Secreted Effector Proteins of Poplar Leaf Spot and Stem Canker Pathogen Sphaerulina musiva Manipulate Plant Immunity and Contribute to Virulence in Diverse Ways.

Fungal effectors play critical roles in manipulating plant immune responses and promoting colonization. Sphaerulina musiva is a heterothallic ascomycete fungus that causes Septoria leaf spot and stem canker disease in poplar (Populus spp.) plantations. This disease can result in premature defoliation, branch and stem breakage, increased mortality, and plantation failure. However, little is known about the interaction between S. musiva and poplar. Previous work predicted 142 candidate secreted effector proteins in S. musiva (SmCSEPs), 19 of which were selected for further functional characterization in this study. SmCSEP3 induced plant cell death in Nicotiana benthamiana, while 8 out of 19 tested SmCSEPs suppressed cell death. The signal peptides of these eight SmCSEPs exhibited secretory activity in a yeast signal sequence trap assay. Confocal microscopy revealed that four of these eight SmCSEPs target both the cytoplasm and the nucleus, whereas four predominantly localize to discrete punctate structures. Pathogen challenge assays in N. benthamiana demonstrated that the transient expression of six SmCSEPs promoted Fusarium proliferatum infection. The expression of these six SmCSEP genes were induced during infection. SmCSEP2, SmCSEP13, and SmCSEP25 suppressed chitin-triggered reactive oxygen species burst and callose deposition in N. benthamiana. The candidate secreted effector proteins of S. musiva target multiple compartments in the plant cell and modulate different pattern-triggered immunity pathways. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Rapid New Diagnostic LAMP (Loop-Mediated Isothermal Amplification) Assays to Distinguish Among the Four Lineages of Phytophthora ramorum.

Sudden oak death (SOD) is caused by Phytophthora ramorum, an invasive oomycete pathogen. This pathogen is of major regulatory concern for nurseries, horticulture, and forestry in the United States and around the world. Three of the 12 identified lineages of P. ramorum currently occur in the United States (NA1, NA2, and EU1) affecting wildland forests and nurseries. Rapid identification and lineage determination is essential to accelerate management decisions, detect introductions of new lineages, and control the spread of SOD. The objective of this study was to develop and validate diagnostic tools to rapidly identify P. ramorum and distinguish among the four common lineages of the pathogen and to accelarate management decision making. The loop-mediated isothermal amplification (LAMP) assays developed here are species specific with no cross reaction to common Phytophthora species found in Oregon, California, and Washington. The lineage-specific assays unambiguously distinguish among the four common clonal lineages. These assays are sensitive and able to detect P. ramorum DNA ranging in concentration from 30 to 0.03 ng/µl depending on the assay. These assays work effectively on a variety of sample types including plant tissue, cultures, and DNA. They have been integrated into the SOD diagnostic process in the forest pathology lab at Oregon State University. To date, 190 samples have been correctly identified from over 200 field samples tested for lineage determination. The development of these assays will help managers in forestry and horticulture identify and rapidly respond to new outbreaks of P. ramorum.

Forest health in the Anthropocene: the emergence of a novel tree disease is associated with poplar cultivation.

Plant domestication and movement are large contributors to the success of new diseases. The introduction of new host species can result in accelerated evolutionary changes in pathogens, affecting long-established coevolutionary dynamics. This has been observed in poplars where severe epidemics of pathogens that were innocuous in their natural pathosystems occurred following host domestication. The North American fungus Sphaerulina musiva is responsible for endemic leaf spots on Populus deltoides. We show that the expansion of poplar cultivation resulted in the emergence of a new lineage of this pathogen that causes stem infections on a new host, P. balsamifera. This suggests a host shift since this is not a known host. Genome analysis of this emerging lineage reveals a mosaic pattern with islands of diversity separated by fixed genome regions, which is consistent with a homoploid hybridization event between two individuals that produced a hybrid swarm. Genome regions of extreme divergence and low diversity are enriched in genes involved in host-pathogen interactions. The specialization of this emerging lineage to a new host and its clonal propagation represents a serious threat to poplars and could affect both natural and planted forests. This work provides a clear example of the changes created by the intensification of tree cultivation that facilitate the emergence of specialized pathogens, jeopardizing the natural equilibrium between hosts and pathogens. This article is part of the theme issue 'Infectious disease ecology and evolution in a changing world'.

Draft Genome Resource for Forest Pathogen Coniferiporia weirii - A Facultative Pathogen of Thuja plicata and Callitropsis nootkatensis.

In North America, Coniferiporia weirii causes root and butt rot of western redcedar (Thuja plicata) and yellow-cedar (Callitropsis nootkatensis). There is currently no draft genome for C. weirii. As a result, C. weirii isolate 30910 originally isolated from a Thuja plicata in Idaho, U.S.A., was sequenced using an Illumina HiSeq 3000 sequencing system. The genome was assembled into 24,918 scaffolds with a scaffold N50 length of 53,821 bp. The total size of the genome was estimated to be 42.2 Mb. This included 96% and 95% recovery of basidiomycete complete and single-copy BUSCO genes, respectively. A total of 3.2% of the assessed BUSCO genes were missing and were not recovered. The assembly contained 10,351 predicted protein-coding genes. The estimated mean gene length of the predicted genes was 1,911 bp. While much is known about the biology of this fungus, little is known about its genome. This draft genome provides a baseline resource that will help further understand the population structure, reproductive mode, and evolutionary history of this important forest pathogen.

Repeated Emergence of Sudden Oak Death in Oregon: Chronology, Impact, and Management.

It has been two decades since the first detection of the sudden oak death pathogen Phytophthora ramorum in Oregon forests. Although the epidemic was managed since its first discovery in 2001, at least three invasions of three separate variants (clonal lineages), NA1, EU1, and NA2, are documented to have occurred to date. Control of this epidemic has cost over US$32 million from 2001 to 2020. This is dwarfed by the predicted cost of the closure to the Coos Bay export terminal, estimated at $58 million per year, if the epidemic was allowed to spread unchecked. Management efforts in Oregon have reduced inoculum and limited the spread of the pathogen. An outreach and citizen scientist program has been piloted to help in early detection efforts and search for disease-resistant tanoak. This feature article documents the repeated emergence, impact, costs, and lessons learned from managing this devastating invasive pathogen.

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.

Local Eradication of Phytophthora ramorum Is Effective on Both NA1 and EU1 Lineages in Oregon Tanoak Forests.

Sudden oak death (SOD), caused by the oomycete Phytophthora ramorum, has been actively managed in Oregon since its discovery there in 2001. SOD is a devastating disease affecting an ecologically and culturally important tree species in southwestern Oregon. Initially infested with the NA1 lineage, the more virulent EU1 lineage was discovered in 2015. Management has adapted over time in response to experimental findings and administrative limitations. Current management practices present an opportunity to compare the efficacy of treatment on these lineages by analyzing P. ramorum inoculum at untreated and treated sites. Current treatment includes herbicide treatment on host stems followed by felling, piling, and burning on site. Infested sites were visited between 2018 and 2020 (n = 88), where understory vegetation and soil was collected. Generalized linear modeling demonstrated that treatment had a significant impact on P. ramorum prevalence from vegetation samples, with an average of 33% (± 10%) fewer positive samples at treated sites. Linear mixed-effects modeling of a subpopulation of EU1 sites visited before and after treatment showed a similar effect of treatment, with a 43% (± 15%) reduction in P. ramorum prevalence. Prevalence of P. ramorum in soil was not affected by treatment in either analysis. A third analysis taking into consideration recent wildfire incursion into infested areas revealed that wildfire alone is insufficient to reduce prevalence of P. ramorum. These results strongly suggest that management is successfully reducing P. ramorum inoculum found on understory vegetation, and that treatment remains necessary to reduce the spread of this major forest pathogen.

Metabolomic Patterns of Septoria Canker Resistant and Susceptible Populus trichocarpa Genotypes 24 Hours Postinoculation.

Sphaerulina musiva is an economically and ecologically important fungal pathogen that causes Septoria stem canker and leaf spot disease of Populus species. To bridge the gap between genetic markers and structural barriers previously found to be linked to Septoria canker disease resistance in poplar, we used hydrophilic interaction liquid chromatography and tandem mass spectrometry to identify and quantify metabolites involved with signaling and cell wall remodeling. Fluctuations in signaling molecules, organic acids, amino acids, sterols, phenolics, and saccharides in resistant and susceptible P. trichocarpa inoculated with S. musiva were observed. The patterns of 222 metabolites in the resistant host implicate systemic acquired resistance (SAR), cell wall apposition, and lignin deposition as modes of resistance to this hemibiotrophic pathogen. This pattern is consistent with the expected response to the biotrophic phase of S. musiva colonization during the first 24 h postinoculation. The fungal pathogen metabolized key regulatory signals of SAR, other phenolics, and precursors of lignin biosynthesis that were depleted in the susceptible host. This is the first study to characterize metabolites associated with the response to initial colonization by S. musiva between resistant and susceptible hosts.

First report of Armillaria cepistipes causing root disease on Populus trichocarpa (black cottonwood) in Oregon, USA.

Populus trichocarpa Torr. and Gray (black cottonwood) is an economically and ecologically important tree species native to western North America. It serves as a model tree species in biology and genetics due to its relatively small genome size, rapid growth, and early reproductive maturity (Jansson and Douglas 2007). Black cottonwood is susceptible to root rot caused by at least one species of Armillaria (Raabe 1962), a globally distributed genus that exhibits diverse ecological behaviors (Klopfenstein et al. 2017) and infects numerous woody plant species (Raabe 1962). However, several Armillaria spp. have been isolated from Populus spp. in North America (Mallet 1990), and the most recent report of Armillaria on P. trichocarpa used the now ambiguated name A. mellea (Vahl.) Quel. (see Raabe 1962). In April 2016, mycelial fans and rhizomorphs of an unknown Armillaria species (isolate WV-ARR-3) were collected from P. trichocarpa in a riparian hardwood stand ca. 5.5 km east of Springfield, Oregon, USA (44°3'21.133"N, 122°49'39.935"W). The host was dominant in the canopy, large in diameter (ca. 90-cm dbh) relative to neighboring trees, and exhibited minimal crown dieback (ca. < 5%). A mycelial fan was observed destroying living cambium beneath the inner bark, indicating pathogenicity. The isolate was cultured on malt extract medium (3% malt extract, 3% dextrose, 1% peptone, and 1.5 % agar) and identified as A.cepistipes on the basis of somatic pairing tests and translation elongation factor 1α (tef1) sequences (GenBank Accession No. MK172784). DNA extraction, PCR, and tef1 sequencing followed protocols of Elías-Román et al. (2018). From nine replications of somatic incompatibility tests (18 tester isolates representing six North American Armillaria spp.), the isolate showed high intraspecific compatibility (colorless antagonism) with three A. cepistipes tester isolates (78%), but low compatibility with the other Armillaria spp. (0 - 33%) that occur in the region. Isolate WV-ARR-3 yielded tef1 sequences with a 99% identity to A. cepistipes (GenBank Accession Nos. JF313115 and JF313121). A second isolate (WV-ARR-1; GenBank Accession No. MK172783) with a nearly identical sequence was collected from a maturing P. trichocarpa in a riparian stand ca. 8 km northeast of Monroe, Oregon (44°21'47.57"N, 123°13'14.415"W) along the Willamette River, downstream from the McKenzie river tributary where WV-ARR-3 was collected. Armillaria cepistipes has been reported on Alnus rubra (red alder) in Washington, USA (Banik et al. 1996) and on broad-leaved trees in British Columbia, Canada (Allen et al. 1996). It is generally considered to be a weak pathogen on broad-leaved trees in the Pacific Northwest, but it is also associated with pathogenicity on both coniferous and deciduous trees in Europe (e.g., Lygis et al. 2005). However, a recent phylogenetic study suggested that North American A. cepistipes is phylogenetically distinct from Eurasian A. cepistipes (Klopfenstein et al. 2017), butadditional studies are needed to determine the formal taxonomic status of North American A. cepistipes. To our knowledge, A. cepistipes has not been previously confirmed on P. trichocarpa in the U.S.A. or formally reported as a pathogen of any Populus species in North America. Continued studies are needed to determine the distribution, host range, and ecological role of A. cepistipes in riparian forests of the Pacific Northwest, while monitoring its populations under changing climates.

Molecular Phylogenomics and Population Structure of Phytophthora pluvialis.

Phytophthora pluvialis is an oomycete that was first isolated from soil, water, and tree foliage in mixed Douglas-fir-tanoak forests of the U.S. Pacific Northwest (PNW). It was then identified as the causal agent of red needle cast of radiata pine (Pinus radiata) in New Zealand (NZ). Genotyping-by-sequencing was used to obtain 1,543 single nucleotide polymorphisms across 145 P. pluvialis isolates to characterize the population structure in the PNW and NZ. We tested the hypothesis that P. pluvialis was introduced to NZ from the PNW using genetic distance measurements and population structure analyses among locations between countries. The low genetic distance, population heterozygosity, and lack of geographic structure in NZ suggest a single colonization event from the United States followed by clonal expansion in NZ. The PNW Coast Range was proposed as a presumptive center of origin of the currently known distribution of P. pluvialis based on its geographic range and position as the central cluster in a minimum spanning network. The Coastal cluster of isolates were located at the root of every U.S. cluster and emerged earlier than all NZ clusters. The Coastal cluster had the highest degree of heterozygosity (Hs = 0.254) and median pairwise genetic distance (0.093) relative to any other cluster. Finally, the rapid host diversification between closely related isolates of P. pluvialis in NZ indicate that this pathogen has the potential to infect a broader range of hosts than is currently recognized.

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.

Safe DNA-extraction Protocol Suitable for Studying Tree-fungus Interactions.

We present a safe and low-cost method suitable for DNA extraction from mycelium and tree tissue samples. After sample preparation, the extraction takes about 60 min. Method performance was tested by extracting DNA from various tree tissue samples and from mycelium grown on solid and liquid media. DNA was extracted from juvenile and mature host material (Picea abies, Populus trichocarpa, Pseudotsuga menziesii) infected with different pathogens (Heterobasidion annosum, Heterobasidion parviporum, Leptographium wagenerii, Sphaerulina musiva). Additionally, DNA was extracted from pure cultures of the pathogens and several endophytic fungi. PCR success rate was 100% for young poplar material and fungal samples, and 48-72% for conifer and mature broadleaved plant samples. We recommend using 10-50 mg of fresh sample for the best results. The method offers a safe and low-cost DNA extraction alternative to study tree-fungus interactions, and is a potential resource for teaching purposes.

Variation in Susceptibility of Tanoak to the NA1 and EU1 Lineages of Phytophthora ramorum, the Cause of Sudden Oak Death.

Phytophthora ramorum, the cause of sudden oak death (SOD), kills tanoak (Notholithocarpus densiflorus) trees in southwestern Oregon and California. Two lineages of P. ramorum are now found in wildland forests of Oregon (NA1 and EU1). In addition to the management of SOD in forest ecosystems, disease resistance could be used as a way to mitigate the impact of P. ramorum. The objectives of this study were to (i) characterize the variability in resistance of N. densiflorus among families using lesion length; (ii) determine whether lineage, isolate, family, or their interactions significantly affect variation in lesion length; and (iii) determine whether there are differences among isolates and among families in terms of lesion length. The parameters isolate nested within lineage (isolate[lineage]) and family × isolate(lineage) interaction explained the majority of the variation in lesion length. There was no significant difference between the NA1 and EU1 lineages in terms of mean lesion length; however, there were differences among the six isolates. Lesions on seedlings collected from surviving trees at infested sites were smaller, on average, than lesions of seedlings collected from trees at noninfested sites (P = 0.0064). The results indicate that there is potential to establish a breeding program for tanoak resistance to SOD and that several isolates of P. ramorum should be used in an artificial inoculation assay.

Contrasting the Pathogen Loads in Co-Existing Populations of Phytophthora pluvialis and Nothophaeocryptopus gaeumannii in Douglas Fir Plantations in New Zealand and the Pacific Northwest United States.

The emergence of Phytophthora pluvialis as a foliar pathogen of Douglas fir in New Zealand and the Pacific Northwest United States has raised questions about its interaction with the widespread Swiss needle cast (SNC) disease. During Spring 2017, we repeatedly sampled 30 trees along an environmental gradient in each region and 292 additional trees in a longitudinal transect to assess the P. pluvialis epidemic and the association between P. pluvialis and Nothophaeocryptopus gaeumannii, which are causal agents of SNC. Both pathogens were consistently more abundant in the host's exotic environment in New Zealand. In both areas, the two pathogens co-exist in different spatial scales for regions and needles. The relative abundance of both pathogens was negatively correlated in the Pacific Northwest, where both presumably have co-existed for longer. Our findings confirmed the interaction of P. pluvialis and N. gaeumannii as foliar pathogens of Douglas fir and suggest a within-site spatial variation in the Pacific Northwest.

Increased streamflow in catchments affected by a forest disease epidemic.

Natural disturbances help maintain healthy forested and aquatic ecosystems. However, biotic and abiotic disturbance regimes are changing rapidly. For example, the Swiss needle cast (SNC) epidemic in the Coast Range of Oregon in the U.S. Pacific Northwest has increased in area from 53,050 to 238,705ha over the 1996-2015 period. We investigated whether the hydrologic regime (i.e., annual streamflow, runoff ratio, and magnitude and timing of peak flows and low flows) was affected by SNC in 12 catchments in western Oregon. The catchments ranged in size from 183 to 1834km2 and area affected by SNC from 0 to 90.5%. To maximize the number of catchments included in the study, we analyzed 20years of SNC aerial survey data and 15-26years of stream discharge (Q) and PRISM precipitation (P) and air temperature (Tair) data to test for trends in hydrologic variables for each catchment. As expected, we found that runoff ratios (Q/P) increased in five catchments, all with an area impacted by SNC >10%. This was likely due to the effects of SNC on the hydraulic architecture (i.e., needle retention, sapwood area, sapwood permeability) of affected trees, leading to decreased canopy interception and transpiration losses. Interestingly, two catchments with the greatest area affected by SNC showed no changes in hydrologic regime. The lack of hydrologic response could either be due to compensatory transpiration by vegetation unaffected by the disease or sub-canopy abiotic evaporation, which counteracted reductions in transpiration. This study is the first to illustrate that chronic canopy disturbance from a foliage pathogen can influence catchment scale hydrology.

Ecology and Evolution of the Sudden Oak Death Pathogen Phytophthora ramorum.

The sudden oak and sudden larch death pathogen Phytophthora ramorum emerged simultaneously in the United States on oak and in Europe on Rhododendron in the 1990s. This pathogen has had a devastating impact on larch plantations in the United Kingdom as well as mixed conifer and oak forests in the Western United States. Since the discovery of this pathogen, a large body of research has provided novel insights into the emergence, epidemiology, and genetics of this pandemic. Genetic and genomic resources developed for P. ramorum have been instrumental in improving our understanding of the epidemiology, evolution, and ecology of this disease. The recent reemergence of EU1 in the United States and EU2 in Europe and the discovery of P. ramorum in Asia provide renewed impetus for research on the sudden oak death pathogen.

Association mapping, transcriptomics, and transient expression identify candidate genes mediating plant-pathogen interactions in a tree.

Invasive microbes causing diseases such as sudden oak death negatively affect ecosystems and economies around the world. The deployment of resistant genotypes for combating introduced diseases typically relies on breeding programs that can take decades to complete. To demonstrate how this process can be accelerated, we employed a genome-wide association mapping of ca 1,000 resequenced Populus trichocarpa trees individually challenged with Sphaerulina musiva, an invasive fungal pathogen. Among significant associations, three loci associated with resistance were identified and predicted to encode one putative membrane-bound L-type receptor-like kinase and two receptor-like proteins. A susceptibility-associated locus was predicted to encode a putative G-type D-mannose-binding receptor-like kinase. Multiple lines of evidence, including allele analysis, transcriptomics, binding assays, and overexpression, support the hypothesized function of these candidate genes in the P. trichocarpa response to S. musiva.

The Correlation Between Septoria Leaf Spot and Stem Canker Resistance in Hybrid Poplar.

Sphaerulina musiva is an important fungal pathogen that causes a leaf spot and stem canker disease of hybrid poplar. Stem cankers are widely regarded as the greatest threat to hybrid poplar plantations because of their ability to cause tree mortality; thus, the efforts of breeding programs have been focused on stem canker resistance. To explore the relationship between resistance to leaf spot and stem canker in Populus nigra × P. deltoides hybrids, two experiments were conducted. Initially, comparisons among leaves of different ages indicated that younger leaves were more susceptible to leaf spot infection than older leaves. Correlations between leaf spot severity and stem canker severity for both individual leaves and all leaves averaged together indicated that, in 10 of 11 comparisons, there were no significant correlations. The lack of correlation suggests that deploying genotypes resistant to stem canker may not affect the pathogen population causing leaf spot disease. To our knowledge, this is the first study specifically designed to test the correlation between stem canker resistance and leaf spot resistance by inoculating whole trees with a spore suspension in a controlled environment.

Variation in Resistance of Populus nigra to Sphaerulina musiva in the North-Central United States.

Populus nigra, commonly used in hybrid poplar breeding programs in the north-central United States, is susceptible to Septoria stem canker, caused by Sphaerulina musiva. In this study, two experiments were conducted to (i) characterize the variation in resistance of 47 genotypes of P. nigra collected from seven locations in Europe in terms of cankers per centimeter and disease severity score; (ii) determine whether location, isolate, genotype, or their interactions significantly affect cankers per centimeter and disease severity score; and (iii) examine the correlation of disease severity score between single-isolate and bulk-isolate inoculations. The majority of the variation in resistance for cankers per centimeter was explained by location (72%; P < 0.001) followed by genotype(location) (28%; P < 0.001). In contrast, the majority of the variation in disease severity score was explained by genotype-location (51%; P < 0.001) followed by location (26%; P = 0.025). The disease severity score model also indicated the presence of a significant isolate effect (P = 0.034) and genotype(location) × isolate interaction (P = 0.004). The correlation coefficients for disease severity score indicated a significant range of correlations (r = 0.871 to r = 0.952) when correlating single-isolate to bulk-isolate inoculations.

Genotype-by-sequencing of the plant-pathogenic fungi Pyrenophora teres and Sphaerulina musiva utilizing Ion Torrent sequence technology.

Genetic and genomics tools to characterize host-pathogen interactions are disproportionately directed to the host because of the focus on resistance. However, understanding the genetics of pathogen virulence is equally important and has been limited by the high cost of de novo genotyping of species with limited marker data. Non-resource-prohibitive methods that overcome the limitation of genotyping are now available through genotype-by-sequencing (GBS). The use of a two-enzyme restriction-associated DNA (RAD)-GBS method adapted for Ion Torrent sequencing technology provided robust and reproducible high-density genotyping of several fungal species. A total of 5783 and 2373 unique loci, 'sequence tags', containing 16,441 and 9992 single nucleotide polymorphisms (SNPs) were identified and characterized from natural populations of Pyrenophora teres f. maculata and Sphaerulina musiva, respectively. The data generated from the P. teres f. maculata natural population were used in association mapping analysis to map the mating-type gene to high resolution. To further validate the methodology, a biparental population of P. teres f. teres, previously used to develop a genetic map utilizing simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers, was re-analysed using the SNP markers generated from this protocol. A robust genetic map containing 1393 SNPs on 997 sequence tags spread across 15 linkage groups with anchored reference markers was generated from the P. teres f. teres biparental population. The robust high-density markers generated using this protocol will allow positional cloning in biparental fungal populations, association mapping of natural fungal populations and population genetics studies.