P.W. Reeser's research while affiliated with Oregon State University and other places

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

Phytophthora ramorum, the cause of Sudden Oak Death, is an invasive pathogen present in parts of coastal California and southwestern Oregon forests. The majority of these forest infestations have all been caused by the NA1 clonal lineage. In 2015, the EU1 lineage of P. ramorum was isolated from a tanoak (Notholithocarpus densiflorus) tree located in a mixed‐conifer forest of Curry County, Oregon. In order to evaluate the threat of the EU1 lineage relative to the established NA1 lineage to Oregon forests a series of experiments were conducted comparing aggressiveness and sporulation of NA1 and EU1 isolates on logs and seedlings in the growth chamber and forest. There was no difference in lesion size on logs inoculated with NA1 and EU1 isolates for any of the tree species tested. Across all seedling experiments differences among isolates within lineage, in terms of both aggressiveness and sporulation, were more commonly observed then differences among lineages. Site to site variation in tanoak sporulation, as measured by bucket baiting, appears to be correlated with the number of P. ramorum positive seedlings detected at each site.

Sudden oak death caused by Phytophthora ramorum has been actively managed in Oregon since the early 2000's. 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 tested if any of the lineages were introduced more than once. Infested regions of the forest were sampled between 2013-2018 (n = 903) and strains were genotyped at 15 microsatellite loci. Most genotypes observed were transient, with 272 of 358 unique genotypes emerging one year and disappearing the next. Diversity of EU1 was very low and isolates were spatially clustered (< 8 km apart), suggesting a single EU1 introduction. Some forest isolates are genetically similar to isolates collected from a local nursery in 2012, suggesting 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 over 30 km2. Principal component analysis supported two to four independent NA1 introductions. The NA1 and EU1 epidemics infest the same area but show disparate demographics owing to initial introductions of the lineages spaced 10 years apart. Comparing these epidemics provides novel insights into patterns of emergence of clonal pathogens in forest ecosystems.

Societal Impact Statement Non‐native pathogens and pests cause high mortality to tree species globally and may imperil the future viability of associated forest ecosystems. Phytophthora lateralis , an oomycete, causes Port‐Orford‐cedar root disease and is a major cause of mortality in the ecologically and economically important conifer species Chamaecyparis lawsoniana (Port‐Orford‐cedar). The P. lateralis resistance program shows promise to help stabilize C. lawsoniana in its native range of northwestern California and southwestern Oregon, USA, and serves as a leading example of disease resistance breeding in forest trees Summary A non‐native, invasive pathogen, Phytophthora lateralis , has caused extensive mortality within the native range, northern California and southern Oregon USA, of Chamaecyparis lawsoniana (Port‐Orford‐cedar), as well as in horticultural and amenity plantings in the USA and Europe. Restoration of affected sites is contingent upon development of populations with genetic resistance. Naturally occurring genetic resistance has been identified in C. lawsoniana , and an active selective breeding program seeks to characterize and increase resistance levels. Two seedling root dip inoculation trials, assessed for mortality for nearly three years each, are used to examine the types and levels of genetic resistance in C. lawsoniana . Most seedlings utilized in these trials are progeny of crosses from parent trees that exhibited apparent resistance to the disease in earlier trials. Seedling trials suggest that both qualitative major gene and quantitative disease resistance occurs in C. lawsoniana . Both types of resistance to P. lateralis appear to be present at levels high enough to be immediately useful for restoration and reforestation. The data suggest that the qualitative resistance is conditioned by a single major gene (designated here as Pla ), but nothing is known about the number of genes involved in quantitative disease resistance. Seedling progeny from resistant parent trees in containerized seed orchards are now being used for restoration and reforestation. Resistant seedlings or clones could also be used to re‐establish C. lawsoniana in urban forests.

Phytophthora ramorum, cause of sudden oak death, has been distributed widely across the United States in horticultural situations, but is not established in forests outside of California and Oregon. Here, it has triggered widespread concern and, especially in Oregon, an intensive disease management programme. Now, we provide the first systematic evaluation of the efficacy of that effort. This paper evaluates four measures of the efficacy of Sudden Oak Death (SOD) local eradication treatments: inoculum availability; inoculum from tree species other than tanoak; disease spread from treated areas; and cumulative infested area with and without treatment. We conclude that local treatments demonstrably reduce local inoculum levels. Eradication of SOD from infested sites is difficult but not impossible. The disease usually does not persist after cutting infected trees but spread on the landscape continues because the pathogen may be present on undetected new infections for a year or two before whole tree symptoms are visible. This limits early detection and coupled with delays in completing eradication treatments, prolongs the chances for long‐distance aerial dispersal.

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.

Overview Phytophthora pluvialis Reeser, Sutton, and E. Hansen was first discovered in streams, soil, and rain traps in Curry County, Oregon, USA, in areas dominated by native forest of mixed tanoak and Douglas-fir (Reeser et al., 2013). On rare occasions it was isolated from lesions on tanoak bark or twigs, but was only weakly pathogenic when inoculated into tanoak stems. In New Zealand, Phytophthora pluvialis was found to cause red needle cast on radiata pine (Dick et al., 2014). InOregon the species is now associated with Douglas-fir, and experimentally has been shown to cause needle cast and twig cankers on seedling trees, both artificially inoculated and exposed beneath the canopy of Douglas-fir plantations (Hansen et al., 2014). The pathogen has not been associated with canker formation or infection of the harvested stem of radiata pine (Hood et al., 2014) .

Initially reported in California as the causal agent of sudden oak death (SOD), efforts to limit spread of Phytophthora ramorum in Oregon natural forests have concentrated on quarantine regulations and eradication of the pathogen from infested areas. P. ramorum has four clonal lineages: NA1; NA2; EU1; and EU2 (Grünwald et al. 2012; Van Poucke et al. 2012). Forest infestations in Oregon have been limited to the NA1 clonal lineage, whereas EU1, NA1, and NA2 clonal lineages have all been found in U.S. nurseries (Kamvar et al. 2015; Prospero et al. 2007). In February 2015, in response to an aerial survey, P. ramorum was isolated from a dying Notholithocarpus densiflorus tree in the South Fork Pistol River drainage of Curry Co., Oregon. The isolated strain was identified as P. ramorum based on presence of chlamydospores, characteristic hyphae, and sporangial morphology. Microsatellite genotyping at 14 loci (Vercauteren et al. 2011) and comparison with reference cultures revealed that these isolates belonged to the EU1 clonal lineage. Subsequently, five more isolates were obtained from the original tree stump and the EU1 lineage was confirmed. Microsatellite alleles of the forest EU1 isolates were nearly identical to EU1 isolates collected in 2012 from a nearby nursery during routine P. ramorum nursery monitoring, except for one allele at locus PrMS145a. Interestingly, several isolates differed at locus ILVOPrMS131a within both the 2015 forest and the 2012 nursery findings with identical allele frequencies in each population for this locus. These data provide inconclusive support for the introduction of EU1 into Curry Co. from the 2012 populations found in nurseries, given that no direct match was found probably owing to the paucity of EU1 samples from nurseries. These results provide further evidence that multiple distinct P. ramorum introduction events into the Curry Co. forest are a critical component of the epidemic (Kamvar et al. 2015). The impact of the EU1 clonal lineage of P. ramorum on Oregon natural forests is uncertain, but it may result in potential sexual reproduction given that EU1 is of A1 mating type while the prior population consisted of NA1 A2 mating type individuals. While sexual populations of P. ramorum have not been observed in nature or were aberrant in the laboratory, the presence of both A1 and A2 mating types makes the potential for sexual recombination more likely. The EU1 forest infestation is undergoing eradication treatments. Additional monitoring is necessary to determine if the EU1 clonal lineage occurs elsewhere in Curry Co. forests. © 2016, American Phytopathological Society. All rights reserved.