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

Article Diversity of Phytophthora Species Detected in Disturbed and Undisturbed British Soils Using High-Throughput Sequencing Targeting ITS rRNA and COI mtDNA Regions Blanca B. Landa 1,†, Luis F. Arias-Giraldo 1,† , Béatrice Henricot 2, Miguel Montes-Borrego 1, Lucas A. Shuttleworth 3and Ana Pérez-Sierra 4,*   Citation: Landa, B.B.; Arias-Giraldo, L.F.; Henricot, B.; Montes-Borrego, M.; Shuttleworth, L.A.; Pérez-Sierra, A. Diversity of Phytophthora Species Detected in Disturbed and Undisturbed British Soils Using High-Throughput Sequencing Targeting ITS rRNA and COI mtDNA Regions. Forests 2021,12, 229. https://doi.org/10.3390/f12020229 Academic Editor: Rimvydas Vasaitis Received: 11 January 2021 Accepted: 12 February 2021 Published: 17 February 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Spanish National Research Council (CSIC), Institute for Sustainable Agriculture, Alameda del Obispo s/n, 14004 Córdoba, Spain; [email protected] (B.B.L.); [email protected] (L.F.A.-G.); [email protected] (M.M.-B.) 2Forest Research, Northern Research Station, Roslin EH25 9SY, UK; [email protected] 3NIAB EMR, East Malling, Kent ME19 6BJ, UK; [email protected] 4Forest Research, Alice Holt Lodge, Farnham, Surrey GU10 4LH, UK *Correspondence: [email protected] † These authors contributed equally to this work. Abstract: 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. Keywords: metabarcoding; forest diseases; oomycetes; soilborne pathogens 1. Introduction In recent years, several outbreaks caused by Phytophthora (P. ramorum,P. kernoviae, P lateralis,P. pseudosyringae and P. austrocedri) have emerged in Britain and also worldwide to cause significant mortality on a range of woody hosts [ 1 – 7 ]. As we witness the devastating impact of Phytophthora on the natural ecosystem, an increasing number of studies have looked at species composition in diverse environments such as nurseries [ 8 – 11 ], agricultural Forests 2021,12, 229. https://doi.org/10.3390/f12020229 https://www.mdpi.com/journal/forests Forests 2021,12, 229 2 of 26 and urban environments [ 12 – 14 ], and semiand natural environments [ 15 – 20 ]. As a result, many new species have been recovered with a lot of these not formally described yet. In some cases, these newly discovered species are endemic to limited geographic areas, and their potential impact on the environment remains to be elucidated [ 16 , 20 – 22 ]. Approximately 180 Phytophthora species have been described with an estimate of 326 species in total covering 12 phylogenetic clades [12]. The spread of Phytophthora has been linked to human activities such as plant movement/trade and other pathways such as soil on shoes, machinery, and water [ 23 – 27 ]. The soil environment plays an integral role in the spread and establishment of Phytophthora pathogens, which can persist long term in soil in the form of resilient thick-walled spores. Waterlogged soils may also harbor free-swimming zoospores, the main mechanism by which Phytophthora infects plants. A better knowledge of the diversity of Phytophthora and the mechanisms of spread from site to site is of great importance in developing management and mitigation strategies for these diseases. The identification of Phytophthora species is mainly based on the sequencing of the Internal Transcribed Spacer (ITS) region [ 28 – 30 ]. The ITS region is considered as the best available genetic marker to identify Phytophthora to clade level, since almost all known Phytophthora taxa have been sequenced for the ITS region [ 31 ]. New technologies such as next-generation sequencing (NGS) using Illumina or 454 technologies base their identification on the ITS region [ 21 , 32 – 35 ]. These new techniques have demonstrated greater depth in detecting Phytophthora communities than traditional cultural methods followed by Sanger sequencing. However, those studies also highlighted the ongoing difficulty of differentiating some closely related Phytophthora species which cannot be discriminated based on their ITS sequence. In such cases, species identification has been limited to species complexes or clusters. To improve the accuracy of DNA sequence-based identification, many markers have been potentially identified [ 36 – 38 ]. Several studies have indicated that DNA barcoding with the cytochrome c oxidase subunit I (COI or cox1) is an approach that can be used as a practical option to identify Phytophthora due to its highest resolution within the genus [ 31 , 39 , 40 ]. It is also complementary to ITS analyses because it uses the mitochondrial genome instead of nuclear DNA and therefore cannot identify hybrids, which are increasingly found in the Phytophthora community [17,41–43]. As part of the research effort to identify Phytophthora species that are a potential risk for the wider environment, this study is looking at the presence of Phytophthora in UK sites that we catalogue as “disturbed” sites (sites frequently visited by the public, with recent and new plantings and a link to nurseries) and from semi-natural forest and woodland sites (with little disturbance or management, i.e., “undisturbed”). Since 1600, most woods in Britain have had some human interference such as cultural activity or felling; therefore, the term “semi-natural” forest is more accurately used for those settings classified as “undisturbed”. Based on the information already published, we hypothesize that Phytophthora diversity will be greater in “disturbed” sites than “undisturbed” sites. In addition, for the first time, we test the applicability of the COI genetic marker in a metabarcoding study to identify Phytophthora communities and compare its discriminative power with the ITS. 2. Materials and Methods 2.1. Soil Sampling and Phytophthora Isolation From May to November of 2016 soil samples were collected from England, Scotland and Wales from nine “disturbed” sites (called DS1 to DS9) including three gardens/arboreta sites open to the public (DS1, DS3, DS9), four woodlands or recreational parks sites frequently visited by hikers, bikers, and children parties (DS2, DS5, DS7, DS8), and two sites with recent and new plantings and a link to nurseries (DS4, DS6). We also sampled five more “natural” forest and woodland sites (with little disturbance or management, i.e., “undisturbed” called US1 to US5) (Figure 1; Table 1). Forests 2021,12, 229 3 of 26 Forests 2021, 12, x FOR PEER REVIEW 3 of 25 five more “natural” forest and woodland sites (with little disturbance or management, i.e., “undisturbed” called US1 to US5) (Figure 1; Table 1). Figure 1. Map of Britain showing the sites where samples were collected for this study. Table 1. Description of site and locations sampled in the study with nearest hosts and health status, and the Phytophthora/Nothophytophthora species identified by using Internal Transcribed Spacer (ITS) and cytochrome c oxidase I (COI) regions or by isolation from soil. Site Code (Site Type) Location Surveyed Location and Nearest Hosts Signs of IllHealth Tree Status ITS COI Isolation DS1 (Arboreta) DS1-1 Mixed Pinus species and Abies Healthy P. austrocedri Clade 8 Phytophthora sp. uncultured 5b P. cryptogea DS1-2 Mixed new planting Asian species (broadleaved trees and conifers) Healthy P. austrocedri - - DS1-3 Heathland Healthy P. austrocedri - - DS1-4 Pinus sp. Declining P. austrocedri - P. cinnamomi DS1-5 Acid grassland, mainly conifers Healthy P. austrocedri - P. cinnamomi DS1-6 Cedrus sp. Healthy P. austrocedri - P. cinnamomi DS1-7 New planting Betula, Nothofagus, Quercus Healthy P. austrocedri, P. pseudosyringae - - Figure 1. Map of Britain showing the sites where samples were collected for this study. Table 1. Description of site and locations sampled in the study with nearest hosts and health status, and the Phytophthora/Nothophytophthora species identified by using Internal Transcribed Spacer (ITS) and cytochrome c oxidase I (COI) regions or by isolation from soil. Site Code (Site Type) Location Surveyed Location and Nearest Hosts Signs of Ill-Health Tree Status ITS COI Isolation DS1 (Arboreta) DS1-1 Mixed Pinus species and Abies Healthy P. austrocedri Clade 8 Phytophthora sp. uncultured 5b P. cryptogea DS1-2 Mixed new planting Asian species (broadleaved trees and conifers) Healthy P. austrocedri - - DS1-3 Heathland Healthy P. austrocedri - - DS1-4 Pinus sp. Declining P. austrocedri -P. cinnamomi DS1-5 Acid grassland, mainly conifers Healthy P. austrocedri -P. cinnamomi DS1-6 Cedrus sp. Healthy P. austrocedri -P. cinnamomi DS1-7 New planting Betula, Nothofagus, Quercus Healthy P. austrocedri, P. pseudosyringae - - DS1-8 Mixed broadleaved trees and conifers new plantings Healthy P. austrocedri New Clade Phytophthora sp. uncultured 12b P. megasperma Forests 2021,12, 229 4 of 26 Table 1. Cont. Site Code (Site Type) Location Surveyed Location and Nearest Hosts Signs of Ill-Health Tree Status ITS COI Isolation DS1-9 Mixed broadleaved trees and conifers new plantings Healthy - - - DS1-10 Acid grassland, conifer and broadleaved trees, new plantings Healthy - - P. cinnamomi DS2 (Woodland/Recreational Park) DS2-1 Fraxinus Declining - - - DS2-2 Quercus Dead trees Clade 7 Phytophthora sp. uncultured 3a and 21a P. europaea - DS2-3 Fraxinus Dead trees P. cactorum, P. syringae, P. cinnamomi P. cactorum - DS2-4 Quercus Declining P. cactorum, P. syringae, Clade 7 Phytophthora sp. uncultured 21a P. uliginosa, Clade 1 Phytophthora sp. uncultured 2b, New Clade Phytophthora sp. uncultured 12b - DS2-5 Larix Dead - - - DS2-6 Betula Dead - - - DS2-7 Quercus Declining - - - DS2-8 Acer campestre Declining P. cactorum, P. megasperma/ crassamura, P. ramorum, P. rubi/fragariae, Clade 7 Phytophthora sp. uncultured 5a and 6a Clade X Phytophthora sp. uncultured 11b - DS2-9 Acer and Fraxinus Declining P. plurivora/ citricola, P. syringae - - DS2-10 Larix in area with Corylus,Fraxinus and Quercus Healthy P. capsici, P. obscura, P. plurivora/ citricola,P. rubi, P. siskiyouensis, P. syringae, Clade 2 Phytophthora sp. uncultured 17a -P. plurivora DS3 (Garden) DS3-1 Border with herbaceous plants and shrubs, mixed planting Healthy Clade 3 Phytophthora sp. uncultured 10a -P. plurivora DS3-2 Kalmia,Quercus and Rhododendron Healthy - - DS3-3 Quercus, Rhododendron and Sorbus Healthy Clade 11 Phytophthora sp. uncultured 19a - Forests 2021,12, 229 5 of 26 Table 1. Cont. Site Code (Site Type) Location Surveyed Location and Nearest Hosts Signs of Ill-Health Tree Status ITS COI Isolation DS3-4 Area with silt from a stream with no trees in the last 8 years surrounded by Acer, Larix and Quercus Healthy P. obscura, Clade 7 Phytophthora sp. uncultured 1a -P. cinnamomi DS3-5 Border, the old kitchen garden, herbaceous plants and shrubs Healthy P. primulae, P. syringae P. primulae P. chlamydospora DS3-6 Burning site for the garden NA P. obscura, P. rubi/fragariae -P. plurivora DS3-7 Quercus and Rhododendron Healthy P. rubi/fragariae - - DS3-8 Composting area of the garden NA Clade 1 Phytophthora sp. uncultured 14a, Clade 11 Phytophthora sp. uncultured 18a and 19a -P. plurivora DS3-9 Mature Fagus, Fraxinus and Quercus Healthy - - P. cinnamomi DS3-10 Mature Quercus and Rhododendron Healthy - - - DS4 (New plantings and a link to nurseries) DS4-1 Young Sorbus Dead - - - DS4-2 Young Quercus Declining P. obscura - - DS4-3 Young Salix Declining P. austrocedri, P. plurivora/ citricola, P. siskiyouensis - - DS4-4 Young Juniperus Declining - - - DS4-5 Young Salix Declining - P. syringae, New Clade Phytophthora sp. uncultured 16b - DS4-6 Young Sorbus Declining P. ramorum - - DS4-7 Young Betula Declining - - - DS4-8 Young Prunus Declining - - - DS4-9 Young Crataegus Declining - New Clade Phytophthora sp. uncultured 12b - DS4-10 Young Corylus Healthy - - - DS5 (Woodland/Recreational Park) DS5-1 Betula Declining - - - DS5-2 Betula Declining - - - DS5-3 Quercus Declining P. siskiyouensis - - DS5-4 Sorbus Declining P. plurivora/ citricola, P. syringae P. plurivora P. plurivora DS5-5 Abies Dead - - - Forests 2021,12, 229 6 of 26 Table 1. Cont. Site Code (Site Type) Location Surveyed Location and Nearest Hosts Signs of Ill-Health Tree Status ITS COI Isolation DS5-6 Quercus robur Declining P. plurivora/ citricola - - DS5-7 Pinus sylvestris Dead - - - DS5-8 Pinus sylvestris Dead - - - DS5-9 Fagus Declining - - - DS5-10 Fagus Healthy - - - DS6 (New plantings and a link to nurseries) DS6-1 Sorbus aucuparia Healthy - - - DS6-2 Betula Healthy - - - DS6-3 Juniperus communis Healthy - - - DS6-4 Sorbus aucuparia Rust - - - DS6-5 Alnus Healthy - - - DS6-6 Pinus sylvestris Healthy P. kernoviae - - DS6-7 Betula Declining - - - DS6-8 Alnus Healthy - - - DS6-9 Alnus Declining - - - DS6-10 Prunus Declining - - - DS7 (Woodland/Recreational Park) DS7-1 Alnus Dead P. austrocedri, Clade 11a Phytophthora sp. uncultured 18a and 19a P. quercina, New Clade Phytophthora sp. uncultured 14b, New Clade Phytophthora sp. uncultured 16b - DS7-2 Alnus Declining P. plurivora/ citricola,P. pseudosyringae, P. syringae, Clade 7 Phytophthora sp. uncultured 1a, Clade 3 Phytophthora sp. uncultured 11a P. plurivora, P. syringae - DS7-3 Young Abies grandis in Alnus and Betula forest Healthy P. cinnamomi, P. uniformis, Clade 7 Phytophthora sp. uncultured 1a and 16a Clade 7 Phytophthora sp. uncultured 7b and 8b, New Clade Phytophthora sp. uncultured 16b, Nothophytophthora sp. 4 - DS7-4 Quercus in mature woodland Healthy P. plurivora/ citricola - - Forests 2021,12, 229 7 of 26 Table 1. Cont. Site Code (Site Type) Location Surveyed Location and Nearest Hosts Signs of Ill-Health Tree Status ITS COI Isolation DS7-5 Crataegus in mature woodland Dead P. capsici, P. castaneae, P. cinnamomi, P. fallax, P. foliorum, P. obscura, P. plurivora/ citricola,P. rubi/ fragariae, P. siskiyouensis, Clade 2 Phytophthora sp. uncultured 17a - - DS8 (Woodland/Recreational Park) DS8-1 Larix Dead - Nothophytophthora sp. 2 - DS8-2 Fraxinus Dead P. syringae P. syringae, New Clade Phytophthora sp. uncultured 13b, Nothophytophthora sp. 2 - DS8-3 Alnus Healthy P. pseudosyringae, P. syringae, Clade 7 Phytophthora sp. uncultured 6a, Clade 3 Phytophthora sp. uncultured 11a P. pseudosyringae, P. syringae - DS8-4 Pinus nigra Healthy Clade 7 Phytophthora sp. uncultured 7a Nothophytophthora sp. 5 - DS8-5 Fraxinus stump, decayed tree in mature woodland NA P. capsici, P. castaneae, P. fallax, P. foliorum, P. obscura, P. plurivora/ citricola, P. rubi/fragariae, P. syringae, Clade 7 Phytophthora sp. uncultured 5a, Clade 6 Phytophthora sp. uncultured 8a, Clade 1 Phytophthora sp. uncultured 13a P. syringae, Clade 1 Phytophthora sp. uncultured 1b, Clade 6 Phytophthora sp. uncultured 4b, Clade 7 Phytophthora sp. uncultured 6b - Forests 2021,12, 229 8 of 26 Table 1. Cont. Site Code (Site Type) Location Surveyed Location and Nearest Hosts Signs of Ill-Health Tree Status ITS COI Isolation DS9 (Arboreta) DS9-1 Chamaecyparis thyoides,Picea brachytyla Healthy P. austrocedri, P. cinnamomi, P. plurivora/ citricola New Clade Phytophthora sp. uncultured 16b P. chlamydospora DS9-2 Grassland, Parrotia persica,Pseudotsuga menziesii Healthy P. syringae Nothophytophthora sp. 7 P. chlamydospora, P. ramorum DS9-3 Quercus cerris Healthy - - - DS9-4 Pinus sylvestris Healthy P. primulae P. primulae - DS9-5 Quercus robur Healthy P. fallax, P. foliorum, P. idaei, P. plurivora/ citricola, Clade 1 Phytophthora sp. uncultured 12a and 13a, Clade 2Phytophthora sp. uncultured 17a New Clade Phytophthora sp. uncultured 15b, New Clade Phytophthora sp. uncultured 17b - DS9-6 Taxus baccata Decline P. syringae, Clade 6 Phytophthora sp. uncultured 15a -P. gonapodyides DS9-7 Mixed species, new plantings: Acer, Cornus,Calocedrus, Fraxinus Healthy P. austrocedri New Clade Phytophthora sp. uncultured 12b, New Clade Phytophthora sp. uncultured 17b - DS9-8 Cedrus atlantica Healthy - - - DS9-9 Quercus petraea, Quercus ilex Healthy P. foliorum, P. rubi/fragariae, Clade 11 Phytophthora sp. uncultured 18a and 19a -P. plurivora DS9-10 Prumnopitys andina, Pinus contorta, boundary with Larix Healthy P. primulae New Clade Phytophthora sp. uncultured 16b - DS9-11 Pseudotsuga menziesii Healthy P. austrocedri - - US1 (Undisturbed site 1) US1-1 Larix Dead - - - US1-2 Sorbus aucuparia Declining P. obscura, P. ramorum P. ramorum - US1-3 Crataegus Declining P. syringae - - US1-4 Betula Dead P. ramorum - - US1-5 Betula Declining - - - US1-6 Sorbus aucuparia Dead P. plurivora/ citricola, Clade 2 Phytophthora sp. uncultured 17a - - US1-7 Quercus Healthy - - - Forests 2021,12, 229 9 of 26 Table 1. Cont. Site Code (Site Type) Location Surveyed Location and Nearest Hosts Signs of Ill-Health Tree Status ITS COI Isolation US1-8 Malus Declining P. syringae, Clade 6 Phytophthora sp. uncultured 15a P. syringae, P. gonapodyides - US1-9 Fraxinus Declining P. fallax, P. plurivora/citricola, P. rubi/fragariae, P. siskiyouensis, P. syringae - - US1-10 Alnus Healthy P. plurivora/citricola, P. pseudosyringae, P. syringae - - US1-11 Alnus Healthy P. cactorum, P. syringae - - US2 (Undisturbed site 2) US2-1 Betula pubescens Declining Clade 7 Phytophthora sp. uncultured 21a - - US2-2 Juniperus communis Dead P. kernoviae - - US2-3 Mature Pinus sylvestris Healthy P. cactorum - - US2-4 Sorbus aucuparia Healthy P. syringae, Clade 7 Phytophthora sp. uncultured 2a - - US2-5 Betula pubescens Declining Clade 11 Phytophthora sp. uncultured 19a - - US2-6 Pinus sylvestris Healthy - - - US2-7 Pinus sylvestris Healthy - - - US2-8 Quercus Healthy - New Clade Phytophthora sp. uncultured 12b, New Clade Phytophthora sp. uncultured 16b - US2-9 Quercus Declining - - - US2-10 Juniperus communis Declining - - - US3 (Undisturbed site 3) US3-1 Fraxinus Declining P. cactorum, P. syringae P. syringae - US3-2 Corylus Declining P. cactorum, P. capsici, P. fallax, P. foliorum, P. plurivora/ citricola, P. siskiyouensis, P. syringae P. plurivora, P. syringae, Clade 7 Phytophthora sp. uncultured 9b, Nothophytophthora sp. 6b P. plurivora US3-3 Fraxinus windblown trees NA P. cactorum, P. plurivora/ citricola, P. primulae, P. syringae P. cactorum, P. plurivora/ citricola, P. syringae, Nothophytophthora sp. 1 - Forests 2021,12, 229 16 of 26 spp., Saprolegnia spp., and Aphanomyces spp.) and other unidentified oomycetes (23%) were also identified (Supplementary Materials Figure S3). 3.3. Identification of Phytophthora Phylotypes in Britain by NGS From all the reads identified as Phytophthora using the ITS region, 79 ASVs were identified. Of those, 20 ASVs were assigned to known species/phylotypes clusters of Phytophthora spp., and 21 ASVs were assigned to unknown/uncultured species of Phytophthora.Phytophthora spp. were detected in 79 locations of the 132 on 14 studied sites (Table 1, Supplementary Materials Table S1 and Figure S4). In this case, the databases of CPSM and BOLD were used for taxonomy assignation and comparison, and when a discrepancy was found, the procedure described by Idphy using the Genbank database was also performed. Most of the ASV taxonomic assignations were in agreement between the two databases used and corresponded to the following species: P. austrocedri, P. cactorum,P. capsici,P. castaneae,P. cinnamomi,P. fallax,P. foliorum,P. idaei,P. kernoviae, P. lacustris,P. obscura,P. primulae,P. pseudosyringae,P. siskiyouensis,P. ramorum,P. syringae and P. uniformis. The following few species did not agree with the databases or could not be reliably discriminated: P. megasperma/crassamura,P. plurivora/citricola and P. rubi/fragariae ( Table 1 , Supplementary Materials Table S1 and Figure S4). The other 21 ASVs were named as Phytophthora sp. uncultured 1a to 21a. Their sequences showed high similarity with sequences of already known/undescribed Phytophthora species such as P. alni/uniformis, P. cambivora,P. capsici/glovera,P. europaea/megasperma,P. iranica/clandestina,P. melonis/sinensis, P. quercina/P. sp. ohioensis,P. sojae and P. uliginosa, but their ITS sequence homology was below 99%, and the phylogenetic reconstruction also showed them to be very close but as independent branches (Supplementary Materials Figure S4). Most of these unidentified ASVs belonged to Clade 7, and the remaining belonged to Clades 1, 2, 3, 6, and 11. In addition, a new species of Nothophytophthora (ASV-56),placed in the same branch of the phylogenetic tree and showing more than 99% identity with N. vietnamensis and N. intricata, was only detected in one location from US4 (Table 1, Supplementary Materials Table S1 and Figure S4). Results from NGS analysis of COI sequences from British soil samples revealed a total of 54 ASVs clustered within the genus Phytophthora that were assigned to 12 known Phytophthora species and 17 unknown Phytophthora phylotypes (named Phytophthora sp. uncultured from 1b to 17b), which were identified in 12 out of the 14 sites of the study (Table 1; Supplementary Materials Table S2 and Figure S5). The taxonomy assignation was obtained after comparing the two curated Phytophthora databases with results from GenBank using the procedure described by Idphy (https://idtools.org/id/phytophthora/molecular.php, accessed on September 2019). The main species detected using COI sequences that were in agreement with the three databases were P. cactorum,P. cinnamomi,P. europaea,P. gonapodyides,P. megasperma,P. plurivora,P. pseudosyringae,P. primulae,P. quercina,P. ramorum, P. syringae and P. uliginosa. The 17 uncultured phylotypes did not show a high percentage of similarity to any of the species present in the database, since homology in the COI sequences was below 99% or could not be reliably discriminated with any reference sequence using the ML phylogenetic reconstruction (Table 1and Supplementary Materials Figure S5). It is important to highlight that 14 ASVs classified as Phytophthora spp. (Phytophthora uncultured 12b to 17b) showed a homology of sequences between 93 and 95% with known reference sequences such as P. agathidicida,P. castaneae,P. heveae,P. litchii/himalsilva and P. nierderhauserii/quercetorum. Moreover, these were clustered, although with low bootstrap support, in a new independent group according to phylogenetic analysis, which may indicate that they belong to a new unidentified clade. In addition, eight ASVs were assigned to the genus Nothophytophthora (Nothophytophthora spp. from 1 to 8) and were detected at the following sites DS7, DS8, DS9, US3, and US4 (Table 1and Supplementary Materials Figure S5). Eight Phytophthora species were detected in the soil samples when using both ITS and COI regions including P. cactorum,P. cinnamomi,P. megasperma,P. plurivora/citricola, Forests 2021,12, 229 17 of 26 P. primulae,P. pseudosyringae,P. ramorum and P. syringae. On the other hand, 12 species were only detected when using the ITS region including P. austrocedri,P. capsici,P. castaneae, P. fallax,P. foliorum,P. idaei,P. kernoviae,P. lacustris,P. obscura,P. rubi/fragariae,P. siskiyouensis and P. uniformis, whereas four species were detected only when using the COI region including P. europaea, P. gonapodyides, P. quercina, and P. uliginosa (Table 1, Figure 2and Supplementary Materials Tables S1 and S2). Forests 2021, 12, x FOR PEER REVIEW 15 of 25 Figure 2. Occurrence of Phytophthora spp. and Nothophytophthora spp. in British soils in the different location sampled in “disturbed” or “undisturbed” sites determined based on next-generation sequencing (NGS) analyses of the ITS and COI regions. Phytophthora syringae was the most abundant and most prevalent species among all samples for both ITS and COI. This species was detected in six or 10 of the 14 studied sites and in 32 or 16 locations sampled when using the ITS or COI region, respectively, and both in “disturbed” and “undisturbed” sites (Table 1, Figure 2; Supplementary Materials Tables S1 and S2). It also represents, with eight or four associated ASVs, 35% (4.363) or 26% (83.642) of the total reads detected when using ITS (Supplementary Materials Table S1) or COI (Supplementary Materials Table S2), respectively. Other Phytophthora spp. that were also found as most prevalent when using the ITS region included P. plurivora, which was detected in 10 sites at 19 locations, and P. primulae, which was detected in three sites and five locations (Table 1, Figure 2); when using the COI region, P. plurivora and P. primulae were detected at four and three sites, respectively with a similar number of locations (Table 1, Figure 2). When using the ITS region, several Phytophthora spp. appeared also to be widespread, including P. cactorum, P. obscura, P. pseudosyringae, P. rubi/fragariae, and P. siskiyouensis. These were detected in at least five sites and a number of locations ranging from six to 19 locations (Figure 2; Supplementary Materials Table S1). Interestingly, when using the COI region, two unknown Phytophthora spp. (New Clade Phytophthora uncultured 12b and 16b) appeared as the most abundant species being detected in five and four Figure 2. Occurrence of Phytophthora spp. and Nothophytophthora spp. in British soils in the different location sampled in “disturbed” or “undisturbed” sites determined based on next-generation sequencing (NGS) analyses of the ITS and COI regions. Phytophthora syringae was the most abundant and most prevalent species among all samples for both ITS and COI. This species was detected in six or 10 of the 14 studied sites and in 32 or 16 locations sampled when using the ITS or COI region, respectively, and both in “disturbed” and “undisturbed” sites (Table 1, Figure 2; Supplementary Materials Tables S1 and S2). It also represents, with eight or four associated ASVs, 35% (4.363) or 26% (83.642) of the total reads detected when using ITS (Supplementary Materials Table S1) or COI (Supplementary Materials Table S2), respectively. Other Phytophthora spp. that were also found as most prevalent when using the ITS region included P. plurivora, which was detected in 10 sites at 19 locations, and P. primulae, which was detected in three sites and five locations (Table 1, Figure 2); when using the COI region, P. plurivora and P. primulae were detected at four and three sites, respectively with a similar number of locations Forests 2021,12, 229 18 of 26 (Table 1, Figure 2). When using the ITS region, several Phytophthora spp. appeared also to be widespread, including P. cactorum,P. obscura,P. pseudosyringae,P. rubi/fragariae and P. siskiyouensis. These were detected in at least five sites and a number of locations ranging from six to 19 locations (Figure 2; Supplementary Materials Table S1). Interestingly, when using the COI region, two unknown Phytophthora spp. (New Clade Phytophthora uncultured 12b and 16b) appeared as the most abundant species being detected in five and four sites and in five and six locations, respectively (Figure 2; Supplementary Materials Table S2). 3.4. Identification of Phytophthora by Isolation Eight Phytophthora species (P. cactorum,P. chlamydospora,P. cinnamomi,P. cryptogea, P. gonapodyides,P. megasperma,P. plurivora and P. ramorum) were isolated from the sampled soils at five of the “disturbed” sites (DS1, DS2, DS3, DS5, and DS9) and two of the “undisturbed” sites (US3 and US4) (Table 1). Only on two of the locations were the following species confirmed by ITS, COI, and isolation: P. plurivora on DS5 (DS5-4) and US3 (US3-2). At one of the sites, P. plurivora was isolated at site DS2 (DS2-10) and only confirmed by ITS, and P. cactorum was isolated at US3 (US3-9) and confirmed only by ITS. However, there were sites where Phytophthora species were isolated, and they were not detected by NGS. This was the case at the sites DS1 (P. cryptogea,P. cinnamomi and P. megasperma), DS2 (P. plurivora), DS3 (P. cinnamomi,P. chlamydospora and P. plurivora), DS9 (P. chlamydospora, P. gonapodyides,P. plurivora and P. ramorum), US3 (P. chlamydospora and P. plurivora) and US4 (P. chlamydospora,P. gonapodyides and P. plurivora). 3.5. Phytophthora spp. Community Composition Differs between Disturbed and Undisturbed Sites PCoA of Bray–Curtis and Jaccard distances showed a trend to differentiate Phytophthora communities according to the disturbance of the sites (i.e., “disturbed” versus “undisturbed”) (Figure 3). Thus, when using both the ITS or COI regions, there was a significant effect of soil disturbance on the composition of Phytophthora species found on those soils, both for Bray–Curtis (PERMANOVA, F> 2.687, p< 0.009) and Jaccard (PERMANOVA, F< 2.982, p< 0.003) distances (Supplementary Materials Table S3). However, the dispersion of the species communities across samples differed significantly for Bray–Curtis distance-based matrices (F> 4.497, p< 0.045) but not for the Jaccard distances (F< 3.427, p> 0.080 ), which indicated that for Bray–Curtis ordination (abundance), there is a heterogeneous dispersion, whereas that does not occur for Jaccard ordination (presence/absence) (Supplementary Materials Table S3). Concerning differences between “disturbed“ and “undisturbed” sites, a higher number of Phytophthora spp. were detected at “disturbed“ sites, both when using ITS (36 species) or COI (26 species) as compared to “undisturbed“ sites, where 29 and 20 species were detected when using the ITS and COI region, respectively (Figure 2; Supplementary Materials Tables S1 and S2). When using the ITS region, of the total of 41 Phytophthora species identified, 13 were only detected in “disturbed” soils (P. austrocedri,P. castaneae,P. idaei,P. uniformis, and the uncultured Phytophthora 1a, 3a, 6a, 7a, 10a, 12a, 13a, 14a, and 18a) and five were detected in “undisturbed” soils (P. lacustris, and the uncultured Phytophthora 2a, 9a, 4a, and 20a) (Figure 2; Supplementary Materials Table S1). The unique Nothophytophthora sp. identified was detected in one location at the “undisturbed” site US4. The remaining 23 were detected in both types of soils. Phytophthora austrocedri was only detected at “disturbed” sites and showed a remarkably high prevalence at one of the sampled sites (DS1) (Figure 2; Supplementary Materials Table S1). From the total of 29 Phytophthora species identified when using the COI gene, 13 Phytophthora spp. were detected only in “disturbed” soils (P. europaea,P. quercina,P. uliginosa, and several uncultured Phytophthora spp. from Clade 1 (Phytophthora sp. uncultured 1b, 2b), Clade 6 (Phytophthora sp. uncultured 4b), Clade 7 (Phytophthora sp. uncultured 6b, 7b, 8b), Clade 1 (Phytophthora sp. uncultured 5b), Clade X (Phytophthora sp. uncultured 11b), and from the new candidate Clade (Phytophthora sp. uncultured 15b and 17b)) Forests 2021,12, 229 19 of 26 (Figure 2; Supplementary Materials Table S2). On the other hand, seven were located exclusively in “undisturbed” soils (P. cinnamomi,P. gonapodyides,P. megasperma,P. ramorum, and two uncultured Phytophthora spp. from Clade 1 and one from Clade 7). From the eight Nothophytophthora spp. detected, four were detected at “disturbed” sites (DS7, DS8, and DS9) and a similar number were detected at “undisturbed” sites (US2 and US3) (Figure 2; Supplementary Materials Table S2). Forests 2021, 12, x FOR PEER REVIEW 17 of 25 Figure 3. Principal coordinates analysis (PCoA) of Bray–Curtis and Jaccard distance matrices from Phytophthora spp. and Nothophytophthora spp. community composition data obtained using COI and ITS markers. PCoA were plotted in the form of a spider diagram with “legs” joining samples that belong to the same cluster (centroid). Shapes on the figure legend correspond to the locations and colors to the sites (“disturbed” and “undisturbed”) sampled. Concerning differences between “disturbed“ and “undisturbed” sites, a higher number of Phytophthora spp. were detected at “disturbed“ sites, both when using ITS (36 species) or COI (26 species) as compared to “undisturbed“ sites, where 29 and 20 species were detected when using the ITS and COI region, respectively (Figure 2; Supplementary Materials Tables S1 and S2). When using the ITS region, of the total of 41 Phytophthora species identified, 13 were only detected in “disturbed” soils (P. austrocedri, P. castaneae, P. idaei, P. uniformis, and the uncultured Phytophthora 1a, 3a, 6a, 7a, 10a, 12a, 13a, 14a, and 18a) and five were detected in “undisturbed” soils (P. lacustris, and the uncultured Phytophthora 2a, 9a, 4a, and 20a) (Figure 2; Supplementary Materials Table S1). The unique Nothophytophthora sp. identified was detected in one location at the “undisturbed” site US4. The remaining 23 were detected in both types of soils. Phytophthora austrocedri was only detected at “disturbed” sites and showed a remarkably high prevalence at one of the sampled sites (DS1) (Figure 2; Supplementary Materials Table S1). From the total of 29 Phytophthora species identified when using the COI gene, 13 Phytophthora spp. were detected only in “disturbed” soils (P. europaea, P. quercina, P. uliginosa, and several uncultured Phytophthora spp. from Clade 1 (Phytophthora sp. uncultured 1b, 2b), Clade 6 (Phytophthora sp. uncultured 4b), Clade 7 (Phytophthora sp. uncultured 6b, 7b, 8b), Clade 1 (Phytophthora sp. uncultured 5b), Clade X (Phytophthora sp. uncultured 11b), and from the new candidate Clade (Phytophthora sp. uncultured 15b and 17b)) (Figure 2; Supplementary Materials Table S2). On the other hand, seven were located exclusively in “undisturbed” soils (P. cinnamomi, P. gonapodyides, P. megasperma, P. ramorum, and two Figure 3. Principal coordinates analysis (PCoA) of Bray–Curtis and Jaccard distance matrices from Phytophthora spp. and Nothophytophthora spp. community composition data obtained using COI and ITS markers. PCoA were plotted in the form of a spider diagram with “legs” joining samples that belong to the same cluster (centroid). Shapes on the figure legend correspond to the locations and colors to the sites (“disturbed” and “undisturbed”) sampled. 4. Discussion Although it is not known how many Phytophthora species are already present in British soils, at least 57 species have been detected in the UK [ 65 , 66 ]. Techniques such as high-throughput sequencing (HTS) technologies or NGS have been used recently to detect alien species in soil samples. While some of these techniques have been used to detect communities (all organisms) in environmental samples [ 33 , 67 , 68 ], others have been targeting a selected group of them. In the case of Phytophthora, there are several studies that have been targeting the ITS region, and they also have shown the lack of discrimination of Phytophthora species within some clades such as clades 2 and 3 [ 32 , 35 , 69 ]. Some authors have indicated the higher resolution of COI as compared to ITS within the genus Phytophthora [ 31 , 39 , 40 ]. However, there are no previous NGS studies comparing the Phytophthora community in soils using both ITS and COI. This is the first study where additionally to the ITS region, the COI region was targeted in order to investigate if the detection of oomycetes in soils could be improved and the discrimination of Phytophthora within clades could be better resolved. Forests 2021,12, 229 20 of 26 To test the consistency of PCR amplification of Phytophthora present in the samples and compare the efficiency of ITS versus COI primers, we included control DNA mixes containing 10 Phytophthora spp. We found considerable variability in the number of Illumina reads generated per species that also differed between both markers. With ITS, we were able to amplify all the species included in the control samples except P. boehmeriae. A similar result was recently found by Riddell et al. [ 35 ] with the same species of Phytophthora. On the contrary, when using the COI region, we were not able to detect P. foliorum. One potential explanation to account for the lack of amplification of those Phytophthora sequences or a different amplification efficiency among the remaining ones may be due to the presence of several base mismatches in the primers used in the second round of PCR, which can induce some PCR bias, since the Ct values of the DNA dilutions of each Phytophthora spp. included in the control reactions were in general consistent across each species used in the DNA control mixture [35]. Although the ITS region has proven valuable in Phytophthora diagnostics, the intraspecific and intra-genomic sequence variation of this region is an important consideration for the accurate identification of several species, emphasizing the need for the further validation and refinement of reference databases. In our study, we found that, as expected, the COI region was able to better differentiate several Phytophthora spp. from clades that are difficult to discriminate when using the ITS region. However, as a drawback, several oomycete sequences and sequences from other Phyla were detected using the COI region, which generated a lower number of Phytophthora reads compared to the ITS. Nevertheless, we were able to confirm the presence of several Phytophthora spp. in British soils using both markers. It would be interesting to test whether other genes that have shown good for discrimination of Phytophthora spp. isolates when using pure cultures [ 31 , 37 ] may be used in NGS analysis and provide complementary results to that obtained for COI and ITS. Thus, a total of 20 ASVs 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) were assigned to known Phytophthora species using the ITS and COI, respectively. Of those, only eight Phytophthora species were detected by both ITS and COI: P. cactorum,P. cinnamomi,P. megasperma, P. plurivora,P. primulae,P. pseudosyringae,P. ramorum and P. syringae. The results on these species were in agreement in 21 of the sampled locations at the different sites. No shared Phytophthora species were detected at four of the studied sites, and there were 51 locations where no Phytophthora was amplified by ITS or COI. Only in one site, US3, ITS and COI were in agreement for all the locations, and at the site US4, they were in agreement in seven of the 10 locations. NGS analysis provides evidence of the presence of Phytophthora DNA, but re-sampling the sites to obtain living cultures from soil or from host material should be attempted to confirm their presence. It is especially important for the new records of Britain. Special care should be taken for processing samples, since soil storage could decrease the viability of propagules and might diminish the success of pathogen isolation. The results from this study also detected one and eight ASVs belonging to Nothophytophthora spp., when using the ITS or COI markers, respectively. This is the first report of species of Nothophytophthora detected through NGS, and to date, species within this new genus have not been detected in Britain. In this study, Nothophytophthora species were detected at five sites. Only one Nothophytophthora sp. was detected with ITS and COI at site US4 (US4-10), whereas with COI, eight Nothophytophthora spp. (1–8) were detected in five sites: DS7 (Nothophytophthora sp. 4), DS8 (Nothophytophthora sp. 2 and sp. 5), DS9 (Nothophytophthora sp. 7), US3 (Nothophytophthora sp. 1 and sp. 6), and US4 (Nothophytophthora sp. 3 and sp. 8). Nothophytophthora was described recently as a new sister genus to Phytophthora from natural ecosystems [ 70 ]. Pathogenicity of Nothophytophthora species still needs to be determined, but their association with symptomatic plants suggest they might be pathogenic. Resampling at these sites would be recommended to obtain isolates of these new species. This study also compares which Phytophthora species are present at “disturbed” sites where diseases caused by these pathogens are common and at “undisturbed” sites Forests 2021,12, 229 21 of 26 where neither Phytophthora nor diseased plants associated with Phytophthora have been recorded previously. This study shows that Phytophthora species within clade 8 were the most commonly identified by either ITS and COI (P. austrocedri,P. foliorum,P. obscura,P. primulae,P. ramorum and P. syringae) followed by Phytophthora species within clade 7 (P. cinnamomi,P. europaea, P. uniformis,P. uliginosa). The abundance of Phytophthora species within clade 8 in “disturbed” soils in Britain was also reported by Riddell et al. [ 35 ]. In their study, they also detected P. lateralis, which is a very significant pathogen of Chamaecyparis lawsoniana in the US and has been recently detected in the UK [ 4 ]. However, this species was not detected in this study. On the other hand, in this study, P. foliorum, also from clade 8, was detected in three “disturbed” sites (DS7, DS8, DS9) and in two “undisturbed” sites (US3, US5), and this species was not detected in their study. Phytophthora foliorum was only detected in the UK in 2016 on Rhododendron ponticum in Scotland [ 71 ]. This species was described causing leaf blight on azaleas in the US in 2006 [ 72 ] and has been found in nurseries in Europe [ 8 ]. Clades 7 and 8 contain very important pathogens threatening forest and woodlands tree species worldwide. Of the six species detected in clade 8, four of them (P. austrocedri, P. foliorum,P. obscura and P. ramorum) were only described between 2002 and 2012 and are considered recently introduced in UK. At present, P. austrocedri and P. ramorum are causing great environmental and economic losses in the UK. Phytophthora austrocedri was only discovered in Britain in 2011 causing dieback and mortality in northern England and Scotland on Juniperus communis, which is one of the three native conifers in the UK [73–75] . This species was described from southern Argentina where it is responsible for the mortality of their native cypress Austrocedrus chilensis [ 76 ]. The lack of genetic diversity of P. austrocedri in the UK and their aggressiveness on a native host support the hypothesis that P. austrocedri has been recently introduced into Britain [ 74 , 75 ]. In this study, this species was detected at four of the “disturbed” sites (DS1, DS4, DS7, and DS9) but not at any of the “undisturbed” sites, which would support the above hypothesis. Phytophthora ramorum was described in Germany [ 77 ] and is responsible for sudden oak death in US. In the UK, this is a regulated organism and was initially discovered on Viburnum tinus in the south of England [ 78 ], and then, it was recorded mainly on Rhododendron or other ornamentals in nurseries, plant retail outlets, gardens, parks, and woodlands [ 79 – 83 ]. However, it was in 2009 that P. ramorum was observed causing mortality in mature and juvenile plantations of commercially grown Japanese larch (Larix kaempferi), and since then, it has been causing great economical losses. Other tree species have also been affected by P. ramorum, Fagus sylvatica,Nothofagus obliqua,Castanea sativa,Betula pendula,Tsuga heterophylla and Pseudotsuga menziesii amongst others [ 84 ]. In this study P. ramorum was detected at two of the “disturbed” sites (DS2 and DS4), and at three of the “undisturbed” sites (US1, US3, and US5). This is surprising as this species is to be expected mainly at “disturbed sites” where new plantings have occurred recently or at sites in close proximity to affected larch. Further investigations and isolations from soil samples need to be attempted at these ”undisturbed” sites. However, it is interesting that P. ramorum was isolated from DS9 from a site where the disease was eradicated in 2009. The affected plant back in 2009 was a Rhododendron, which was removed and burned. That area was grassed over, and there are not woody hosts growing there. However, P. ramorum was isolated from the soil underneath the grass, confirming that the pathogen can survive at least nine years in the soil after eradication. These results indicate that re-sampling the sites where some Phytophthora outbreaks have occurred would be advisable to test whether viable propagules of the pathogen are still present in those soils. Phytophthora obscura was described in the USA in 2012 [ 85 ] on foliage of Kalmia latifolia, in substrate of Pieris, and in soil around Aesculus hippocastanum in Germany. This species was detected at three sites in British soils [ 35 ]. In this study, it was detected at five “disturbed” sites (DS2, DS3, DS4, DS7, and DS8) and at one “undisturbed” site (US1). More information is needed regarding this species and the possible threat to British trees. Forests 2021,12, 229 22 of 26 In this study, Phytophthora phylotypes that could not be assigned to known Phytophthora species were identified by both ITS (22) and COI (17). Some of these species could be identified as known Phytophthora species if the cutting point was lowered, but the approach taken in this study was conservative, and only sequences with ≥ 99% similarity were identified to the species level. When the similarity was below 99%, phylogenetic trees were constructed to place them in their clades and show their genetic distances within the clades. As a result of this approach, some of these species might be known species, and some of them could be new to science. For example, Clade 1 Phytophthora sp. uncultured 12 has a high similarity with species detected in at least two previous studies [ 32 , 86 ], or Clade 6 Phytophthora sp. uncultured 9 has a high similarity to species detected by Reeser et al. [87] . Therefore, it is important to try to isolate these species to confirm the results obtained here and to study their biological features and their pathogenicity. In Britain, since the mid-1990s, new or invasive Phytophthora species have been described affecting trees in different environments. Two of these species were novel and were detected in the UK before being discovered in other countries. This was the case for example of Phytophthora alni, which was described in 1993 from riparian alder (Alnus glutinosa) in Britain [ 88 ] and P. kernoviae, which was discovered in 2003 in the southwest of England on Fagus sylvatica and Rhododendron ponticum (Brasier et al., 2005). Phytophthora kernoviae is also a regulated organism in Britain and was detected in this study at site DS6 (young plantation) and US2, which is in close proximity to DS6. These two sites are next to a nursery, and inspection of the nursery would be recommended. The closest host plant for a soil sample from DS6 was a healthy young Pinus sylvestris, and for US2, it was a mature dead Juniperus communis. In the latter, P. austrocedri was expected, but none were detected at this site. However, the detection of P. kernoviae close to a healthy Scots pine needs to be followed up, as recently, P. pluvialis and P. kernoviae have been associated with needle diseases of Pinus radiata in New Zealand [89], and this could be an emerging problem on pine. The detection of species previously unknown in Britain and detected in this study were P. castaneae, P. capsici, and P. fallax. These species were only detected with the ITS primers and not with COI. They are host specific and have been described mainly on Castanea, Solanaceae, and Eucalyptus, respectively [ 90 , 91 ]. Confirmation of their presence at the sites is recommended. 5. Conclusions This study shows that Phytophthora species are present at “disturbed” and “undisturbed” sites in British soils independent of the presence of declined trees. As we hypothesized, more Phytophthora species were detected at “disturbed“ than at “undisturbed“ sites, and this was confirmed by NGS of both ITS and COI regions. This is the first study using COI to study the Phytophthora community in soils, and the results presented showed that COI primers were less specific than the ITS when targeting Phytophthora species. However, COI amplified more Oomycota and a wider range of organisms than when using ITS primers. This is the first detection of Nothophytophthora species in British soils, where it was detected at both “disturbed” and “undisturbed” sites, and the first detection of Nothophytophthora species using NGS analysis with both ITS and COI regions. Of the known Phytophthora species detected in this study, only three are not known to be present in the UK. Therefore, re-sampling of these sites to confirm their presence by isolation would be recommended not only for these three species but for all the potential new Phytophthora species detected by both ITS and COI regions. Supplementary Materials: The following are available online at https://www.mdpi.com/1999-490 7/12/2/229/s1. Figure S1: Frequency of total samples ( A ) and locations ( B ) sampled in “disturbed” and “undisturbed” soils showing positive PCR amplification using primers targeting ITS or COI region. Figure S2: Krona chart showing the relative abundance and diversity from phylum to genus level of all ASVs obtained by NGS using the ITS marker. Figure S3: Krona chart showing the relative abundance and diversity from the phylum to genus level of all ASVs obtained by NGS Forests 2021,12, 229 23 of 26 using the COI marker. Figure S4: Maximum-likelihood tree of ITS sequences obtained in this study and reference sequences inferred in IQ-TREE with the GTR + I + G4 model. Values at branches correspond to bootstraps support. Figure S5: Maximum-likelihood tree of COI sequences obtained in this study and reference sequences inferred in IQ-TREE with the GTR + I + G4 model. Values at branches correspond to bootstraps support. Table S1: Distribution of the different Amplicon Sequence Variants of Phytophthora/Nothophytophthora spp. and number of reads identified using the ITS marker across the 14 sites “disturbed” and “undisturbed” sites sampled in this study; Table S2: Distribution of the different Amplicon Sequence Variants of Phytophthora/Nothophytophthora spp. and number of reads identified using the COI marker across the 14 sites “disturbed” and “undisturbed” sites sampled in this study; Table S3: Permutational multivariate analysis of variance (PERMANOVA) and permutational analysis of multivariate dispersion (Betadisper) results based on Bray–Curtis and Jaccard distances using Hellinger data transformation, for Phytophthora/Nothophytophthora spp. community composition data using ITS and COI marker genes. Author Contributions: Conception and design of the research, B.B.L., B.H. and A.P.-S.; soil sampling, B.H. and A.P.-S.; isolation and soil processing, B.H., L.A.S. and A.P.-S.; acquisition of data, B.B.L., M.M.-B., B.H. and A.P.-S.; analysis and interpretation of data, L.F.A.-G., B.B.L., B.H., M.M.-B. and A.P.-S.; statistical and bioinformatics analysis, L.F.A.-G. and B.B.L.; writing the manuscript, L.F.A.-G., B.B.L., B.H. and A.P.-S. All authors have read and agreed to the published version of the manuscript. Funding: This work has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 635646, POnTE (Pest Organisms Threatening Europe ). Data Availability Statement: The raw sequence data have been deposited in the Sequence Read Archive (SRA) database at the NCBI under BioProject accession number PRJNA690943 and PRJNA691575. Acknowledgments: From Forest Research (FR), we would like to thank Alex Lewis for producing the map for Figure 1and Caroline Gorton for her help processing some of the soil samples. We would like to thank Liz Richardson for helping on site selection, and we thank Forestry England and other site owners for allowing FR to collect the soil samples and for being part of this study. We would also like to thank Treena Burgess (Centre of Phytophthora Science and Management; Murdoch University, WA, Australia) for providing two curated Phytophthora databases for ITS and COI sequences. Conflicts of Interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References 1. Brasier, C.M.; Beales, P.A.; Kirk, S.A.; Denman, S.; Rose, J. Phytophthora kernoviae sp. nov., an invasive pathogen causing bleeding stem lesions on forest trees and foliar necrosis of ornamentals in the UK. 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