The effect of inoculation with on survival of seed and seedlings of 19 populations of was examined under environmentally controlled conditions, with four treatments (0, 50, 10, 10 spores ml). A single seed source of was included as a positive control. Germination (emergence of the plumule above the compost) and health of seedlings was assessed daily, for 85 days. Spore density had a significant effect on germination: at 50 spores ml, only germination of a Northeast Scotland population was reduced. Treatment with 1000 spores ml, however, reduced germination of six populations of and of Survival of emerged seedlings also varied with inoculum dose. Approximately 75% of seedlings survived 85 days after germination after inoculation with 50 spores ml. Seedlings of all populations were killed within 12-16 days of germination by the 10 and 10 spores ml treatments. Emerged seedlings of the Austrian populations showed the highest susceptibility to following treatment with 50 spores ml, although 15% of seedlings of one Austrian population (AU3) survived to the end of the experiment (85 days after germination). There was no clear pattern in survival rates of the seedlings from other populations treated with 1000 or 1 million spores ml due to death of all emerged seedlings within a short period. Variations in susceptibility of different populations of to may be used in future selection and breeding programmes to reduce the impact of the pathogen as it spreads over wider areas in Europe and Eurasia.

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 ( 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 based on characteristic morphology of hyphae, sporangia, and chlamydospores (Werres et al. 2001). Positive identification as 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 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 infestation. Follow-up ground surveys on adjacent lands have identified over 100 -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 establishment, justifying continued monitoring for 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.

We describe a method for inoculating rachises of (European or common ash) with which is faster than previous methods and allows associated foliar symptoms to be assessed on replicate leaves. A total of ten ash seedlings were inoculated with five isolates of and lesion development assessed over four weeks. A five-point disease progress scale of symptom development was developed from no lesion (0), lesion on rachis (1), "pre-top dead," with curling of distal leaflets and bending of the rachis (2), top dead, with wilting and death of distal leaflets (3) to leaf abscission (4). The method revealed variation in aggressiveness of isolates and may be suitable for assessing the resistance of and other species to dieback. The in vitro growth rate of isolates was highly correlated with both disease progress and the length of rachis lesions on susceptible plants, indicating that it can be used as a preliminary step in selecting isolates with high aggressiveness for use in resistance screening.

is a fungus associated with oak wilt and deemed to cause extensive oak mortality in South Korea. Since the discovery of this fungus on a dead Mongolian oak () in 2004, the mortality continued to spread southwards in South Korea. Despite continued expansion of the disease and associated significant impacts on forest ecosystems, information is lacking about the origin and genetic diversity of . Restriction-site-Associated DNA (RAD) sequencing was used to assess genetic diversity and population structure among five populations (provinces) of in South Korea. In total, 179 single nucleotide polymorphisms (SNPs) were identified among 2,639 RAD loci across the nuclear genome of the 54 isolates (0.0012 SNPs per bp), which displayed an overall low expected heterozygosity and no apparent population structure. The low genetic diversity and no apparent population structure among South Korean populations of this ambrosia beetle-vectored fungus supports the hypothesis that this fungus was introduced to South Korea.

Since the early 1990s, European ash (Fraxinus excelsior L.) has been affected by a lethal disease caused by the ascomycete fungus, Hymenoscyphus pseudoalbidus, originally known under the name of its anamorph, Chalara fraxinea (2,4). Pathogenicity of H. pseudoalbidus was demonstrated by inoculations on young trees (3). This emerging pathogen induces necrosis of leaf rachises, leaf wilting and shedding, bark necrosis, and wood discoloration as well as shoot, twig, and branch dieback. First observed in Poland, ash dieback now occurs in many parts of Europe. Since 2009, a survey of ash dieback caused by H. pseudoalbidus has been conducted in Wallonia (southern Belgium). Sampling units were selected to take the occurrence of ash stands and the potential points of entry of the pathogen into the country (nurseries, sawmills, rivers, and roads) into account. While the disease was not detected in 2009, young, naturally regenerated trees displaying typical symptoms of ash dieback were found in June 2010 in Silly, a village in the province of Hainaut. Symptomatic trees were located along a road in front of a large ash stand. Examination of shoots with bark necrosis from three symptomatic trees yielded positive results on the basis of a real time PCR test developed in our laboratory for the detection of H. pseudoalbidus (1). To confirm the molecular identification, fungal isolation from discolored wood onto malt extract agar supplemented with 100 mg liter of streptomycin sulfate was attempted. After 18 days at 20 to 22°C in the dark, slow-growing, dull white colonies with gray patches, resembling those of C. fraxinea, had formed. The nuclear ribosomal internal transcribed spacer region (ITS) was amplified with primers ITS1 and ITS4 (4) and partly sequenced (GenBank Accession No. FR667687). A BLASTn search in GenBank revealed that the sequence of the Belgian isolate (452 bp) displayed 100% identity with sequences of a H. pseudoalbidus isolate from Switzerland (GenBank Accession No GU586932). In contrast, the sequence showed some mismatches with that of the closely related and probably strictly saprotrophic fungus, Hymenoscyphus albidus (GenBank Accession No GU586891.1). The strain was deposited as reference material in the Fungal Biology collection (CBS 128012). To our knowledge, this is the first report of ash dieback caused by H. pseudoalbidus in Belgium. The discovery of this aggressive tree pathogen in Wallonia documents its further westward spread in Europe. In the future, we expect that H. pseudoalbidus will continue its range expansion into areas that have so far not been affected by ash dieback. References: (1) A. Chandelier et al. For. Pathol. 40:87, 2010. (2) T. Kowalski. For. Pathol. 36:264, 2006. (3) T. Kowalski and O. Holdenrieder. For. Pathol. 39:1, 2009. (4) V. Queloz et al. For. Pathol. Online publication. doi:10.1111/j.1439-0329.2010.00645.x, 2010.

European ash (Fraxinus excelsior), also known as common ash, occurs naturally inland in lower areas of southeastern Norway and along the southern coast of the country. It is important both as a forest and ornamental tree. During the last decade, dieback has become a disastrous disease on F. excelsior in many European countries. The anamorphic fungus Chalara fraxinea T. Kowalski (1), described for the first time from dying ash trees in Poland, is now considered the cause of ash dieback (2). In May of 2008, C. fraxinea was isolated from 1.5 m high diseased F. excelsior in a nursery in Østfold County in southeastern Norway. Symptoms included wilting, necrotic lesions around leaf scars and side branches, and discoloration of the wood. From symptomatic branches, small pieces (approximately 1 cm) were excised in the transition area between healthy and discolored wood. After surface sterilization (10 s in 70% ethanol + 90 s in NaOCl), the pieces were air dried for 1 min in a safety cabinet, cut into smaller pieces, and placed on media. The fungus was isolated on potato dextrose agar (PDA) and water agar (WA). On PDA, the cultures were tomentose, light orange, and grew slowly (21 mm mean colony diameter after 2 weeks at room temperature). Typical morphological features of C. fraxinea developed in culture. Brownish phialides (14.8 to 30.0 [19.5] × 2.5 to 5.0 [4.1] μm, n = 50) first appeared in the center of the colonies on the agar plugs that had been transferred. The agar plugs were 21 days old when phialides were observed. Abundant sporulation occurred 3 days later. Conidia (phialospores) extruded apically from the phialides and formed droplets. Conidia measured 2.1 to 4.0 (3.0) × 1.4 to 1.9 (1.7) μm (n = 50). The first-formed conidia from each phialide were different in size and shape from the rest by being longer (6 μm, n = 10) and more narrow in the end that first appeared at the opening of the phialide. Internal transcribed spacer sequencing confirmed that the morphological identification was correct (Accession No. EU848544 in GenBank). A pathogenicity test was carried out in June of 2008 by carefully removing one leaf per plant on 10 to 25 cm high F. excelsior trees (18 trees) and placing agar plugs from a 31-day-old C. fraxinea culture (isolate number 10636) on the leaf scars and covering with Parafilm. After 46 days, isolations were carried out as described above from discolored wood that had developed underneath necrotic lesions in the bark and subsequently caused wilting of leaves. All the inoculated plants showed symptoms, and C. fraxinea was successfully reisolated. No symptoms were seen on uninoculated control plants (eight trees) that had received the same treatment except that sterile PDA agar plugs had been used. References: (1) T. Kowalski. For. Pathol. 36:264, 2006. (2) T. Kowalski and O. Holdenrieder, For. Pathol. Online publication, doi: 10.1111/j.1439-0329.2008.00565.x, 2008.

During 2006 and 2007, declining mature beech trees (Fagus sylvatica) were recorded in two stands in the Natural Park of Monti Cimini in central Italy. Symptoms included crown thinning and the presence of bleeding lesions on the main roots and lower stem. Incidence of decline was approximately 5%. Samples of necrotic bark tissue were collected, cut into 5 mm long segments, plated on PARPNH, and incubated at 20°C (1). Phytophthora isolates were obtained from necrotic tissues of 25% of the sampled declining trees. Colonies were rosaceous on potato dextrose agar (PDA) and homothallic. Papillate, ovoid-to-obpyriform, caducous sporangia (mean 38 × 26.2 μm) were produced in soil extract. Oospores were plerotic (mean diameter of 22 to 32 μm) and antheridia paragynous. Optimum growth temperature was 23 to 25°C, minimum 6 to 8°C and maximum 30 to 33°C. A portion of the internal transcribed spacer sequence has been deposited in the NCBI database (GenBank Accession No. FJ183724). A BLAST search of the NCBI database revealed Phytophthora cactorum, Accession No. EU194384, as the closest match with 100% sequence similarity. Pathogenicity of two isolates, PFE3 and IFB-CAC 38, collected from distressed beech trees was tested using a soil infestation test (10 beech seedlings per isolate and control) and an under the bark inoculation method (10 twigs per isolate and controls, wounded and noninoculated taken from a declining beech tree) (2). After 2 weeks at 20°C, twigs and seedlings inoculated with each isolate developed extensive necrotic lesions around the inoculation sites and the collar, respectively, and P. cactorum could be reisolated from all lesions. Controls showed no symptoms. P. cactorum is widespread in declining beech forests in central Europe (1). In Italy, P. cactorum occurs in soils of chestnut and oak forests and was isolated from collar and root lesions of declining walnut trees (3). To our knowledge, this is the first report of P. cactorum being associated with declining beech trees in Italy. References: (1) T. Jung. Forest Pathol. Online publication. doi:10.1111/j.1439-0329.2008.00566.x, 2008. (2) T. Jung et al. Eur. J. For. Pathol. 26:253, 1996. (3) A. M. Vettraino et al. Plant Pathol. 52:491, 2003.

Tanoak (Lithocarpus densiflorus) is a principal host of Phytophthora ramorum, cause of sudden oak death (SOD), in the western United States (1). In the course of SOD surveys in southwestern Oregon, other Phytophthora species were encountered to be causing stem cankers on tanoak that were indistinguishable from those caused by P. ramorum. In Oregon, SOD is subject to quarantine and eradication. Aerial surveys are flown two or more times a year to locate symptomatic tanoaks, which are then examined from the ground to determine the cause of death. Isolations on selective media were attempted from all trees with stem cankers typical of Phytophthora. Phytophthora species were identified by morphological features and DNA sequencing of either internal transcribed spacer (ITS) or the mitochondrial COX spacer region. ITS sequences were compared with validated GenBank records, and COX spacer sequences were compared with known reference isolates in the OSU collection. From 2001 through 2006, Phytophthora spp. were isolated from 482 of 1,057 tanoak stem cankers sampled. P. ramorum was isolated from 359 cankers, P. nemorosa was isolated from 102 cankers, P. gonapodyides was isolated from six cankers, P. cambivora was isolated from four cankers (all A1 mating type), P. siskiyouensis was isolated from four cankers, P. pseudosyringae was isolated from two cankers, P. cinnamomi was isolated from one canker (mating type A2), and P. taxon "Pgchlamydo" was isolated from one canker. Three cankers yielded isolates that were not identified but were closely related to P. pseudosyringae based on ITS sequence. No Phytophthora spp. were cultured from the remaining cankers. One isolate from each species identified (except P. ramorum and P. pseudosyringae) was tested for pathogenicity on tanoak stems (11.4 to 16.0 cm DBH) in the field. A 5-mm-diameter plug from the margin of a V8 agar culture was placed in a hole in the bark, covered with wet cheesecloth, and sealed with aluminum foil and duct tape. Each isolate was inoculated into five different stems. Each stem received three different isolates and an agar control. After 4 weeks, bark was removed to reveal lesion development. Lesions were measured (length by width), and pieces from four points on the lesion margin were plated in selective media to reisolate. P. cambivora, P. cinnamomi, P. gonapodyides, P. nemorosa, P. siskiyouensis and P. taxon "Pgchlamydo" all caused substantial lesions in inoculated tanoak trees (average area 11.5 to 18.6 cm). In all cases, the species used for inoculation was recovered on reisolation from lesion margins. Control inoculations caused necrotic areas averaging 0.2 cm. Isolations from these areas were clean. Prior to the recent SOD epidemic, no species of Phytophthora were known as pathogens of tanoak. The discovery of P. ramorum as a pathogen of tanoak in California was quickly followed by the discovery that P. nemorosa and P. pseudosyringae were also associated with tanoak cankers (2). Six years of diagnostic support for survey and detection of P. ramorum in tanoak forests of southwest Oregon has revealed the occurrence, at very low frequency, of at least five additional species of Phytophthora causing stem cankers in tanoak. References: (1) D. M. Rizzo et al. Ann. Rev. Phytopathol. 43:309, 2005. (2) A. C. Wickland et al. For. Pathol. Online publication. DOI:10.1111/j.1439-0329.2008.00552.x), 2008.