Bacterial Leaf Streak Diseases of Plants: Symptom Convergence in Monocot Plants by Distant Pathogenic Xanthomonas Species.

Bacterial leaf streak (BLS) is a disease of monocot plants caused by Xanthomonas translucens on small grains, X. vasicola on maize and sorghum, and X. oryzae on rice. These three pathogens cause remarkably similar symptomology in their host plants. Despite causing similar symptoms, BLS pathogens are dispersed throughout the larger Xanthomonas phylogeny. Each aforementioned species includes strain groups that do not cause BLS and instead cause vascular disease. In this commentary, we hypothesize that strains of X. translucens, X. vasicola, and X. oryzae convergently evolved to cause BLS due to shared evolutionary pressures. We examined the diversity of secreted effectors, which may be important virulence factors for BLS pathogens and their evolution. We discuss evidence that differences in gene regulation and abilities to manipulate plant hormones may also separate BLS pathogens from other Xanthomonas species or pathovars. BLS is becoming an increasing issue across the three pathosystems. Overall, we hope that a better understanding of conserved mechanisms used by BLS pathogens will enable researchers to translate findings across production systems and guide approaches to control this (re)emerging threat.

DNA Sequence-Based Identification of Fusarium: A Work in Progress.

Accurate species-level identification of an etiological agent is crucial for disease diagnosis and management because knowing the agent's identity connects it with what is known about its host range, geographic distribution, and toxin production potential. This is particularly true in publishing peer-reviewed disease reports, where imprecise and/or incorrect identifications weaken the public knowledge base. This can be a daunting task for phytopathologists and other applied biologists that need to identify Fusarium in particular, because published and ongoing multilocus molecular systematic studies have highlighted several confounding issues. Paramount among these are: (i) this agriculturally and clinically important genus is currently estimated to comprise more than 400 phylogenetically distinct species (i.e., phylospecies), with more than 80% of these discovered within the past 25 years; (ii) approximately one-third of the phylospecies have not been formally described; (iii) morphology alone is inadequate to distinguish most of these species from one another; and (iv) the current rapid discovery of novel fusaria from pathogen surveys and accompanying impact on the taxonomic landscape is expected to continue well into the foreseeable future. To address the critical need for accurate pathogen identification, our research groups are focused on populating two web-accessible databases (FUSARIUM-ID v.3.0 and the nonredundant National Center for Biotechnology Information nucleotide collection that includes GenBank) with portions of three phylogenetically informative genes (i.e., TEF1, RPB1, and RPB2) that resolve at or near the species level in every Fusarium species. The objectives of this Special Report, and its companion in this issue (Torres-Cruz et al. 2022), are to provide a progress report on our efforts to populate these databases and to outline a set of best practices for DNA sequence-based identification of fusaria.

FUSARIUM-ID v.3.0: An Updated, Downloadable Resource for Fusarium Species Identification.

Species within Fusarium are of global agricultural, medical, and food/feed safety concern and have been extensively characterized. However, accurate identification of species is challenging and usually requires DNA sequence data. FUSARIUM-ID (http://isolate.fusariumdb.org/blast.php) is a publicly available database designed to support the identification of Fusarium species using sequences of multiple phylogenetically informative loci, especially the highly informative ∼680-bp 5' portion of the translation elongation factor 1-alpha (TEF1) gene that has been adopted as the primary barcoding locus in the genus. However, FUSARIUM-ID v.1.0 and 2.0 had several limitations, including inconsistent metadata annotation for the archived sequences and poor representation of some species complexes and marker loci. Here, we present FUSARIUM-ID v.3.0, which provides the following improvements: (i) additional and updated annotation of metadata for isolates associated with each sequence, (ii) expanded taxon representation in the TEF1 sequence database, (iii) availability of the sequence database as a downloadable file to enable local BLAST queries, and (iv) a tutorial file for users to perform local BLAST searches using either freely available software, such as SequenceServer, BLAST+ executable in the command line, and Galaxy, or the proprietary Geneious software. FUSARIUM-ID will be updated on a regular basis by archiving sequences of TEF1 and other loci from newly identified species and greater in-depth sampling of currently recognized species.

Host-generalist fungal pathogens of seedlings may maintain forest diversity via host-specific impacts and differential susceptibility among tree species.

Host-specialized pathogens are credited with the maintenance of tropical forest diversity under the Janzen-Connell hypothesis. Yet, in diverse forests, selection may favor pathogens with broad host ranges, given their passive dispersal and the relative rarity of tree species. We surveyed the host associations of potential pathogens isolated from symptomatic seedlings in forests in Panama and used inoculations to assess the pathogenicity and host ranges of 27 fungal isolates, and differences among tree species in susceptibility. Thirty-one of the 33 nonsingleton operational taxonomic units (OTUs) isolated from seedlings are multi-host. All 31 multi-host OTUs exhibit low to moderate specialization, and we observed phylogenetically overdispersed host use for 19 OTUs. The pathogenicity of 10 isolates was experimentally confirmed; nine caused disease in seedlings in multiple families. However, the outcome of infection differs among tree species susceptible to a given multi-host pathogen. Furthermore, some tree species were seemingly resistant to all fungi tested, while others were susceptible to multiple fungi. Tree species adapted to environments with lower disease pressure were most likely to exhibit disease. Our results suggest that generalist pathogens contribute to the maintenance of local and regional forest diversity via host-specific impacts and the exclusion of disease-sensitive trees from disease-prone habitats.

Current Understanding of the History, Global Spread, Ecology, Evolution, and Management of the Corn Bacterial Leaf Streak Pathogen, Xanthomonas vasicola pv. vasculorum.

Bacterial leaf streak of corn, caused by Xanthomonas vasicola pv. vasculorum, has been present in South Africa for over 70 years, but is an emerging disease of corn in North and South America. The only scientific information pertaining to this disease on corn came from work done in South Africa, which primarily investigated host range on other African crops, such as sugarcane and banana. As a result, when the disease was first reported in the United States in 2016, there was very limited information on where this pathogen came from, how it infects its host, what plant tissue(s) it is capable of infecting, where initial inoculum comes from at the beginning of each crop season, how the bacterium spreads from plant to plant and long distance, what meteorological variables and agronomic practices favor disease development and spread, how many other plant species X. vasicola pv. vasculorum is capable of infecting or using as alternate hosts, and if the bacterium will be able to persist in all corn growing regions of the United States. There were also no rapid diagnostic assays available which initially hindered prompt identification prior to the development of molecular diagnostic tools. The goal of this synthesis is to review the history of X. vasicola pv. vasculorum and bacterial leaf streak in South Africa and its movement to North and South America, and highlight the recent research that has been done in response to the emergence of this bacterial disease.

Occurrence in Seeds and Potential Seed Transmission of Xanthomonas vasicola pv. vasculorum in Maize in the United States.

This paper reports original evidence regarding the potential role of seed transmission of Xanthomonas vasicola pv. vasculorum in the epidemiology of bacterial leaf streak (BLS) in maize. We evaluated the occurrence of the pathogen on seeds from diseased fields and its subsequent transmission to seedlings. In 2016 and 2017, X. vasicola pv. vasculorum was detected by TaqMan PCR from 22 of 41 maize seed lots harvested from naturally infected fields in Colorado, Nebraska, and Iowa. However, many of the PCR-positive samples did not yield culturable X. vasicola pv. vasculorum colonies. The highest levels of seed contamination were detected in dent maize and popcorn from NE and CO. Seed transmission was evaluated in greenhouse grow-outs from eight seed lots, totaling more than 14,000 plants. Putative seed transmission events from naturally contaminated seed lots, estimated from PCR results, occurred at a frequency between 0.1 and 0.5% in 10-seedling pooled samples and at a frequency of 2.7% from individual plant assays. However, no seedling symptoms were observed during these assays and live X. vasicola pv. vasculorum colonies were not recovered from PCR-positive seedlings. In contrast, seed transmission was readily demonstrated from artificially contaminated seed lots, including typical symptoms and recovery of live bacteria. Seed transmission consistently occurred from seeds soaked in bacterial suspensions with concentrations of ≥106 CFU/ml, suggesting that a threshold population of the bacterium is necessary for the development of BLS symptoms and recovery of live bacteria. The low bacterial populations on naturally contaminated seeds apparently were not sufficient to result in diseased seedlings.

Genomics-Informed Molecular Detection of Xanthomonas vasicola pv. vasculorum Strains Causing Severe Bacterial Leaf Streak of Corn.

Xanthomonas vasicola pv. vasculorum (syn. X. campestris pv. vasculorum) was initially identified as the causal agent of bacterial leaf streak of corn in South Africa. The pathovar vasculorum causes disease on sugarcane and corn, but a subset of these strains was noted for its increased disease severity in corn. This subset was reclassified as X. campestris pv. zeae in the early 1990s and was found to have slightly different biochemical and genetic properties than isolates from sugarcane. There has been an emergence of X. campestris pv. zeae-like strains of X. vasicola pv. vasculorum in both the United States and Argentina since 2010. We performed whole genome sequencing on U.S. isolates to confirm their identity. Informed by comparative genomics, we then developed specific TaqMan qPCR and loop-mediated isothermal amplification (LAMP) assays for the detection of this specific subset of X. vasicola pv. vasculorum strains. The qPCR 4909 assay was tested against 27 xanthomonads (diverse representation), 32 DNA extractions from corn leaves confirmed as positive or negative for the bacterium, 41 X. vasicola pv. vasculorum isolates from corn in the United States and Argentina, and 31 additional bacteria associated with corn, sugarcane, or sorghum. In all cases the assay was shown to be specific for the X. vasicola pv. vasculorum isolates that cause more severe disease on corn. We then tested the LAMP 166 assay against the 27 xanthomonads and 32 corn leaf DNA samples, and we found this assay was also specific for this subset of X. vasicola pv. vasculorum isolates. We also developed a live/dead cells distinction protocol using propidium monoazide prior to DNA extraction for analyzing seed washes using these assays. These two detection assays can be useful for both diagnosticians and researchers to specifically identify the X. vasicola pv. vasculorum isolates that cause more severe symptoms on corn.

Genomic Acquisitions in Emerging Populations of Xanthomonas vasicola pv. vasculorum Infecting Corn in the United States and Argentina.

Xanthomonas vasicola pv. vasculorum is an emerging bacterial plant pathogen that causes bacterial leaf streak on corn. First described in South Africa in 1949, reports of this pathogen have greatly increased in the past years in South America and in the United States. The rapid spread of this disease in North and South America may be due to more favorable environmental conditions, susceptible hosts and/or genomic changes that favored the spread. To understand whether genetic mechanisms exist behind the recent spread of X. vasicola pv. vasculorum, we used comparative genomics to identify gene acquisitions in X. vasicola pv. vasculorum genomes from the United States and Argentina. We sequenced 41 genomes of X. vasicola pv. vasculorum and the related sorghum-infecting X. vasicola pv. holcicola and performed comparative analyses against all available X. vasicola genomes. Time-measured phylogenetic analyses showed that X. vasicola pv. vasculorum strains from the United States and Argentina are closely related and arose from two introductions to North and South America. Gene content comparisons identified clusters of genes enriched in corn X. vasicola pv. vasculorum that showed evidence of horizontal transfer including one cluster corresponding to a prophage found in all X. vasicola pv. vasculorum strains from the United States and Argentina as well as in X. vasicola pv. holcicola strains. In this work, we explore the genomes of an emerging phytopathogen population as a first step toward identifying genetic changes associated with the emergence. The acquisitions identified may contain virulence determinants or other factors associated with the spread of X. vasicola pv. vasculorum in North and South America and will be the subject of future work.

Transfer of Xanthomonas campestris pv. arecae and X. campestris pv. musacearum to X. vasicola (Vauterin) as X. vasicola pv. arecae comb. nov. and X. vasicola pv. musacearum comb. nov. and Description of X. vasicola pv. vasculorum pv. nov.

We present an amended description of the bacterial species Xanthomonas vasicola to include the causative agent of banana Xanthomonas wilt, as well as strains that cause disease on Areca palm, Tripsacum grass, sugarcane, and maize. Genome-sequence data reveal that these strains all share more than 98% average nucleotide with each other and with the type strain. Our analyses and proposals should help to resolve the taxonomic confusion that surrounds some of these pathogens and help to prevent future use of invalid names.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Emergence of white pine needle damage in the northeastern United States is associated with changes in pathogen pressure in response to climate change.

The defoliation of the eastern white pine (Pinus strobus) across the northeastern United States is an escalating concern threatening the ecological health of northern forests and economic vitality of the region's lumber industry. First documented in the spring of 2010 affecting 24 328 hectares in the state of Maine, white pine needle damage (WPND) has continued to spread and is now well established in all New England states. While causal agents of WPND are known, current research is lacking in both sampling distribution and the specific environmental factor(s) that affect the development and spread of this disease complex. This study aims to construct a more detailed distribution map of the four primary causal agents within the region, as well as utilize long-term WPND monitoring plots and data collected from land-based weather stations to develop a climatic model to predict the severity of defoliation events in the proceeding year. Sampling results showed a greater distribution of WPND than previously reported. WPND was generally found in forest stands that compromised >50% eastern white pine by basal area. No single species, nor a specific combination of species had a dominating presence in particular states or regions, thus supporting the disease complex theory that WPND is neither caused by an individual species nor by a specific combination of species. In addition, regional weather data confirmed the trend of increasing temperature and precipitation observed in this region with the previous year's May, June, and July rainfall being the best predictor of defoliation events in the following year. Climatic models were developed to aid land managers in predicting disease severity and accordingly adjust their management decisions. Our results clearly demonstrate the role changing climate patterns have on the health of eastern white pine in the northeastern United States.

Priority of Lophophacidium over Canavirgella: taxonomic status of Lophophacidium dooksii and Canavirgella banfieldii, causal agents of a white pine needle disease.

In 2009 unusual white pine needle discoloration was observed in eastern Canada and northeastern USA. While the symptoms were similar in most pine stands, the disease was diagnosed as Canavirgella banfieldii in several locations and Dooks needle blight caused by Lophophacidium dooksii in others. Because of the similarities in symptom development and morphological characters of the causal agents, it was suspected that C. banfieldii and L. dooksii are either the same or closely related species. To test this hypothesis we examined several collections representing C. banfieldii and L. dooksii, including the two type specimens. Phylogenetic analyses of nuc internal transcribed spacer rDNA sequences confirm the synonymy of C. banfieldii with the earlier described L. dooksii and provide the first evidence of the close evolutionary relationship of L. dooksii to other pine pathogens.

Fungal community dissimilarity predicts plant-soil feedback strength in a lowland tropical forest.

Soil microbes impact plant community structure and diversity through plant-soil feedbacks. However, linking the relative abundance of plant pathogens and mutualists to differential plant recruitment remains challenging. Here, we tested for microbial mediation of pairwise feedback using a reciprocal transplant experiment in a lowland tropical forest in Panama paired with amplicon sequencing of soil and roots. We found evidence that plant species identity alters the microbial community, and these changes in microbial composition alter subsequent growth and survival of conspecific plants. We also found that greater community dissimilarity between species in their arbuscular mycorrhizal and nonpathogenic fungi predicted increased positive feedback. Finally, we identified specific microbial taxa across our target functional groups that differentially accumulated under conspecific settings. Collectively, these findings clarify how soil pathogens and mutualists mediate net feedback effects on plant recruitment, with implications for management and restoration.

Synchrospora gen. nov., a New Peronosporaceae Genus with Aerial Lifestyle from a Natural Cloud Forest in Panama.

During a survey of Phytophthora diversity in Panama, fast-growing oomycete isolates were obtained from naturally fallen leaves of an unidentified tree species in a tropical cloud forest. Phylogenetic analyses of sequences from the nuclear ITS, LSU and ßtub loci and the mitochondrial cox1 and cox2 genes revealed that they belong to a new species of a new genus, officially described here as Synchrospora gen. nov., which resided as a basal genus within the Peronosporaceae. The type species S. medusiformis has unique morphological characteristics. The sporangiophores show determinate growth, multifurcating at the end, forming a stunted, candelabra-like apex from which multiple (8 to >100) long, curved pedicels are growing simultaneously in a medusa-like way. The caducous papillate sporangia mature and are shed synchronously. The breeding system is homothallic, hence more inbreeding than outcrossing, with smooth-walled oogonia, plerotic oospores and paragynous antheridia. Optimum and maximum temperatures for growth are 22.5 and 25-27.5 °C, consistent with its natural cloud forest habitat. It is concluded that S. medusiformis as adapted to a lifestyle as a canopy-dwelling leaf pathogen in tropical cloud forests. More oomycete explorations in the canopies of tropical rainforests and cloud forests are needed to elucidate the diversity, host associations and ecological roles of oomycetes and, in particular, S. medusiformis and possibly other Synchrospora taxa in this as yet under-explored habitat.

Two new species of Pythium, P. schmitthenneri and P. selbyi pathogens of corn and soybean in Ohio.

Two new species of Pythium, pathogens of corn and soybean in Ohio, are described. Pythium schmitthenneri sp. nov. and Pythium selbyi sp. nov. both have morphological and sequence characteristics that place them in clade E1 of the genus Pythium. Morphology and sequence analysis of the ITS1-5.8S-ITS2 regions of these species were different from previously described species. The ITS region of Pythium schmitthenneri was 99.9% similar to P. acrogynum and 99.8% similar to P. hypogynum. All three species are characterized by globose to limoniform sporangia and plerotic oospores. Pythium schmitthenneri has mostly diclinous antheridia, compared to the strictly hypogynous antheridia of P. acrogynum and P. hypogynum. The temperature for growth of P. schmitthenneri is below 4 C to 32 C, and optimum growth is 18-25 C compared to 31-34 C for P. hypogynum. The ITS region of P. selbyi was 97.1% similar to P. longandrum and 97.5% similar to P. longisporangium. All three species are characterized by globose sporangia, mostly plerotic oospores, with one to two oospores per oogonium, and hypogynous or monoclinous antheridia. The temperature for growth of P. selbyi is below 4 to 32 C, with an optimum 18-25 C. These new species were widely dispersed throughout the soybean- and corn-producing regions in Ohio, making their characterization critical for managing the Pythium complex that causes seedling and root-rot disease in Ohio soybean and corn fields.

A large accessory genome and high recombination rates may influence global distribution and broad host range of the fungal plant pathogen Claviceps purpurea.

Pangenome analyses are increasingly being utilized to study the evolution of eukaryotic organisms. While pangenomes can provide insight into polymorphic gene content, inferences about the ecological and adaptive potential of such organisms also need to be accompanied by additional supportive genomic analyses. In this study we constructed a pangenome of Claviceps purpurea from 24 genomes and examined the positive selection and recombination landscape of an economically important fungal organism for pharmacology and agricultural research. Together, these analyses revealed that C. purpurea has a relatively large accessory genome (~ 38%), high recombination rates (ρ = 0.044), and transposon mediated gene duplication. However, due to observations of relatively low transposable element (TE) content (8.8%) and a lack of variability in genome sizes, prolific TE expansion may be controlled by frequent recombination. We additionally identified that within the ergoline biosynthetic cluster the lpsA1 and lpsA2 were the result of a recombination event. However, the high recombination rates observed in C. purpurea may be influencing an overall trend of purifying selection across the genome. These results showcase the use of selection and recombination landscapes to identify mechanisms contributing to pangenome structure and primary factors influencing the evolution of an organism.

Building More Resilient Culture Collections: A Call for Increased Deposits of Plant-Associated Bacteria.

Biological collections preserve our past, while helping protect our future and increase future knowledge. Plant bacterial culture collections are our security for domestic and global biosecurity. This feature article will provide an introduction to the global position of plant bacterial collections. The role of collections in monitoring plant pathogenic bacteria will be explored through the presentation of five cases studies. These case studies demonstrate why culture collections were imperative for the outcome in each situation. We discuss what we believe should be the best practices to improve microbial preservation and accessioning rates, and why plant bacterial culture collections must increase deposits to be prepared for future emerging pathogens. This is not only the case for global culture collections, but on a much bigger scale, our future scientific successes, our biosecurity decisions and responses, and our knowledge are contingent upon preserving our valuable bacterial strains. It is hoped that once you read this article, you will see the need to deposit your strains in registered public collections and make a concerted effort to build better bacterial culture collections with us.

Microbial diversity declines in warmed tropical soil and respiration rise exceed predictions as communities adapt.

Perturbation of soil microbial communities by rising temperatures could have important consequences for biodiversity and future climate, particularly in tropical forests where high biological diversity coincides with a vast store of soil carbon. We carried out a 2-year in situ soil warming experiment in a tropical forest in Panama and found large changes in the soil microbial community and its growth sensitivity, which did not fully explain observed large increases in CO2 emission. Microbial diversity, especially of bacteria, declined markedly with 3 to 8 °C warming, demonstrating a breakdown in the positive temperature-diversity relationship observed elsewhere. The microbial community composition shifted with warming, with many taxa no longer detected and others enriched, including thermophilic taxa. This community shift resulted in community adaptation of growth to warmer temperatures, which we used to predict changes in soil CO2 emissions. However, the in situ CO2 emissions exceeded our model predictions threefold, potentially driven by abiotic acceleration of enzymatic activity. Our results suggest that warming of tropical forests will have rapid, detrimental consequences both for soil microbial biodiversity and future climate.

Phyllachora species infecting maize and other grass species in the Americas represents a complex of closely related species.

The genus Phyllachora contains numerous obligate fungal parasites that produce raised, melanized structures called stromata on their plant hosts referred to as tar spot. Members of this genus are known to infect many grass species but generally do not cause significant damage or defoliation, with the exception of P. maydis which has emerged as an important pathogen of maize throughout the Americas, but the origin of this pathogen remains unknown. To date, species designations for Phyllachora have been based on host associations and morphology, and most species are assumed to be host specific. We assessed the sequence diversity of 186 single stroma isolates collected from 16 hosts representing 15 countries. Samples included both herbarium and contemporary strains that covered a temporal range from 1905 to 2019. These 186 isolates were grouped into five distinct species with strong bootstrap support. We found three closely related, but genetically distinct groups of Phyllachora are capable of infecting maize in the United States, we refer to these as the P. maydis species complex. Based on herbarium specimens, we hypothesize that these three groups in the P. maydis species complex originated from Central America, Mexico, and the Caribbean. Although two of these groups were only found on maize, the third and largest group contained contemporary strains found on maize and other grass hosts, as well as herbarium specimens from maize and other grasses that include 10 species of Phyllachora. The herbarium specimens were previously identified based on morphology and host association. This work represents the first attempt at molecular characterization of Phyllachora species infecting grass hosts and indicates some Phyllachora species can infect a broad range of host species and there may be significant synonymy in the Phyllachora genus.

Whole-Genome Comparisons of Ergot Fungi Reveals the Divergence and Evolution of Species within the Genus Claviceps Are the Result of Varying Mechanisms Driving Genome Evolution and Host Range Expansion.

The genus Claviceps has been known for centuries as an economically important fungal genus for pharmacology and agricultural research. Only recently have researchers begun to unravel the evolutionary history of the genus, with origins in South America and classification of four distinct sections through ecological, morphological, and metabolic features (Claviceps sects. Citrinae, Paspalorum, Pusillae, and Claviceps). The first three sections are additionally characterized by narrow host range, whereas section Claviceps is considered evolutionarily more successful and adaptable as it has the largest host range and biogeographical distribution. However, the reasons for this success and adaptability remain unclear. Our study elucidates factors influencing adaptability by sequencing and annotating 50 Claviceps genomes, representing 21 species, for a comprehensive comparison of genome architecture and plasticity in relation to host range potential. Our results show the trajectory from specialized genomes (sects. Citrinae and Paspalorum) toward adaptive genomes (sects. Pusillae and Claviceps) through colocalization of transposable elements around predicted effectors and a putative loss of repeat-induced point mutation resulting in unconstrained tandem gene duplication coinciding with increased host range potential and speciation. Alterations of genomic architecture and plasticity can substantially influence and shape the evolutionary trajectory of fungal pathogens and their adaptability. Furthermore, our study provides a large increase in available genomic resources to propel future studies of Claviceps in pharmacology and agricultural research, as well as, research into deeper understanding of the evolution of adaptable plant pathogens.

Genome-wide SNP identification in Fraxinus linking genetic characteristics to tolerance of Agrilus planipennis.

Ash (Fraxinus spp.) is one of the most widely distributed tree genera in North America. Populations of ash in the United States and Canada have been decimated by the introduced pest Agrilus planipennis (Coleoptera: Buprestidae; emerald ash borer), having negative impacts on both forest ecosystems and economic interests. The majority of trees succumb to attack by A. planipennis, but some trees have been found to be tolerant to infestation despite years of exposure. Restriction site-associated DNA (RAD) sequencing was used to sequence ash individuals, both tolerant and susceptible to A. planipennis attack, in order to identify single nucleotide polymorphism (SNP) patterns related to tolerance and health declines. de novo SNPs were called using SAMtools and, after filtering criteria were implemented, a set of 17,807 SNPs were generated. Principal component analysis (PCA) of SNPs aligned individual trees into clusters related to geography; however, five tolerant trees clustered together despite geographic location. A subset of 32 outlier SNPs identified within this group, as well as a subset of 17 SNPs identified based on vigor rating, are potential candidates for the selection of host tolerance. Understanding the mechanisms of host tolerance through genome-wide association has the potential to restore populations with cultivars that are able to withstand A. planipennis infestation. This study was successful in using RAD-sequencing in order to identify SNPs that could contribute to tolerance of A. planipennis. This was a first step toward uncovering the genetic basis for host tolerance to A. planipennis. Future studies are needed to identify the functionality of the loci where these SNPs occur and how they may be related to tolerance of A. planipennis attack.