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.

Risk of Epidemic Development in Nurseries from Soil Inoculum of Phytophthora ramorum.

Soilborne inoculum arising from buried, infested leaf debris may contribute to the persistence of Phytophthora ramorum at recurrently positive nurseries. To initiate new epidemics, inoculum must not only survive but also produce sporangia during times conducive to infection at the soil surface. To assess this risk, we performed two year-long experiments in a soil plot at the National Ornamentals Research Site at Dominican University of California. Inoculated rhododendron leaf disks were buried at a depth of 5 or 15 cm in the early summer of 2014 or 2015. Inoculum was baited at the soil surface with noninfested leaf disks (2014 only) and then retrieved to assess pathogen viability and sporulation capacity every 5 weeks. Two 14-week-long trials were conducted in 2016. We were able to consistently culture P. ramorum over all time periods. Soil incubation rapidly reduced the capacity of inoculum to sporulate, especially at 5 cm; however, sporulation capacity increased with the onset of seasonally cooler temperatures. P. ramorum was baited most frequently between November and January, especially from inoculum buried at 5 cm 1 day before the baiting period; in January we also baited P. ramorum from inoculum buried at 15 cm the previous June. We validate prior observations that P. ramorum poses a greater risk after exposure to cooler temperatures and provide evidence that infested leaf debris plays a role in the perpetuation of P. ramorum in nurseries. This work provides novel insights into the survival and epidemic behavior of P. ramorum in nursery soils.

Root Rot of Juniperus and Microbiota by Phytophthora lateralis in Oregon Horticultural Nurseries.

Widespread symptoms of root rot and mortality on Juniperus communis and Microbiota decussata were observed in two horticultural nurseries in Oregon, leading to the isolation of a Phytophthora sp. from diseased roots. Based on morphology and sequencing the internal transcribed spacer ITS1-5.8S-ITS2 region, isolates were identified as the invasive pathogen Phytophthora lateralis, causal agent of Port-Orford-cedar (POC; Chamaecyparis lawsoniana) root disease. Additional sequencing of the cytochrome c oxidase subunit 1 and 2 genes identified all isolates as belonging to the PNW lineage. Utilizing recovered isolates plus a POC-wildlands isolate and susceptible POC as controls, we completed Koch's postulates on potted Juniperus and Microbiota plants. Nursery isolates were more aggressive than the forest isolate, which was used in the POC resistance breeding program. Increased aggressiveness was confirmed using a branch stem dip assay with four POC clones that differed in resistance, although no isolate completely overcame major-gene resistance. Isolates were sensitive to mefenoxam, a fungicide commonly used to suppress Phytophthora spp. growth in commercial nurseries. Although POC resistance is durable against these more aggressive nursery isolates, the expanded host range of P. lateralis challenges POC conservation through the continued movement of P. lateralis by the nursery industry.

Film-Forming Polymers and Surfactants Reduce Infection and Sporulation of Phytophthora ramorum on Rhododendron.

Phytophthora ramorum, cause of sudden oak death and ramorum leaf blight, can persist undetected in infested nurseries. Many conventional fungicides are effective in reducing or delaying symptom expression but some may confound visual detection of infected plants. We tested film-forming polymers (FFPs) and surfactants for their ability to reduce infection and sporulation of P. ramorum on rhododendron. FFPs (Anti-Stress, Moisturin, Nature Shield, Nu-Film, and Vapor Gard) and surfactants (Tergitol, Zonix, and an unregistered AGAE product) were screened in detached-leaf assays. Anti-Stress, Nu-Film, Zonix, and a Nu-Film-Zonix mixture were additionally tested for durability, protection against exposure to infested water, and a reduction in sporulation. FFP effectiveness was retained for at least 3 weeks of exposure to overhead irrigation and rain. Relative to controls, foliar treatments protected rhododendron branches exposed to infested water. No treatments prevented symptom development when applied postinfection but leaves treated with Anti-Stress, Zonix, and the Nu-Film-Zonix mixture produced significantly fewer sporangia relative to controls. Application of FFPs and surfactants to quarantined, potentially infected plants offers a management tool for reducing infection and sporulation but not symptom expression, thereby limiting disease spread without interfering with disease detection.

Temporal Epidemiology of Sudden Oak Death in Oregon.

An effort to eradicate Phytophthora ramorum, causal agent of sudden oak death, has been underway since its discovery in Oregon forests. Using an information-theoretical approach, we sought to model yearly variation in the size of newly infested areas and dispersal distance. Maximum dispersal distances were best modeled by spring and winter precipitation 2 years before detection, and infestation size the year prior. Infestation size was best modeled by infestation size and spring precipitation the year prior. In our interpretation, there is a 2-year delay between the introduction of inoculum and onset of mortality for a majority of sites. The year-long gap in between allows ample time for the production of inoculum contributing to the spread of P. ramorum. This is supported by epidemic development following changes in eradication protocols precipitated by an outbreak in 2011, attributable to a 2009 treatment delay and an uncharacteristically wet spring in 2010. Posteradication, we have observed an increase in the total area of new outbreaks and increased frequency in dispersal distances greater than 4 km. Although the eradication program has not eliminated P. ramorum from Oregon forests, it has likely moderated this epidemic, emphasizing the need for prompt treatment of future invasive forest pathogens.