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'.

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.

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.

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.