand or loam), and the third to test the detection of species (P. cinnamomi, P. multivora, or P. pseudocryptogea). The specificity of different plant species baits was compared within and between the methods. Substrate type influenced isolation in all methods; however, the CPSM method was superior regardless of substrate, albeit slower than one of the other methods for one substrate. Comparing bait species between the three methods, Quercus ilex was the most attractive bait for P. cinnamomi, particularly in the CPSM method. The choice of protocol affected the isolation associated with each bait type. Overall, the multiple bait system used by CPSM was shown to provide the most sensitive and reliable detection of Phytophthora species from soil samples.

The mode of persistence of Phytophthora cinnamomi, a highly aggressive soil‐ and water‐borne pathogen, remains unclear. This study investigated the survival of viable oospores and chlamydospores of P. cinnamomi when present as free propagules in untreated soil, or in soil subject to four exogenous treatments: smoke water, fish emulsion and two fungicides (ridomil and furalaxyl). The exogenous treatments were applied under moist and dry soil conditions. Spore viability was determined by the thiazolyl blue tetrazolium bromide (MTT) staining technique, with a qPCR assay used to compare general patterns of decline. Over 96% of oospores lost viability over a period of 48 weeks irrespective of soil moisture conditions. The mean percentage viability for oospores decreased from 91% at time zero to 72, 35, 20 and 1% after 6, 12, 24 and 48 weeks, respectively. Reduction in viability of chlamydospores was more rapid than oospores, with viability declining from 92% to zero after 12 weeks. There was no significant difference between untreated soil and the exogenous treatments. The RNA‐based qPCR assay indicated a strong presence of viable oospores of P. cinnamomi up to week 12 for moist soil and week 3 for dry soil, but thereafter failed to detect RNA even though viable oospores could be detected by MTT staining. Based on the MTT staining, this study indicated that viability of P. cinnamomi oospores may be entirely lost within 1 year and that of chlamydospores within 3 months for the soil type tested. Therefore, oospores and chlamydospores when existing as free propagules in soil appear unlikely to be involved in the long‐term survival of P. cinnamomi.

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.

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.

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.

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.