Phytophthora Communities Research Articles

Impacts of phosphite treatment on Phytophthora community assemblages and inoculum abundances in Phytophthora-infected forest soil

Phytophthora are causing declines in forest tree species worldwide and the chemical control treatment, phosphite, is the only treatment consistently shown to provide some protection to natural ecosystems from Phytophthora diseases. Phosphite inhibits Phytophthora growth and sporulation whilst boosting defence responses in the plant host. It is unclear, however, the extent of the impact of phosphite on Phytophthora species assemblages and inoculum abundances in soil around trees following treatment within natural ecosystems. In New Zealand, kauri (Agathis australis), an endemic and threatened foundation species, suffers from a dieback disease primarily caused by Phytophthora agathidicida. Phosphite is applied by trunk injection to kauri and has been shown to improve resin ‘bleed’ symptoms from basal trunk lesions and to promote recovery of thinned canopies. Phytophthora community and inoculum abundance were investigated in response to phosphite treatments at two field sites (Huia and Waitoki) in infected kauri stands in Auckland, New Zealand. At Huia, soil sampling and tree health surveying were conducted in November 2023 on trees treated with phosphite in 2012 as part of an earlier study. At Waitoki, the response to phosphite treatment was monitored 6 and 18 months following treatment. Phytophthora species were detected using soil baiting and metabarcoding of environmental DNA (eDNA) from soil and quantified with qPCR of root and soil DNA. Three species were detected with soil baiting (P. agathidicida, P. cinnamomi, and P. multivora) and two additional species with metabarcoding (P. pseudocryptogea, and an unknown clade 7 species similar to P. europaea). Phytophthora cinnamomi was the most abundant species, followed by P. agathidicida. Both species were more likely to occur together than by chance alone and were associated with declining tree health. The P. europaea-like species was in approximately 50% of the samples and was less likely to occur in roots with poorer health, or in association with P. agathidicida. The abundance of P. agathidicida inoculum was lower in the soil around the phosphite-treated trees than around the untreated control trees 1.5 years after treatment at Waitoki. Phosphite halted the lateral expansion of basal resin bleeds, and resin viscosity was reduced. Not only did phosphite treatments improve kauri dieback symptoms, but the phosphite treatments potentially had a direct impact on the epidemiology of the disease by reducing inoculum load around treated trees, with direct implications for disease management as an effective way to protect uninfected areas and minimise the spread of inoculum from infested zones.

Full-length ITS amplicon sequencing resolves Phytophthora species in surface waters used for orchard irrigation in California's San Joaquin Valley.

Diverse Phytophthora species, including many important plant pathogens, have been widely detected among surface water irrigation sources. In the past decade, metabarcoding has been used to characterize waterborne Phytophthora populations. Metabarcoding typically involves amplification of portions of the nuclear ribosomal internal transcribed spacer (ITS)1 or ITS2 from Phytophthora species, followed by indexed high throughput sequencing. However, full-length sequences of the entire ITS region are required for resolution of many Phytophthora species. We used metabarcoding with PacBio sequencing of full-length ITS amplicons to analyze populations of Phytophthora in waterways of the Stockton East Water District (SEWD) in the northern San Joaquin Valley of California. This approach yielded species-level resolution of many members of the Phytophthora community. Results were compared to those obtained by using ITS1 or ITS2 regions alone and were found to provide superior species resolution for P. pini, P. capsici, and P. gregata. Samples were collected throughout the 2021 irrigation season from five waterways across the SEWD. Thirty-eight Phytophthora species were detected in the waterways, including tree-crop pathogens P. acerina, P. cactorum, P. pini, P. ×cambivora, P. niederhauserii, P. mediterranea, and P. taxon walnut. These pathogenic species were detected throughout the SEWD during most of the irrigation season. The results demonstrated the utility of full-length ITS amplicon sequencing for identifying Phytophthora species in environmental samples and suggested that some disease risk may be incurred by orchardists irrigating with SEWD water. Additional epidemiological studies will be required to critically evaluate this risk.

Climate acts as an environmental filter to plant pathogens.

Climate shapes the distribution of plant-associated microbes such as mycorrhizal and endophytic fungi. However, the role of climate in plant pathogen community assembly is less understood. Here, we explored the role of climate in the assembly of Phytophthora communities at >250 sites along a latitudinal gradient from Spain to northern Sweden and an altitudinal gradient from the Spanish Pyrenees to lowland areas. Communities were detected by ITS sequencing of river filtrates. Mediation analysis supported the role of climate in the biogeography of Phytophthora and ruled out other environmental factors such as geography or tree diversity. Comparisons of functional and species diversity showed that environmental filtering dominated over competitive exclusion in Europe. Temperature and precipitation acted as environmental filters at different extremes of the gradients. In northern regions, winter temperatures acted as an environmental filter on Phytophthora community assembly, selecting species adapted to survive low minimum temperatures. In southern latitudes, a hot dry climate was the main environmental filter, resulting in communities dominated by drought-tolerant Phytophthora species with thick oospore walls, a high optimum temperature for growth, and a high maximum temperature limit for growth. By taking a community ecology approach, we show that the establishment of Phytophthora plant pathogens in Europe is mainly restricted by cold temperatures.

Phytophthora in Horticultural Nursery Green Waste—A Risk to Plant Health

Phytophthora is a genus of destructive plant pathogens. Certain species are damaging to native ecosystems, forestry, and the horticultural sector, and there is evidence of their dissemination in plant imports. Horticultural nurseries are central nodes of the plant trade and previous studies have found a high diversity of Phytophthora associated with plant nursery stock. It was subsequently hypothesized that green waste disposal sites in nurseries could harbour diverse Phytophthora communities and act as a pathogen reservoir and conduit, facilitating further Phytophthora infection of nursery stock and its spread into the wider environment. This project identified Phytophthora species associated with green waste at three Scottish nurseries by sampling material from waste piles, water run-off from piles, and roots from discarded plants. Species were identified using a baiting method and sequencing of environmental DNA. Plant nursery green waste was shown to harbour diverse and varied Phytophthora species assemblages, with differences among nurseries reflecting biosecurity management practices. Eighteen Phytophthora species were detected in the samples, including the highly destructive pathogens P. ramorum and P. austrocedri. Results suggest that the improved management of waste, for example through effective on-site composting, is essential to reduce the risk of Phytophthora pathogens spreading from nurseries into the wider environment.

An open-access T-BAS phylogeny for emerging Phytophthora species.

Phytophthora species cause severe diseases on food, forest, and ornamental crops. Since the genus was described in 1876, it has expanded to comprise over 190 formally described species. There is a need for an open access phylogenetic tool that centralizes diverse streams of sequence data and metadata to facilitate research and identification of Phytophthora species. We used the Tree-Based Alignment Selector Toolkit (T-BAS) to develop a phylogeny of 192 formally described species and 33 informal taxa in the genus Phytophthora using sequences of eight nuclear genes. The phylogenetic tree was inferred using the RAxML maximum likelihood program. A search engine was also developed to identify microsatellite genotypes of P. infestans based on genetic distance to known lineages. The T-BAS tool provides a visualization framework allowing users to place unknown isolates on a curated phylogeny of all Phytophthora species. Critically, the tree can be updated in real-time as new species are described. The tool contains metadata including clade, host species, substrate, sexual characteristics, distribution, and reference literature, which can be visualized on the tree and downloaded for other uses. This phylogenetic resource will allow data sharing among research groups and the database will enable the global Phytophthora community to upload sequences and determine the phylogenetic placement of an isolate within the larger phylogeny and to download sequence data and metadata. The database will be curated by a community of Phytophthora researchers and housed on the T-BAS web portal in the Center for Integrated Fungal Research at NC State. The T-BAS web tool can be leveraged to create similar metadata enhanced phylogenies for other Oomycete, bacterial or fungal pathogens.

The Never-Ending Presence of Phytophthora Species in Italian Nurseries.

Plant trade coupled with climate change has led to the increased spread of well-known and new Phytophthora species, a group of fungus-like organisms placed in the Kingdom Chromista. Their presence in plant nurseries is of particular concern because they are responsible for many plant diseases, with high environmental, economic and social impacts. This paper offers a brief overview of the current status of Phytophthora species in European plant nurseries. Focus was placed on Italian sites. Despite the increasing awareness of the risk of Phytophthora spread and the management strategies applied for controlling it, the complexity of the Phytophthora community in the horticulture industry is increasing over time. Since the survey carried out by Jung et al. (2016), new Phytophthora taxa and Phytophthora-host associations were identified. Phytophthorahydropathica, P. crassamura, P. pseudocryptogea and P. meadii were reported for the first time in European plant nurseries, while P. pistaciae, P. mediterranea and P. heterospora were isolated from Italian ornamental nurseries. Knowledge of Phytophthora diversity in plant nurseries and the potential damage caused by them will help to contribute to the development of early detection methods and sustainable management strategies to control Phytophthora spread in the future.

Comparison of Primers for the Detection of Phytophthora (and Other Oomycetes) from Environmental Samples.

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.

DNA Metabarcoding and Isolation by Baiting Complement Each Other in Revealing Phytophthora Diversity in Anthropized and Natural Ecosystems.

Isolation techniques supplemented by sequencing of DNA from axenic cultures have provided a robust methodology for the study of Phytophthora communities in agricultural and natural ecosystems. Recently, metabarcoding approaches have emerged as new paradigms for the detection of Phytophthora species in environmental samples. In this study, Illumina DNA metabarcoding and a conventional leaf baiting isolation technique were compared to unravel the variability of Phytophthora communities in different environments. Overall, 39 rhizosphere soil samples from a natural, a semi-natural and a horticultural small-scale ecosystem, respectively, were processed by both baiting and metabarcoding. Using both detection techniques, 28 out of 39 samples tested positive for Phytophthora. Overall, 1,406,613 Phytophthora internal transcribed spacer 1 (ITS1) sequences and 155 Phytophthora isolates were obtained, which grouped into 21 taxa, five retrieved exclusively by baiting (P. bilorbang; P. cryptogea; P. gonapodyides; P. parvispora and P. pseudocryptogea), 12 exclusively by metabarcoding (P. asparagi; P. occultans; P. psycrophila; P. syringae; P. aleatoria/P. cactorum; P. castanetorum/P. quercina; P. iranica-like; P. unknown sp. 1; P. unknown sp. 2; P. unknown sp. 3; P. unknown sp. 4; P. unknown sp. 5) and four with both techniques (P. citrophthora, P. multivora, P. nicotianae and P. plurivora). Both techniques complemented each other in describing the variability of Phytophthora communities from natural and managed ecosystems and revealing the presence of rare or undescribed Phytophthora taxa.

Diversity of Phytophthora Species Detected in Disturbed and Undisturbed British Soils Using High-Throughput Sequencing Targeting ITS rRNA and COI mtDNA Regions

Disease outbreaks caused by introduced Phytophthora species have been increasing in British forests and woodlands in recent years. A better knowledge of the Phytophthora communities already present in the UK is of great importance when developing management and mitigation strategies for these diseases. To do this, soils were sampled in “disturbed” sites, meaning sites frequently visited by the public, with recent and new plantings or soil disturbances versus more “natural” forest and woodland sites with little disturbance or management. Phytophthora diversity was assessed using high-throughput Illumina sequencing targeting the widely accepted barcoding Internal Transcribed Spacer 1 (ITS1) region of rRNA and comparing it with the mitochondrial cytochrome c oxidase I (COI) gene. Isolation of Phytophthora was run in parallel. Nothophytophthora spp. and Phytophthora spp. were detected in 79 and 41 of the 132 locations of the 14 studied sites when using ITS or COI, respectively. A total of 20 Phytophthora amplicon sequence variants (ASVs) were assigned to known Phytophthora species from eight clades (1a, 2, 2b, 3a, 5, 6b, 7a, 8b, 8c, 8d, 10a, and 10b) and 12 ASVs from six clades (1a, 2c, 3a, 3b, 6b, 7a, 8b, 8c, and 8d) when using ITS or COI, respectively. Only at two locations were the results in agreement for ITS, COI, and isolation. Additionally, 21 and 17 unknown Phytophthora phylotypes were detected using the ITS and COI, respectively. Several Phytophthora spp. within clades 7 and 8, including very important forest pathogens such as P. austrocedri and P. ramorum, were identified and found more frequently at “disturbed” sites. Additionally, eight ASVs identified as Nothophytophthora spp. were detected representing the first report of species within this new genus in Britain. Only three species not known to be present in Britain (P. castaneae, P. capsici, and P. fallax) were detected with the ITS primers and not with COI. To confirm the presence of these or any potential new Phytophthora species, sites should be re-sampled for confirmation. Additionally, there is a need to confirm if these species are a threat to British trees and try to establish any eradication measures required to mitigate Phytophthora spread in Britain.

Phytophthora and vascular plant species distributions along a steep elevation gradient

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.

The Challenges of Managing Phytophthora Root Rot in the Nursery Industry

For nearly 100 years, Phytophthora root rot (PRR) has been a severe problem in nurseries. Early surveys found only one or two Phytophthora species on any one host. However, recent surveys show that nurseries are impacted by increasingly complex Phytophthora communities that vary by host, nursery, and region. Individual community members have diverse biological characteristics and differ in their responses to disease control measures. These differences may shift community composition toward members that are less sensitive to treatment, thereby decreasing overall disease control. Together, this suggests that PRR is better approached as a disease complex rather than as a disease caused by a single entity. Yet most experiments use only a single Phytophthora isolate and therefore overlook the potential for other community members to be less responsive to treatment. Successful control is further limited by a lack of data on the disease losses and soilborne inoculum levels encountered in the nursery industry, which are essential for establishing risks for infection and pathogen movement and for evaluating disease control efficacy. Focused surveys with intensive sampling are needed to better characterize the Phytophthora communities occurring on major nursery crops. Experiments should utilize a representative set of species and isolates to ensure treatments are effective. The presence of diverse Phytophthora communities in the nursery industry makes it less likely that any one disease control tactic will be broadly effective. Instead, a combination of approaches that take into account the individual weaknesses of each community member will likely be necessary to achieve long-term PRR control.

Diversity of Phytophthora Communities across Different Types of Mediterranean Vegetation in a Nature Reserve Area

Research Highlights: Protected natural areas are a reservoir of Phytophthora species and represent the most suitable sites to study their ecology, being less disturbed by human activities than other environments. Background and Objectives: The specific objective of this study was to correlate the diversity and distribution of Phytophthora species with the vegetation in aquatic, riparian and terrestrial habitats within a protected area in Eastern Sicily, Southern Italy. Materials and Methods: Environmental samples (water and soil) were sourced from two streams running through the reserve and six different types of vegetation, including Platano-Salicetum pedicellatae, the Sarcopoterium spinosum community, Myrto communis-Pistacietum lentisci, Pistacio-Quercetum ilicis,Oleo-Quercetum virgilianae and a gallery forest dominated by Nerium oleander (Natura 2000 classification of habitats). Phytophthora species were recovered from samples using leaf baiting and were classified on the basis of morphological characteristics and sequencing of internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA). Results: As many as 11 Phytophthora species, within five different ITS clades, were identified, including P. asparagi, P. bilorbang, P. cryptogea, P. gonapodyides, P. lacustris, P. multivora, P. nicotianae, P. oleae, P. parvispora, P. plurivora and P. syringae. No Phytophthora species were found in the Sarcopoterium spinosum comm. Phytophthora asparagi, P. lacustris and P. plurivora were the prevalent species in the other five plant communities, but only P. plurivora was present in all of them. Overall aquatic species from clade 6 (100 out of 228 isolates) were the most common; they were recovered from all five types of vegetation, streams and riparian habitats. Phytophthora populations found in the Platano-Salicetum pedicellatae and Oleo-Quercetum virgilianae show the highest diversity, while no correlation was found with the physicochemical characteristics of the soil. Conclusions: The vegetation type and the aquatic or terrestrial habitat were identified as major environmental factors correlated with the diversity of Phytophthora communities in this reserve.

Surprising low diversity of the plant pathogen Phytophthora in Amazonian forests.

The genus Phytophthora represents a group of plant pathogens with broad global distribution. The majority of them cause the collar and root-rot of diverse plant species. Little is known about Phytophthora communities in forest ecosystems, especially in the Neotropical forests where natural enemies could maintain the huge plant diversity via negative density dependence. We characterized the diversity of soil-borne Phytophthora communities in the North French Guiana rainforest and investigated how they are structured by host identity and environmental factors. In this little-explored habitat, 250 soil cores were sampled from 10 plots hosting 10 different plant families across three forest environments (Terra Firme, Seasonally Flooded and White Sand). Phytophthora diversity was studied using a baiting approach and metabarcoding (High-Throughput Sequencing) on environmental DNA extracted from both soil samples and baiting-leaves. These three approaches revealed very similar communities, characterized by an unexpected low diversity of Phytophthora species, with the dominance of two cryptic species close to Phytophthora heveae. As expected, the Phytophthora community composition of the French Guiana rainforest was significantly impacted by the host plant family and environment. However, these plant pathogen communities are very small and are dominated by generalist species, questioning their potential roles as drivers of plant diversity in these Amazonian forests.

Association of Phytophthora with Declining Vegetation in an Urban Forest Environment.

Urban forests consist of various environments from intensely managed spaces to conservation areas and are often reservoirs of a diverse range of invasive pathogens due to their introduction through the nursery trade. Pathogens are likely to persist because the urban forest contains a mixture of native and exotic plant species, and the environmental conditions are often less than ideal for the trees. To test the impact of different land management approaches on the Phytophthora community, 236 discrete soil and root samples were collected from declining trees in 91 parks and nature reserves in Joondalup, Western Australia (WA). Sampling targeted an extensive variety of declining native trees and shrubs, from families known to be susceptible to Phytophthora. A sub-sample was set aside and DNA extracted for metabarcoding using Phytophthora-specific primers; the remaining soil and root sample was baited for the isolation of Phytophthora. We considered the effect on the Phytophthora community of park class and area, soil family, and the change in canopy cover or health as determined through sequential measurements using remote sensing. Of the 236 samples, baiting techniques detected Phytophthora species from 24 samples (18 parks), while metabarcoding detected Phytophthora from 168 samples (64 parks). Overall, forty-four Phytophthora phylotypes were detected. Considering only sampling sites where Phytophthora was detected, species richness averaged 5.82 (range 1–21) for samples and 9.23 (range 2–24) for parks. Phytophthora multivora was the most frequently found species followed by P. arenaria, P. amnicola and P. cinnamomi. While park area and canopy cover had a significant effect on Phytophthora community the R2 values were very low, indicating they have had little effect in shaping the community. Phytophthora cinnamomi and P. multivora, the two most invasive species, often co-occurring (61% of samples); however, the communities with P. multivora were more common than those with P. cinnamomi, reflecting observations over the past decade of the increasing importance of P. multivora as a pathogen in the urban environment.

Diversity of woody-host infecting Phytophthora species in public parks and botanic gardens as revealed by metabarcoding, and opportunities for mitigation through best practice

The diversity of Phytophthora species in soils collected from 14 highly disturbed sites in northern Britain, including botanic gardens, arboreta, public parks and other amenity woodland sites, was analysed using a molecular technique known as DNA metabarcoding. This technique enables the identification of multiple species present in a single environmental sample based on a DNA ‘barcode’ unique to each species. The genus Phytophthora was targeted in this study due to its increasing impact on Britain’s forests and woodlands over thelast 20 years. The introduction and spread of new Phytophthora species into Britain has been strongly associated with the movement of traded containerised plants, with a number of Phytophthora outbreaks reported on host trees located in public gardens and parks that had recently undergone planting or landscape regeneration schemes. This study was undertaken to assess the extent to which these highly disturbed sites with extensive planting regimes act as harbours for woody-host infecting Phytophthora species. A total of 23 Phytophthora species, the majority of which are known to be pathogens of woody hosts, were detected across the 14 sites sampled. These included four quarantine-regulated pathogens and four species notpreviously recorded in Britain. Also detected were three as-yet undescribed Phytophthora species and nine oomycete sequences with no clear match to any known genus. There was no effect of geographical location, elevation, underlying soil type, host family or host health status on the Phytophthora assemblages at each site, suggesting that the Phytophthora communities detected are likely to comprise introduced species associated with planting programmes. P. austrocedri and P. pseudosyringae were two of the most abundant Phytophthoraspecies detected, both of which cause serious damage to trees and are regarded as fairly recent introductions to Britain. The practical implications of the findings in terms of mitigating Phytophthora introduction, spread and impact at botanic gardens, arboreta and urban parks are discussed.

Phytophthora Species from Xinjiang Wild Apple Forests in China

Phytophthora species are well-known destructive forest pathogens, especially in natural ecosystems. The wild apple (Malus sieversii (Ledeb.) Roem.) is the primary ancestor of M. domestica (Borkh.) and important germplasm resource for apple breeding and improvement. During the period from 2016 to 2018, a survey of Phytophthora diversity was performed at four wild apple forest plots (Xin Yuan (XY), Ba Lian (BL), Ku Erdening (KE), and Jin Qikesai (JQ)) on the northern slopes of Tianshan Mountain in Xinjiang, China. Phytophthora species were isolated from baiting leaves from stream, canopy drip, and soil samples and were identified based on morphological observations and the rDNA internal transcribed spacer (ITS) sequence analysis. This is the first comprehensive study from Xinjiang to examine the Phytophthora communities in wild apple forests The 621 resulting Phytophthora isolates were found to reside in 10 different Phytophthora species: eight known species (P. lacustris being the most frequent, followed by P. gonapodyides, P. plurivora, P. gregata, P. chlamydospora, P. inundata, P. virginiana, and P. cactorum) and two previously unrecognized species (P. sp. CYP74 and P. sp. forestsoil-like). The highest species richness of Phytophthora occurred at BL, followed by XY. P. lacustris was the dominant species at BL, XY, and JQ, while P. gonapodyides was the most common at KE. In the present paper, the possible reasons for their distribution, associated implications, and associated diseases are discussed.

Comparison and validation of Oomycetes metabarcoding primers for Phytophthora high throughput sequencing

Oomycetes are eukaryotic plant pathogens that require health monitoring. High-throughput sequencing (HTS) methods replace progressively cultivation-based approaches in soil surveys of Oomycetes, but very little control has been done from synthetic communities. Indeed, several potential biases do exist and need to be assessed for Oomycetes communities. We created a mock community by mixing DNA from 24 Phytophthora species. We amplified two barcode regions with Oomycete-specific primers before HTS. With this aim, we used three primer sets in nested PCR amplification, targeting the ITS-1 region or the RAS gene region. The three nested PCR strategies proved to be a reliable qualitative approach, identifying approximately 95% of the species after Illumina Miseq sequencing and bioinformatic analysis. However, quantitative proportions of each species showed distortions compared to the original mixture of the mock. In addition, we compared the two ITS primer sets on soil environmental DNA sampled from temperate forests. The ‘oom18S-ITS7/18ph2f-5.8S-1R’ primer set, more specific to Phytophthora, was able to detect seven Phytophthora species, confirming what was expected for temperate forests. Using the ‘DC6-ITS7/oom18S-ITS7’ primer set that covers the broader Peronosporaceans, we detected only one Phytophthora species among the dominance of Pythium and Phytopythium species. We concluded that ‘oom18S-ITS7/18ph2f-5.8S-1R’ primer set is a reliable tool for the qualitative description of environmental Phytophthora communities.

Waterborne Phytophthora Species Occurrence and Diversity in the Valley of the Rák Brook

Abstract This paper reports on a two-year monitoring of Phytophthora species occurring in the catchment area of the Rák Brook near Sopron. P. gonapodyides, P. lacustris, P. plurivora and P. pseudosyringae were found in the course of surveys completed in the vegetation period of 2011 and 2012. Diversity profiles and cluster analysis were calculated in order to compare the Phytophthora communities detected at different sites and times. Seasonal differences were observed in the species compositions. Temperature data and basic hydrological parameters were found to determine the presence or absence of waterborne Phytophthora species in the catchment area of the Rák Brook. Pathogenicity of the Phytophthora species discovered was confirmed and evaluated against sessile oak seedlings.

Contrasting distribution patterns between aquatic and terrestrial Phytophthora species along a climatic gradient are linked to functional traits

Diversity of microbial organisms is linked to global climatic gradients. The genus Phytophthora includes both aquatic and terrestrial plant pathogenic species that display a large variation of functional traits. The extent to which the physical environment (water or soil) modulates the interaction of microorganisms with climate is unknown. Here, we explored the main environmental drivers of diversity and functional trait composition of Phytophthora communities. Communities were obtained by a novel metabarcoding setup based on PacBio sequencing of river filtrates in 96 river sites along a geographical gradient. Species were classified as terrestrial or aquatic based on their phylogenetic clade. Overall, terrestrial and aquatic species showed contrasting patterns of diversity. For terrestrial species, precipitation was a stronger driver than temperature, and diversity and functional diversity decreased with decreasing temperature and precipitation. In cold and dry areas, the dominant species formed resistant structures and had a low optimum temperature. By contrast, for aquatic species, temperature and water chemistry were the strongest drivers, and diversity increased with decreasing temperature and precipitation. Within the same area, environmental filtering affected terrestrial species more strongly than aquatic species (20% versus 3% of the studied communities, respectively). Our results highlight the importance of functional traits and the physical environment in which microorganisms develop their life cycle when predicting their distribution under changing climatic conditions. Temperature and rainfall may be buffered differently by water and soil, and thus pose contrasting constrains to microbial assemblies.

EDNA from roots: a robust tool for determining Phytophthora communities in natural ecosystems.

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.