First report of the NA2 clonal lineage of the sudden oak death pathogen, Phytophthora ramorum, infecting tanoak in Oregon forests.

Phytophthora ramorum Werres, de Cock & Man in't Veld, causal agent of sudden oak death (SOD) and ramorum leaf blight, is comprised of four clonal lineages in its invasive ranges of North America and Europe (Grünwald et al. 2012, Van Poucke et al. 2012). Of these, three - the NA1, NA2, and EU1 lineages - are found in U.S. nurseries, but only two, the NA1 and EU1 lineages, have been found infecting trees in North American forests (Grünwald et al. 2012, 2016). In the spring of 2021, tanoak (Notholithocarpus densiflorus Manos, Cannon & Oh) displaying symptoms consistent with SOD were detected north of Port Orford (Curry County, Oregon). Symptoms were canopy dieback and blackened petiole and stem lesions on tanoak sprouts. The pathogen isolated on PAR (CMA plus 200 ml/L ampicillin, 10 mg/L rifamycin, 66.7 mg/L PCNB) selective media was determined to be P. ramorum based on characteristic morphology of hyphae, sporangia, and chlamydospores (Werres et al. 2001). Positive identification as P. ramorum was obtained with a lineage-specific LAMP assay targeting an NA2 orphan gene, indicating the presence of the NA2 lineage. NA2 was confirmed by sequencing a portion of the cellulose binding elicitor lectin (CBEL) gene using CBEL5U and CBEL6L primers (Gagnon et al. 2014). Sequences (GenBank accessions MZ733981 and MZ733982) were aligned against reference sequences for all lineages (Gagnon et al. 2014) confirming the presence of NA2. Lineage determination as NA2 was further confirmed at eleven SSR loci (ILVOPrMS145, PrMS39, PrMS9C3, ILVOPrMS79, KI18, KI64, PrMS45, PrMS6, ILVOPrMS131, KI82ab, and PrMS43) using the methods of Kamvar et al. (2015). We completed Koch's postulates using potted tanoaks, wound-inoculated at the midpoint of 1-year old stems with either hyphal plugs or non-colonized agar (n=4 per treatment). Tanoaks were maintained in a growth chamber (20°C-day / 18°C-night temperatures) with regular watering and an 18-photoperiod using F32T8 fluorescent bulbs (Phillips, Eindhoven, The Netherlands). After 7 days, brown to black lesions 1.2 to 2.9 cm in length were observed on the inoculated stems, from which P. ramorum was subsequently re-isolated; no symptoms were observed on the controls, and no pathogens were recovered when plating the wound sites in PAR. This is the first detection of the NA2 lineage causing disease in forests worldwide. The outbreak was found on private and public lands in forests typical to the SOD outbreak in Oregon (mixed conifer and tanoak), and was 33 km north of the closest known P. ramorum infestation. Follow-up ground surveys on adjacent lands have identified over 100 P. ramorum-positive tanoak trees, from which additional NA2 isolates have been recovered from bole cankers. NA2 is thought to be more aggressive than the NA1 lineage (Elliott et al. 2011), which has been present in Curry County since the mid-1990s (Goheen et al. 2017). Eradication of the NA2 lineage is being pursued to slow its further spread and prevent overlap with existing NA1 and EU1 populations. The repeated introductions of novel lineages into the western United States native plant communities highlights the vulnerability of this region to Phytophthora establishment, justifying continued monitoring for P. ramorum in nurseries and forests. References • Elliott, M, et al. 2011. For. Path. 41:7. https://doi.org/10.1111/j.1439-0329.2009.00627.x • Gagnon, M.-C., et al. 2014. Can. J. Plant Pathol. 36:367. https://doi.org/10.1080/07060661.2014.924999 • Goheen, E.M., et al. 2017. For. Phytophthoras 7:45. https://doi: 10.5399/osu/fp.7.1.4030 • Grünwald, N. J., et al. 2012. Trends Microbiol. 20:131. https://doi.org/10.1016/j.tim.2011.12.006 • Grünwald, N. J., et al. 2016. Plant Dis. 100:1024. https://doi.org/10.1094/PDIS-10-15-1169-PDN • Kamvar, Z.N. et al. 2015. Phytopath. 105:982. https://doi.org/10.1094/PHYTO-12-14-0350-FI • Van Poucke, K., et al. 2012. Fungal Biol. 116:1178. https://doi.org/10.1016/j.funbio.2012.09.003 • Werres, S., et al. 2001. Mycol. Res. 105: 1155. https://doi.org/10.1016/S0953-7562(08)61986-3.

Novel Introductions and Epidemic Dynamics of the Sudden Oak Death Pathogen Phytophthora ramorum in Oregon Forests.

Sudden oak death caused by Phytophthora ramorum has been actively managed in Oregon since the early 2000s. To date, this epidemic has been driven mostly by the NA1 clonal lineage of P. ramorum, but an outbreak of the EU1 lineage has recently emerged. Here, we contrast the population dynamics of the NA1 outbreak first reported in 2001 to the outbreak of the EU1 lineage first detected in 2015. We performed tests to determine whether any of the lineages were introduced more than once. Infested regions of the forest were sampled between 2013 and 2018 (n = 903), and strains were genotyped at 15 microsatellite loci. Most genotypes observed were transient, with 272 of 358 unique genotypes emerging during one year and disappearing the next year. The diversity of EU1 was very low and isolates were spatially clustered (less than 8 km apart), suggesting a single EU1 introduction. Some forest isolates are genetically similar to isolates collected from a local nursery in 2012, suggesting the introduction of EU1 from this nursery or simultaneous introduction to both the nursery and latently into the forest. In contrast, the older NA1 populations were more polymorphic and spread more than 30 km2. A principal component analysis supported two to four independent NA1 introductions. The NA1 and EU1 epidemics infest the same area but show disparate demographics because of the initial introductions of the lineages spaced 10 years apart. Comparing these epidemics provides novel insight regarding patterns of emergence of clonal pathogens in forest ecosystems.

Ecology and pathology of Phytophthora ITS clade 3 species in forests in western Oregon, USA.

Phytophthora species are widespread and diverse in forest ecosystems, but little is known about their ecology. We explore ecological attributes of the closely related clade 3 species that occur sympatrically in western North American forests. We address the population structure, pathology, and epidemiology of P. ilicis, P. nemorosa, P. pluvialis, P. pseudosyringae, and P. psychrophila. Phytophthora species were isolated from plant tissues, rainwater falling through the forest canopy, streams, and soils in forests in western Oregon. Species identifications were based on morphology in culture with molecular confirmation using COX spacer and internal transcribed spacer (ITS) sequences. All five clade 3 Phytophthora species are present in western Oregon forests, although P. ilicis (only 1 forest isolate) and P. psychrophila (only 12 isolates) are apparently rare. P. ilicis is known only from holly in horticultural situations and once from a naturalized seedling in an urban forest. The known distribution of P. nemorosa in forest settings coincides with the ranges of its principle hosts, tanoak and myrtlewood, in Oregon and California. Although it is regularly identified from streams within the tanoak range, it has not been recovered from streams beyond that range. P. pluvialis is primarily associated with Douglas-fir canopies. It was identified from scattered locations throughout western Oregon in rain traps beneath Douglas-fir plantations and from diseased needles. P. pseudosyringae is also isolated from tanoak and myrtlewood in southwest Oregon and California, but its distribution, in streams at least, extends throughout much of western Oregon. P. psychrophila in Oregon is known only from rain traps beneath tanoak trees. Little intraspecific variation was detected in the nuclear rDNA ITS of clade 3 species. Variation in the mitochondrial COX spacer region was more frequent, with 2 to 10 haplotypes identified in the clade 3 species, for which we had multiple isolates.

The Phytophthora species assemblage and diversity in riparian alder ecosystems of western Oregon, USA.

Phytophthora species were systematically sampled, isolated, identified and compared for presence in streams, soil and roots of alder (Alnus species) dominated riparian ecosystems in western Oregon. We describe the species assemblage and evaluate Phytophthora diversity associated with alder. We recovered 1250 isolates of 20 Phytophthora species. Only three species were recovered from all substrates (streams, soil, alder roots): P. gonapodyides, the informally described "P. taxon Pgchlamydo", and P. siskiyouensis. P. alni ssp. uniformis along with five other species not previously recovered in Oregon forests are included in the assemblage: P.citricola s.l., P. gregata, P. gallica, P. nicotianae and P. parsiana. Phytophthora species diversity was greatest in downstream riparian locations. There was no significant difference in species diversity comparing soil and unwashed roots (the rhizosphere) to stream water. There was a difference between the predominating species from the rhizosphere compared to stream water. The most numerous species was the informally described "P. taxon Oaksoil", which was mainly recovered from, and most predominant in, stream water. The most common species from riparian forest soils and alder root systems was P. gonapodyides.

Phytophthora borealis and Phytophthora riparia, new species in Phytophthora ITS Clade 6.

Phytophthora borealis and Phytophthora riparia, identified in recent Phytophthora surveys of forest streams in Oregon, California and Alaska, are described as new species in Phytophthora ITS Clade 6. They are similar in growth form and morphology to P. gonapodyides and are predominantly sterile. They present unique DNA sequences, however, and differ in temperature/growth relations and geographic distribution.

Phytophthora beyond agriculture.

Little is known about indigenous Phytophthora species in natural ecosystems. Increasing evidence, however, suggests that a diverse, trophically complex Phytophthora community is important in many forests. The number of described species has steadily increased, with a dramatic spike in recent years as new species have been split from old and new species have been discovered through exploration of new habitats. Forest soil, streams, and the upper canopies of trees are now being explored for Phytophthora diversity, and a new appreciation for the ecological amplitude of the genus is emerging. Ten to twenty species are regularly identified in temperate forest surveys. Half or more of this Phytophthora diversity comes from species described since 2000. Taxa in internal transcribed spacer (ITS) Clade 6 are especially numerous in forest streams and may be saprophytic in this habitat. Three ecological assemblages of forest Phytophthora species are hypothesized: aquatic opportunists, foliar pathogens, and soilborne fine-root and canker pathogens. Aggressive invasive species are associated with all three groups.

Phytophthora species in forest streams in Oregon and Alaska.

Eighteen Phytophthora species and one species of Halophytophthora were identified in 113 forest streams in Alaska, western Oregon and southwestern Oregon that were sampled by baiting or filtration of stream water with isolation on selective media. Species were identified by morphology and DNA characterization with single strand conformational polymorphism, COX spacer sequence and ITS sequence. ITS Clade 6 species were most abundant overall, but only four species, P. gonapodyides (37% of all isolates), P. taxon Salixsoil, P. taxon Oaksoil and P. pseudosyringae, were found in all three regions. The species assemblages were similar in the two Oregon regions, but P. taxon Pgchlamydo was absent in Alaska and one new species present in Alaska was absent in Oregon streams. The number of Phytophthora propagules in Oregon streams varied by season and in SW Oregon, where sampling continued year round, P. taxon Salixsoil, P. nemorosa and P. siskiyouensis were recovered only in some seasons.

Phytophthora rosacearum and P. sansomeana, new species segregated from the Phytophthora megasperma "complex".

Phytophthora megasperma sensu lato was a conglomeration of morphologically similar but phylogenetically unrelated species. In this paper we continue the segregation of species from the old P. megasperma complex, formally naming two previously recognized isolate groups. Isolates recovered from rosaceous fruit trees (especially apple and cherry) are in ITS clade 6, related to but distinct from P. megasperma sensu strictu. They are named here Phytophthora rosacearum. They have been referred to previously as the "AC" or "high temperature small oospore" group of P. megasperma. A second group of isolates, earlier called "soybean race non-classifiable", recovered from soybeans in Indiana and other Midwestern states, are morphologically similar to P. megasperma sensu strictu but unrelated to that species, falling in ITS clade 8. They are named here P. sansomeana. Isolates recovered from Douglas-fir seedlings in nurseries in the Pacific Northwest and various weedy hosts in New York State, referred to in earlier work as "P. megasperma DF1", appear to be conspecific with the soybean isolates, although they include certain ITS DNA polymorphisms. Both new species are supported by a combination of new and previously published morphological, growth and molecular data.

Phytophthora siskiyouensis, a new species from soil, water, myrtlewood (Umbellularia californica) and tanoak (Lithocarpus densiflorus) in southwestern Oregon.

An unknown Phytophthora species was recovered in southwestern Oregon from rhododendron and tanoak leaf baits used for monitoring streams and soils for the presence of Phytophthora ramorum, from a blighted shoot of myrtlewood and from tanoak bark cankers. Isolates of this species yielded ITS-DNA sequences that differed substantially from other Phytophthora sequences in GenBank. Morphological features also differed from available descriptions of known Phytophthora species. Based on the combination of unique morphology and unique ITS sequences a new species is proposed. The new species, Phytophthora siskiyouensis, is homothallic with globose to subglobose oogonia, which may be terminal, sessile or laterally intercalary. Antheridia are capitate and mostly paragynous but sometimes amphigynous. Oospores are mostly aplerotic. Sporangia are variable but commonly ovoid to reniform, with apical, subapical or lateral semipapillae (occasionally more than one). Sporangia are terminal, subterminal or occasionally intercalary on unbranched sporangiophores, with basal, subbasal or lateral attachment. Sporangia are weakly deciduous, with variable length pedicels. This combination of characters clearly separates Phytophthora siskiyouensis from other known Phytophthora species.

A climate-based model for predicting geographic variation in swiss needle cast severity in the Oregon coast range.

ABSTRACT Since the early 1990s, Swiss needle cast disease caused by Phaeocryptopus gaeumannii has been increasing in Douglas-fir plantations in the Oregon Coast Range. Considerable variation in disease severity across the affected area often has been noted. We investigated the influence of site microclimate on fungal colonization as a basis for this variation with a combination of seedling inoculation and field studies. Development of P. gaeumannii ascocarps on inoculated seedlings subjected to mist, irrigation, and shading treatments was followed for 10 months. Contrary to expectations, numbers of ascocarps on foliage were negatively correlated with shade and mist and positively correlated with temperature. Numbers of ascocarps on foliage, site temperature, and leaf wetness were monitored over 5 years at nine field sites in the Oregon Coast Range. Factors most highly correlated with ascocarp abundance were winter mean daily temperature and spring cumulative leaf wetness. Predictive models for disease severity on the basis of these correlations were tested against disease and climate data measured at field sites during 2003-2004. A temperature-based disease prediction model was developed in combination with geographical information systems (GIS)-linked climate databases to estimate disease levels across a portion of the Oregon Coast Range. This model can be used for hypothesis testing and as a decision support tool for forest managers.