Three novel species from Phytophthora ITS Clade 9 are described from Australia and South Africa. Phylogenetic analyses were performed using data from nuclear regions (ITS, β-tubulin, heat shock protein 90 and elongation factor) and mitochondrial regions (cytochrome c oxidase subunit 1 and NADH dehydrogenase subunit 1). The three novel species share typical Clade 9 features, including non-papillate, persistent sporangia with broad, flat apices and frequent internal proliferation, and are either homothallic or sterile. Each species is nonetheless separable from related species by a distinct combination of morphological characters and temperature profiles. The molecular and morphological data supported recognition of three novel species: Phytophthora stellaris in Clade 9b, and P. brevifila and P. calida in the newly proposed Clade 9e. Surveys in Australia and South Africa also revealed additional but as yet undescribed taxa, including several single-isolate lineages and a complex of polymorphic isolates closely related to P. honggalleglyana and P. virginiana, likely of hybrid origin. These findings expand the known diversity, morphology and biogeography of Clade 9. Although few Clade 9 species are currently associated with serious plant disease, documenting their diversity is critical for establishing baselines, clarifying species placement and informing future monitoring. By formally describing novel species and recognising unresolved complexes, this study provides an essential reference point for researchers navigating one of the most taxonomically complex clades of Phytophthora.

Cryptotermes brevis is one of the most destructive invasive termites in the subtropics and tropics and is a common biosecurity intercept at the Australian border. Drywood termite species are cryptic and difficult to identify morphologically in situations when soldiers or imagos are unavailable. We developed a novel DNA based loop-mediated isothermal amplification (LAMP) assay to detect C. brevis and differentiate it from other drywood termites. Validated voucher specimens of 30 different drywood termite species were obtained from several insect collections from which DNA was extracted and amplified. The amplicons containing partial mitochondrial 16S rRNA were sequenced and a DNA database was created from which C. brevis LAMP primers were developed, optimized, and tested. The assay was assessed against a range of target and non-target species and found to be specific, successfully amplifying the target specimens of C. brevis in under 30 min. Amplification success was variable against C. brevis faecal pellets due to minute, unmeasurable or degraded DNA. This LAMP test is a new tool for the rapid detection of C. brevis that will enable faster and less destructive management of drywood termite infestations.

Many oomycetes are important plant pathogens that cause devastating diseases in agricultural fields, orchards, urban areas, and natural ecosystems. Limitations and difficulties associated with isolating these pathogens have led to a strong uptake of DNA metabarcoding and mass parallel sequencing. At least 21 primer combinations have been designed to amplify oomycetes, or more specifically, Phytophthora species, from environmental samples. We used the Illumina sequencing platform to compare 13 primer combinations on mock communities and environmental samples. The primer combinations tested varied significantly in their ability to amplify Phytophthora species in a mock community and from environmental samples; this was due to either low sensitivity (unable to detect species present in low concentrations) or a lack of specificity (an inability to amplify some species even if they were present in high concentrations). Primers designed for oomycetes underestimated the Phytophthora community compared to Phytophthora-specific primers. We recommend using technical replicates, primer combinations, internal controls, and a phylogenetic approach for assigning a species identity to OTUs or ASVs. Particular care must be taken if sampling substrates where hybrid species could be expected. Overall, the choice of primers should depend upon the hypothesis being tested.

Several species from the genus Quambalaria (order Microstromatales) cause diseases on eucalypts (Eucalyptus and related genera) both in plantations and natural ecosystems. We developed real-time quantitative PCR (qPCR) assays to rapidly detect and distinguish five Quambalaria species. The design of the species-specific qPCR assay for each species, Q. pitereka (PIT), Q. coyrecup (COR), Q. cyanescens (CYN), Q. pusilla (PUS), and Q. eucalypti (EUC), was based on the ITS region and was evaluated for specificity and sensitivity. The PIT, COR, and CYN qPCR assays could amplify as little as 10 fg µl-1 from pure cultures, whereas PUS and EUC qPCR assays could amplify 100 fg µl-1 of their target species. The PIT, COR, and CYN qPCR assays were further validated using naturally and artificially infected samples of their plant host Corymbia calophylla. These assays will be used for rapid diagnostics and future experiments on the infection process.

AbstractDNA 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.

A diverse Phytophthora community was detected in recent surveys conducted in alpine and subalpine areas, previously considered Phytophthora free. The current study was conducted to determine patterns of Phytophthora species richness and distribution along a steep elevation gradient, and to compare these patterns with those of vascular plant species. Phytophthora and vascular plant species were recorded over a wide range of elevation gradient (410–2125 m) and across a disturbance boundary. Vascular plant species exhibited a monotonic decline with increasing elevation. With the exception of native Phytophthora species isolated by baiting, Phytophthora species richness was invariant in relation to elevation and had higher elevational ranges than vascular plant species. Vascular plants occurred in discrete plant communities with introduced species more frequently recorded in road habitat and native species more frequently recorded in natural vegetation habitat. Both native and introduced Phytophthora species occurred with equal frequency in road and natural vegetation habitat. Phytophthora species were absent from one-third of sample plots and plots with no Phytophthora species were randomly distributed across landscapes. Only two Phytophthora species repeatedly occurred with a particular plant community. Our findings show that Phytophthora species are habitat generalists, being widely distributed across elevation and disturbance gradients, while vascular plant species are mostly habitat specialists, being confined to particular environments within narrow elevation bands. The effect of Phytophthora species on vascular plant species is largely unknown but the fact that Phytophthora species are already present throughout the elevation and disturbance gradients warrants closer examination of plant-pathogen relationships.

Phytophthora cinnamomi causes root and collar rot in many plant species in natural ecosystems and horticulture. A species-specific primer and probe PCIN5 were designed based on a mitochondrial locus encoding subunit 2 of cytochrome c oxidase (cox2). Eight PCR primers, including three forward and five reverse, were designed and tested in all possible combinations. Annealing temperatures were optimized for each primer pair set to maximize both specificity and sensitivity. Each set was tested against P. cinnamomi and two closely related clade 7 species, P. parvispora and P. niederhauseri. From these tests, five primer pairs were selected based on specificity and, with a species-specific P. cinnamomi probe, used to develop quantitative real-time PCR (qPCR) assays. The specificity of the two most sensitive qPCR assays was confirmed using the genomic DNA of 29 Phytophthora isolates, including 17 isolates of 11 species from clade 7, and representative species from nine other clades (all except clade 3). The assay was able to detect as little as 150 ag of P. cinnamomi DNA and showed no cross-reaction with other Phytophthora species, except for P. parvispora, a very closely related species to P. cinnamomi, which showed late amplification at high DNA concentrations. The efficiency of the qPCR protocol was evaluated with environmental samples including roots and associated soil from plants artificially infected with P. cinnamomi. Different RNA isolation kits were tested and evaluated for their performance in the isolation of RNA from environmental samples, followed by cDNA synthesis, and qPCR assay. Finally, a protocol was recommended for determining the presence of P. cinnamomi in recalcitrant environmental samples.

Proper isolation and identification of Phytophthora species is critical due to their broad distribution and huge impact on natural ecosystems throughout the world. In this study, five different sites were sampled and seven methods were compared to determine the Phytophthora community. Three traditional isolation methods were conducted (i) soil baiting, (ii) filtering of the bait water and (iii) isolation from field roots using Granny Smith apples. These were compared to four sources of eDNA used for metabarcoding using Phytophthora-specific primers on (i) sieved field soil, (ii) roots from field, (iii) filtered baiting water and (iv) roots from bait plants grown in the glasshouse in soil collected from these sites. Six Phytophthora species each were recovered by soil baiting using bait leaves and from the filtered bait water. No Phytophthora species were recovered from Granny Smith apples. eDNA extracted from field roots detected the highest number of Phytophthora species (25). These were followed by direct DNA isolation from filters (24), isolation from roots from bait plants grown in the glasshouse (19), and DNA extraction from field soil (13). Therefore, roots were determined to be the best substrate for detecting Phytophthora communities using eDNA.

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.

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 66 Phytophthora 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.

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.

This RIRDC report describes the potential impacts of pruning on the heartwood rot fungal diseases within Indian sandalwood (Santalum album) in Australia associated with pruning. This report is targeted towards Indian sandalwood plantation growers. Whilst the focus of this report is sandalwood, the findings will be applicable to other tropical tree species.

The riparian zone in Western Australia is dominated by Eucalyptus rudis in a similar manner to Alnus spp. in Europe. For the last 20 years the health of these trees have been declining. This is attributed to an endemic leaf sucking Psyllid, however contributing factors could be an increase in the salinity of the waterways or the presence of a root pathogen, both of which would reduce the health of the trees. We sampled 25 sites along different rivers and streams in the southwest of Western Australia. At each site we recorded tree health, determined water quality and filtered water for the isolation of pythiaceous oomycetes. There was considerable variation in water quality (pH and salinity) and the health of the adjacent E. rudus, however the poor health w as not related to low water quality. There was also considerable variation in the number of colony forming units (from 1.33 to 90 L‐1), the proportion of Phytophthora compared to Pythium isolates (from 0‐100%) and the species biodiversity. In general, far more isolates were obtained from low quality water, except for when the pH was greater than 8.5. Water quality did not effect the proportion of Phytophthora isolates. Phytophthora species isolated included P. thermophila, P. fluvialis, P. amnicola and hybrids between these species. Additionally, numerous isolates of P. taxon salixsoil were obtained. Remnant sites closer to the urban area contained predominantly P. thermophila while P. taxon salixsoil predominated on the more southerly sites from remnants within agricultural zones. The link between tree health, water quality and associated pythiaceous populations was not established in this study.

Twenty eight species within Mycosphaerellaceae and Teratosphaeriaceae (includes Mycosphaerella, Teratosphaeria, Pseudocercospora and Sonderhenia) are reported from Eucalyptus and Corymbia in New South Wales and Queensland, Australia, based on field surveys and examination of herbarium specimens and published reports. Teratosphaeria cryptica was the most commonly recorded species, with the widest host range and distribution, followed by Mycosphaerella marksii. Six new species are described: T. keanei, T. coolabuniensis, T. crispata, M. medusae, M. nootherensis and T. praelongispora. New or interesting records for known species are reported, including new records for Australia (T. pluritubularis and T. verrucosiafricana) and new records for Queensland (T. excentrica, T. multiseptata, T. nubilosa, T. suberosa and Ps. pseudoeucalyptorum).

For 30 years, large-scale aerial photography has been used to map the extent of Phytophthora dieback disease in native forests in the southwest of Western Australia, with validation of the observations involving routine testing of soil and root samples for the presence of Phytophthora cinnamomi. In addition to P. cinnamomi, six morpho-species have been identified using this technique: P. citricola, P. megasperma, P. cryptogea, P. drechsleri, P. nicotianae, and P. boehmeriae. In recent years, many new Phytophthora species have been described worldwide, often with similar morphology to existing species; thus, as many of the isolates collected in Western Australia have been difficult to identify based on morphology, molecular identification of the morpho-species is required. Based on amplification of the internal transcribed spacer (ITS) region of the rDNA gene, sequence data of more than 230 isolates were compared with those of existing species and undescribed taxa. P. inundata, P. asparagi, P. taxon PgChlamydo, P. taxon personii, and P. taxon niederhauserii were identified based on sequence data. Phylogenetic analysis revealed that nine potentially new and undescribed taxa can be distinguished. Several of the new taxa are morphologically indistinguishable from species such as P. citricola, P. drechsleri, and P. megasperma. In some cases, the new taxa are closely related to species with similar morphology (e.g., P.sp.4 and P. citricola). However, the DNA sequences of other new taxa such as P.sp.3 and P.sp.9 show that they are not closely related to morphologically similar species P. drechsleri and P. megasperma, respectively. Most of the new taxa have been associated with dying Banksia spp., while P.sp.2 and P.sp.4 have also been isolated from dying Eucalyptus marginata (jarrah). Some taxa (P.sp.3, 6, and 7) appear to have limited distribution, while others like P.sp.4 are widespread.

Phytophthora cinnamomi has had a huge impact on natural ecosystems in Western Australia. For 29 years the extent of the disease in native forests has been mapped based on large-scale aerial photography, with validation of observations involving the routine testing of soil and root samples for the presence of the pathogen. In addition to P. cinnamomi, six other morphological species have been reported from native ecosystems in Western Australia: P. citricola, P. megasperma, P. cryptogea, P. drechsleri, P. nicotianae and P. boehmeriae. Within the collection there were many isolates that were difficult to identify based on morphology and as more Phytophthora species have been described with similar morphology it was realised that molecular identification of some of the morphological species was required. Thus, the internal transcribed spacer (ITS) region of the rDNA gene has been amplified and sequence data compared to that of known species. Based on phylogenetic analysis, nine potentially new and undescribed taxa can be distinguished. In addition P. inundata, P. gonapodyides, and P. sp. asparagi and P. sp. niederhauseria were identified based on sequence data. Several of the new species are morphologically indistinguishable from known species (eg P. citricola, P. drechsleri, P. megasperma). In some cases the new taxa are indeed most closely related to the known species (eg P.sp. 4 and P. citricola). However, the DNA sequences of other new taxa show that they are not closely related to the morphologically similar species (eg P.sp. 3 and P. drechsleri, P.sp. 9 and P. megasperma). Most of the new species have been associated with dying Banksia spp. whilst P.sp. 2 and P.sp. 4 have also been isolated from Eucalyptus marginata (jarrah). Some species (eg P.spp. 3, 6 and 7) appear to have limited distribution, whilst others (eg P.sp.4) are more widespread. Further work is planned to describe the new taxa and to test their pathogenicity.

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.

The Vegetation Health Survey (VHS) at the Department of the Environment, Western Australia has a Phytophthora collection extending back to 1979. Isolates in this collection have been recovered during routine monitoring on natural ecosystems in Western Australia for the presence of Phytophthora cinnamomi. Through molecular reevaluation of this collection we have subsequently described 11 new Phytophthora species and the diseases associated with them and additional descriptions are underway. Elsewhere in Australia, however, there is extremely limited information on Phytophthora diversity within natural ecosystems. Using modern molecular techniques such as Next Generation Sequencing, it is possible to determine Phytophthora species diversity from environmental soil samples. In this study, DNA was extracted from soils obtained from 700 locations around Australia. ITS1 amplicons were generated using Phytophthora specific primers (Scibetta et al. 2012) adapted for NGS by Santi Català and sequenced on a Roche Junior GS platform. For 50 samples roots and rhizosphere soil were extracted separately. Results reveal an astonishing diversity, several new species and very different species profiles when comparing roots and rhizosphere soil from the same location. Species described and known only from Western Australia have an Australia-wide distribution raising intriguing questions in regards to origin and movement of species.