Terpenoids are involved in the expression of systemic-induced resistance in Austrian pine.

Terpenoids are defense metabolites that are induced upon infection or wounding. However, their role in systemic-induced resistance (SIR) is not known. Here, we explored the role of terpenoids in this phenomenon at a very early stage in the interaction between Austrian pine and the tip blight and canker pathogen Diplodia pinea. We induced Austrian pine saplings by either wounding or inoculating the lower stems with D. pinea. The seedlings were then challenged after 12 h, 72 h, or 10 days with D. pinea on the stem 15 cm above the induction. Lesion lengths and terpenoids were quantified at both induction and challenge locations. Key terpenoids were assayed for antifungal activity in in vitro bioassays. SIR increased with time and was correlated with the inducibility of several compounds. α-Pinene and a cluster of ß-pinene, limonene, benzaldehyde, dodecanol, and n-dodecyl acrylate were positively correlated with SIR and were fungistatic in vitro, while other compounds were negatively correlated with SIR and appeared to serve as a carbon source for D. pinea. This study shows that, overall, terpenoids are involved in SIR in this system, but their role is nuanced, depending on the type of induction and time of incubation. We hypothesize that some, such as α-pinene, could serve in SIR signaling.

A glimmer of hope - ash genotypes with increased resistance to ash dieback pathogen show cross-resistance to emerald ash borer.

Plants rely on cross-resistance traits to defend against multiple, phylogenetically distinct enemies. These traits are often the result of long co-evolutionary histories. Biological invasions can force naïve plants to cope with novel, coincident pests, and pathogens. For example, European ash (Fraxinus excelsior) is substantially threatened by the emerald ash borer (EAB), Agrilus planipennis, a wood-boring beetle, and the ash dieback (ADB) pathogen, Hymenoscyphus fraxineus. Yet, plant cross-resistance traits against novel enemies are poorly explored and it is unknown whether naïve ash trees can defend against novel enemy complexes via cross-resistance mechanisms. To gain mechanistic insights, we quantified EAB performance on grafted replicates of ash genotypes varying in ADB resistance and characterized ash phloem chemistry with targeted and untargeted metabolomics. Emerald ash borer performed better on ADB-susceptible than on ADB-resistant genotypes. Moreover, changes in EAB performance aligned with differences in phloem chemical profiles between ADB-susceptible and ADB-resistant genotypes. We show that intraspecific variation in phloem chemistry in European ash can confer increased cross-resistance to invasive antagonists from different taxonomic kingdoms. Our study suggests that promotion of ADB-resistant ash genotypes may simultaneously help to control the ADB disease and reduce EAB-caused ash losses, which may be critical for the long-term stability of this keystone tree species.

Mapping the Environmental Risk of Beech Leaf Disease in the Northeastern United States.

The recently emerged beech leaf disease (BLD) is causing the decline and death of American beech in North America. First observed in 2012 in northeast Ohio, U.S.A., BLD had been documented in 10 northeastern states and the Canadian province of Ontario as of July 2022. A foliar nematode has been implicated as the causal agent, along with some bacterial taxa. No effective treatments have been documented in the primary literature. Irrespective of potential treatments, prevention and prompt eradication (rapid responses) remain the most cost-effective approaches to the management of forest tree disease. For these approaches to be feasible, however, it is necessary to understand the factors that contribute to BLD spread and use them in estimation of risk. Here, we conducted an analysis of BLD risk across northern Ohio, western Pennsylvania, western New York, and northern West Virginia, U.S.A. In the absence of symptoms, an area cannot necessarily be deemed free of BLD (i.e., absence of BLD cannot be certain) due to its fast spread and the lag in symptom expression (latency) after infection. Therefore, we employed two widely used presence-only species distribution models (SDMs), one-class support vector machine (OCSVM), and maximum entropy (Maxent) to predict the spatial pattern of BLD risk based on BLD presence records and associated environmental variables. Our results show that both methods work well for BLD environmental risk modeling purposes, but Maxent outperforms OCSVM with respect to both the quantitative receiver operating characteristics (ROC) analysis and the qualitative evaluation of the spatial risk maps. Meanwhile, the Maxent model provides a quantification of variable contribution for different environmental factors, indicating that meteorological (isothermality and temperature seasonality) and land cover type (closed broadleaved deciduous forest) factors are likely key contributors to BLD distribution. Moreover, the future trajectories of BLD risk over our study area in the context of climate change were investigated by comparing the current and future risk maps obtained by Maxent. In addition to offering the ability to predict where the disease may spread next, our work contributes to the epidemiological characterization of BLD, providing new lines of investigation to improve ecological or silvicultural management. Furthermore, this study shows strong potential for extension of environmental risk mapping over the full American beech distribution range so that proactive management measures can be put in place. Similar approaches can be designed for other significant or emerging forest pest problems, contributing to overall management efficiency and efficacy.

Why do entomologists and plant pathologists approach trophic relationships so differently? Identifying biological distinctions to foster synthesis.

Plant interactions with herbivores and pathogens are among the most widespread ecological relationships, and show many congruent properties. Despite these similarities, general models describing how plant defenses function in ecosystems, and the prioritization of responses to emerging challenges such as climate change, invasive species and habitat alteration, often differ markedly between entomologists and plant pathologists. We posit that some fundamental distinctions between how insects and pathogens interact with plants underlie these differences. We propose a conceptual framework to help incorporate these distinctions into robust models and research priorities. The most salient distinctions include features of host-searching behavior, evasion of plant defenses, plant tolerance to utilization, and sources of insect and microbial population regulation. Collectively, these features lead to relatively more diffuse and environmentally mediated plant-insect interactions, and more intimate and genetically driven plant-pathogen interactions. Specific features of insect vs pathogen life histories can also yield different patterns of spatiotemporal dynamics. These differences can become increasingly pronounced when scaling from controlled laboratory to open ecological systems. Integrating these differences alongside similarities can foster improved models and research approaches to plant defense, trophic interactions, coevolutionary dynamics, food security and resource management, and provide guidance as traditional departments increase collaborations, or merge into larger units.

Anatomical defences against bark beetles relate to degree of historical exposure between species and are allocated independently of chemical defences within trees.

Conifers possess chemical and anatomical defences against tree-killing bark beetles that feed in their phloem. Resins accumulating at attack sites can delay and entomb beetles while toxins reach lethal levels. Trees with high concentrations of metabolites active against bark beetle-microbial complexes, and more extensive resin ducts, achieve greater survival. It is unknown if and how conifers integrate chemical and anatomical components of defence or how these capabilities vary with historical exposure. We compared linkages between phloem chemistry and tree ring anatomy of two mountain pine beetle hosts. Lodgepole pine, a mid-elevation species, has had extensive, continual contact with this herbivore, whereas high-elevation whitebark pines have historically had intermittent exposure that is increasing with warming climate. Lodgepole pine had more and larger resin ducts. In both species, anatomical defences were positively related to tree growth and nutrients. Within-tree constitutive and induced concentrations of compounds bioactive against bark beetles and symbionts were largely unrelated to resin duct abundance and size. Fewer anatomical defences in the semi-naïve compared with the continually exposed host concurs with directional differences in chemical defences. Partially uncoupling chemical and morphological antiherbivore traits may enable trees to confront beetles with more diverse defence permutations that interact to resist attack.

Defence syndromes in lodgepole - whitebark pine ecosystems relate to degree of historical exposure to mountain pine beetles.

Warming climate is allowing tree-killing bark beetles to expand their ranges and access naïve and semi-naïve conifers. Conifers respond to attack using complex mixtures of chemical defences that can impede beetle success, but beetles exploit some compounds for host location and communication. Outcomes of changing relationships will depend on concentrations and compositions of multiple host compounds, which are largely unknown. We analysed constitutive and induced chemistries of Dendroctonus ponderosae's primary historical host, Pinus contorta, and Pinus albicaulis, a high-elevation species whose encounters with this beetle are transitioning from intermittent to continuous. We quantified multiple classes of terpenes, phenolics, carbohydrates and minerals. Pinus contorta had higher constitutive allocation to, and generally stronger inducibility of, compounds that resist these beetle-fungal complexes. Pinus albicaulis contained higher proportions of specific monoterpenes that enhance pheromone communication, and lower induction of pheromone inhibitors. Induced P. contorta increased insecticidal and fungicidal compounds simultaneously, whereas P. albicaulis responses against these agents were inverse. Induced terpene accumulation was accompanied by decreased non-structural carbohydrates, primarily sugars, in P. contorta, but not P. albicaulis, which contained primarily starches. These results show some host species with continuous exposure to bark beetles have more thoroughly integrated defence syndromes than less-continuously exposed host species.

Quantification of hydrogen peroxide in plant tissues using Amplex Red.

Reactive oxygen species (ROS) are by-products of photosynthesis and respiration in plant tissues. Abiotic and biotic stressors also induce the production and temporary accumulation of ROS in plants, including hydrogen peroxide (H2O2), whereby they can act as secondary messengers/chemical mediators in plant defense signaling and lead to programmed cell death. H2O2 acts as a hub for critical information flow in plants. Despite such key roles in fundamental cellular processes, reliable determination of H2O2 levels in plant tissues is hard to achieve. We optimized an Amplex Red-based quantitation method for H2O2 estimation from plant tissue lysate. The standard limit of detection and quantitation was determined as 6 and 18picomol respectively. In this study we also quantified constitutive and/or induced levels of H2O2 in three model plants, Pinus nigra (Austrian pine), Oryza sativa (rice), and Arabidopsis thaliana. Overall, assay sensitivity was in the nmolg-1 FW range. Commonly used additives for H2O2 extraction such as activated charcoal, ammonium sulfate, perchloric acid, polyvinylpolypyrrolidone, and trichloroacetic acid either degraded H2O2 directly or interfered with the Amplex Red assay. Finally, We measured stability of Amplex Red working solution over one month of storage at -80°C and found it to be significantly stable over time. With appropriate modifications, this optimized method should be applicable to any plant tissue.

A Native Parasitic Plant Systemically Induces Resistance in Jack Pine to a Fungal Symbiont of Invasive Mountain Pine Beetle.

Conifer trees resist pest and pathogen attacks by complex defense responses involving different classes of defense compounds. However, it is unknown whether prior infection by biotrophic pathogens can lead to subsequent resistance to necrotrophic pathogens in conifers. We used the infection of jack pine, Pinus banksiana, by a common biotrophic pathogen dwarf mistletoe, Arceuthobium americanum, to investigate induced resistance to a necrotrophic fungus, Grosmannia clavigera, associated with the mountain pine beetle, Dendroctonus ponderosae. Dwarf mistletoe infection had a non-linear, systemic effect on monoterpene production, with increasing concentrations at moderate infection levels and decreasing concentrations at high infection levels. Inoculation with G. clavigera resulted in 33 times higher monoterpene concentrations and half the level of phenolics in the necrotic lesions compared to uninoculated control trees. Monoterpene production following dwarf mistletoe infection seemed to result in systemic induced resistance, as trees with moderate disease severity were most resistant to G. clavigera, as evident from shorter lesion lengths. Furthermore, trees with moderate disease severity had the highest systemic but lowest local induction of α-pinene after G. clavigera inoculation, suggesting a possible tradeoff between systemically- and locally-induced defenses. The opposing effects to inoculation by G. clavigera on monoterpene and phenolic levels may indicate the potential for biosynthetic tradeoffs by the tree between these two major defense classes. Our results demonstrate that interactions between a biotrophic parasitic plant and a necrotrophic fungus may impact mountain pine beetle establishment in novel jack pine forests through systemic effects mediated by the coordination of jack pine defense chemicals.

Progress and gaps in understanding mechanisms of ash tree resistance to emerald ash borer, a model for wood-boring insects that kill angiosperms.

We review the literature on host resistance of ash to emerald ash borer (EAB, Agrilus planipennis), an invasive species that causes widespread mortality of ash. Manchurian ash (Fraxinus mandshurica), which coevolved with EAB, is more resistant than evolutionarily naïve North American and European congeners. Manchurian ash was less preferred for adult feeding and oviposition than susceptible hosts, more resistant to larval feeding, had higher constitutive concentrations of bark lignans, coumarins, proline, tyramine and defensive proteins, and was characterized by faster oxidation of phenolics. Consistent with EAB being a secondary colonizer of coevolved hosts, drought stress decreased the resistance of Manchurian ash, but had no effect on constitutive bark phenolics, suggesting that they do not contribute to increased susceptibility in response to drought stress. The induced resistance of North American species to EAB in response to the exogenous application of methyl jasmonate was associated with increased bark concentrations of verbascoside, lignin and/or trypsin inhibitors, which decreased larval survival and/or growth in bioassays. This finding suggests that these inherently susceptible species possess latent defenses that are not induced naturally by larval colonization, perhaps because they fail to recognize larval cues or respond quickly enough. Finally, we propose future research directions that would address some critical knowledge gaps.

Higher Activities of Defense-Associated Enzymes may Contribute to Greater Resistance of Manchurian Ash to Emerald Ash Borer Than A closely Related and Susceptible Congener.

Emerald ash borer (EAB) is an invasive beetle native to Asia that infests and kills ash (Fraxinus spp.) in North America. Previous experiments indicated that larvae feeding on co-evolved, resistant Manchurian ash (F. mandshurica) have increased antioxidant and quinone-protective enzyme activities compared to larvae feeding on susceptible North American species. Here, we examined mechanisms of host-generated oxidative and quinone-based stress and other putative defenses in Manchurian ash and the closely related and chemically similar, but susceptible, black ash (F. nigra), with and without exogenous application of methyl jasmonate (MeJA) to induce resistance mechanisms. Peroxidase activities were 4.6-13.3 times higher in Manchurian than black ash, although both species appeared to express the same three peroxidase isozymes. Additionally, peroxidase-mediated protein cross-linking activity was stronger in Manchurian ash. Polyphenol oxidase, ß-glucosidase, chitinase, and lipoxygenase activities also were greater in Manchurian ash, but only lipoxygenase activity increased with MeJA application. Phloem H2O2 levels were similar and were increased by MeJA application in both species. Lastly, trypsin inhibitor activity was detected in methanol and water extracts that were not allowed to oxidize, indicating the presence of phenolic-based trypsin inhibitors. However, no proteinaceous trypsin inhibitor activity was detected in either species. In response to MeJA application, Manchurian ash had higher trypsin inhibitor activity than black ash using the unoxidized water extracts, but no treatment effects were detected using methanol extracts. Based on these results we hypothesize that peroxidases, lignin polymerization, and quinone generation contribute to the greater resistance to EAB displayed by Manchurian ash.

Comparative Herbivory Rates and Secondary Metabolite Profiles in the Leaves of Native and Non-Native Lonicera Species.

Non-native plants introduced to new habitats can have significant ecological impact. In many cases, even though they interact with the same community of potential herbivores as their new native competitors, they regularly receive less damage. Plants produce secondary metabolites in their leaves that serve a range of defensive functions, including resistance to herbivores and pathogens. Abiotic factors such as nutrient availability can influence the expression of defensive traits, with some species exhibiting increased chemical defense in low-nutrient conditions. Plants in the genus Lonicera are known to produce a diverse array of these secondary metabolites, yet non-native Lonicera species sustain lower amounts of herbivore damage than co-occurring native Lonicera species in North America. In this study, we searched for evidence of biochemical novelty in non-native species, and quantified its association with resistance to herbivores. In order to achieve this, we evaluated the phenolic and iridoid glycoside profiles in leaves of native and non-native Lonicera species grown under high and low fertilization treatments in a common garden. We then related these profiles to naturally occurring herbivore damage on whole plants in the garden. Herbivore damage was greater on native Lonicera, and chemical profiles and concentrations of selected putative defense compounds varied by species. Geographic origin was an inconsistent predictor of chemical variation in detected phenolics and iridoid glycosides (IGs). Overall, fertilization did not affect herbivore damage or measures of phenolics or IGs, but there were some fertilization effects within species. While we cannot conclude that non-natives were more chemically novel than native Lonicera species, chemical defense profiles and concentrations of specific compounds varied by species. Reduced attraction or deterrence of oviposition, specific direct resistance traits, or a combination of both may contribute to reduced herbivory and competitive advantages for non-native Lonicera in North America.

Effects of water availability on emerald ash borer larval performance and phloem phenolics of Manchurian and black ash.

The invasive emerald ash borer (EAB) beetle is a significant threat to the survival of North American ash. In previous work, we identified putative biochemical and molecular markers of constitutive EAB resistance in Manchurian ash, an Asian species co-evolved with EAB. Here, we employed high-throughput high-performance liquid chromatography with photodiode array detection and mass spectrometry (HPLC-PDA-MS) to characterize the induced response of soluble phloem phenolics to EAB attack in resistant Manchurian and susceptible black ash under conditions of either normal or low water availability, and the effects of water availability on larval performance. Total larval mass per tree was lower in Manchurian than in black ash. Low water increased larval numbers and mean larval mass overall, but more so in Manchurian ash. Low water did not affect levels of phenolics in either host species, but six phenolics decreased in response to EAB. In both ashes, pinoresinol A was induced by EAB, especially in Manchurian ash. Pinoresinol A and pinoresinol B were negatively correlated with each other in both species. The higher accumulation of pinoresinol A in Manchurian ash after attack may help explain the resistance of this species to EAB, but none of the responses measured here could explain increased larval performance in trees subjected to low water availability.

Failure under stress: the effect of the exotic herbivore Adelges tsugae on biomechanics of Tsuga canadensis.

BACKGROUND AND AIMS: Exotic herbivores that lack a coevolutionary history with their host plants can benefit from poorly adapted host defences, potentially leading to rapid population growth of the herbivore and severe damage to its plant hosts. The hemlock woolly adelgid (Adelges tsugae) is an exotic hemipteran that feeds on the long-lived conifer eastern hemlock (Tsuga canadensis), causing rapid mortality of infested trees. While the mechanism of this mortality is unknown, evidence indicates that A. tsugae feeding causes a hypersensitive response and alters wood anatomy. This study investigated the effect of A. tsugae feeding on biomechanical properties at different spatial scales: needles, twigs and branches. METHODS: Uninfested and A. tsugae-infested samples were collected from a common garden experiment as well as from naturally infested urban and rural field sites. Tension and flexure mechanical tests were used to quantify biomechanical properties of the different tissues. In tissues that showed a significant effect of herbivory, the potential contributions of lignin and tissue density on the results were quantified. KEY RESULTS: Adelges tsugae infestation decreased the abscission strength, but not flexibility, of needles. A. tsugae feeding also decreased mechanical strength and flexibility in currently attacked twigs, but this effect disappeared in older, previously attacked branches. Lignin and twig tissue density contributed to differences in mechanical strength but were not affected by insect treatment. CONCLUSIONS: Decreased strength and flexibility in twigs, along with decreased needle strength, suggest that infested trees experience resource stress. Altered growth patterns and cell wall chemistry probably contribute to these mechanical effects. Consistent site effects emphasize the role of environmental variation in mechanical traits. The mechanical changes measured here may increase susceptibility to abiotic physical stressors in hemlocks colonized by A. tsugae. Thus, the interaction between herbivore and physical stresses is probably accelerating the decline of eastern hemlock, as HWA continues to expand its range.

Decreased emergence of emerald ash borer from ash treated with methyl jasmonate is associated with induction of general defense traits and the toxic phenolic compound verbascoside.

The emerald ash borer (EAB; Agrilus planipennis Fairmaire) is causing widespread mortality of ash (Fraxinus spp.) in North America. To date, no mechanisms of host resistance have been identified against this pest. Methyl jasmonate was applied to susceptible North American and resistant Asian ash species to determine if it can elicit induced responses in bark that enhance resistance to EAB. In particular, phenolic compounds, lignin, and defense-related proteins were quantified, and compounds associated with resistance were subsequently tested directly against EAB larvae in bioassays with artificial diet. MeJA application decreased adult emergence in susceptible ash species, comparable to levels achieved by insecticide application. Concentration of the phenolic compound verbascoside sharply increased after MeJA application to green and white ash. When incorporated in an artificial diet, verbascoside decreased survival and growth of EAB neonates in a dose-dependent fashion. Lignin and trypsin inhibitors were also induced by MeJA, and analogs of both compounds reduced growth of EAB larvae in artificial diets. We conclude that the application of MeJA prior to EAB attack has the ability to enhance resistance of susceptible ash trees by inducing endogenous plant defenses, and report evidence that induction of verbascoside is a mechanism of resistance to EAB.

Reserves accumulated in non-photosynthetic organs during the previous growing season drive plant defenses and growth in aspen in the subsequent growing season.

Plants store non-structural carbohydrates (NSC), nitrogen (N), as well as other macro and micronutrients, in their stems and roots; the role of these stored reserves in plant growth and defense under herbivory pressure is poorly understood, particularly in trees. Trembling aspen (Populus tremuloides) seedlings with different NSC and N reserves accumulated during the previous growing season were generated in the greenhouse. Based on NSC and N contents, seedlings were assigned to one of three reserve statuses: Low N-Low NSC, High N-Medium NSC, or High N-High NSC. In the subsequent growing season, half of the seedlings in each reserve status was subjected to defoliation by forest tent caterpillar (Malacosoma disstria) while the other half was left untreated. Following defoliation, the effect of reserves was measured on foliar chemistry (N, NSC) and caterpillar performance (larval development). Due to their importance in herbivore feeding, we also quantified concentrations of phenolic glycoside compounds in foliage. Seedlings in Low N-Low NSC reserve status contained higher amounts of induced phenolic glycosides, grew little, and supported fewer caterpillars. In contrast, aspen seedlings in High N-Medium or High NSC reserve statuses contained lower amounts of induced phenolic glycosides, grew faster, and some of the caterpillars which fed on these seedlings developed up to their fourth instar. Furthermore, multiple regression analysis indicated that foliar phenolic glycoside concentration was related to reserve chemistry (NSC, N). Overall, these results demonstrate that reserves accumulated during the previous growing season can influence tree defense and growth in the subsequent growing season. Additionally, our study concluded that the NSC/N ratio of reserves in the previous growing season represents a better measure of resources available for use in defense and growth than the foliar NSC/N ratios.

Novel Pathogen-Plant Host Interaction: Colletotrichum jiangxiense and Fraxinus americana L. (White Ash) in a Sentinel Garden in China.

Fraxinus americana L. (white ash), a native North American tree commonly cultivated for its ornamental qualities, displayed symptoms of leaf spot disease in a sentinel garden located in Nanjing, Jiangsu, China, in 2022. This disease led to premature leaf shedding, adversely affecting the plant's growth and substantially diminishing its ornamental value. Potential fungal pathogens were isolated from the diseased leaves and the subsequent application of Koch's postulates confirmed the pathogenicity of the fungal isolates (BL-1, BL-2). Through a combination of multi-locus phylogenetic analysis, including ITS, ACT, ApMat, CAL, CHS-1, GAPDH, and TUB2, alongside morphological assessments, the fungus was conclusively identified as Colletotrichum jiangxiense. This represents the first record of C. jiangxiense affecting white ash, highlighting the important role of sentinel gardens in uncovering novel pathogen-plant host interactions.

The Global Forest Health Crisis: A Public-Good Social Dilemma in Need of International Collective Action.

Society is confronted by interconnected threats to ecological sustainability. Among these is the devastation of forests by destructive non-native pathogens and insects introduced through global trade, leading to the loss of critical ecosystem services and a global forest health crisis. We argue that the forest health crisis is a public-good social dilemma and propose a response framework that incorporates principles of collective action. This framework enables scientists to better engage policymakers and empowers the public to advocate for proactive biosecurity and forest health management. Collective action in forest health features broadly inclusive stakeholder engagement to build trust and set goals; accountability for destructive pest introductions; pooled support for weakest-link partners; and inclusion of intrinsic and nonmarket values of forest ecosystems in risk assessment. We provide short-term and longer-term measures that incorporate the above principles to shift the societal and ecological forest health paradigm to a more resilient state.

Interspecific comparison of constitutive ash phloem phenolic chemistry reveals compounds unique to manchurian ash, a species resistant to emerald ash borer.

The emerald ash borer (Agrilus planipennis, EAB) is an invasive wood-borer indigenous to Asia and is responsible for widespread ash (Fraxinus spp.) mortality in the U.S. and Canada. Resistance and susceptibility to EAB varies among Fraxinus spp., which is a result of their co-evolutionary history with the pest. We characterized constitutive phenolic profiles and lignin levels in the phloem of green, white, black, blue, European, and Manchurian ash. Phloem was sampled twice during the growing season, coinciding with phenology of early and late instar EAB. We identified 66 metabolites that displayed a pattern of variation, which corresponded strongly with phylogeny. Previously identified lignans and lignan derivatives were confirmed to be unique to Manchurian ash, and may contribute to its high level of resistance to EAB. Other compounds that had been considered unique to Manchurian ash, including hydroxycoumarins and the phenylethanoids calceolarioside A and B, were detected in closely related, but susceptible species, and thus are unlikely to contribute to EAB resistance of Manchurian ash. The distinct phenolic profile of blue ash may contribute to its relatively high resistance to EAB.

Phytobiome Metabarcoding: A Tool to Help Identify Prokaryotic and Eukaryotic Causal Agents of Undiagnosed Tree Diseases.

Recent advancements in high-throughput sequencing have provided scientists with vastly enhanced tools to diagnose unknown tree diseases. One of these techniques is referred to as metabarcoding, which uses phylogenetically informative reference genes to taxonomically classify short DNA sequences amplified from environmental samples. Using metabarcoding, we are able to compare the microbiota of symptomatic and asymptomatic (including presumably naïve) samples and identify microbe(s) that are only present in symptomatic samples and could therefore be responsible for the undiagnosed disease. Metabarcoding involves two main steps: library preparation and bioinformatic processing. For library preparation, the appropriate reference gene for the organism of interest (i.e., bacteria, phytoplasma, fungi, or other eukaryotes, such as nematodes) is amplified from the DNA extracted from the environmental samples using PCR and prepared for sequencing. The bioinformatic processing includes four major steps: (1) quality check and cleanup on raw reads; (2) classification of the sequences into taxonomically informative groups (ASVs or OTUs); (3) taxonomy assignments based on the reference database; and (4) differential abundance and diversity analyses to identify microbes that are significantly associated with just symptomatic samples and that point toward the putative causal agent of the disease.

Differential response in foliar chemistry of three ash species to emerald ash borer adult feeding.

The emerald ash borer (EAB; Agrilus planipennis Fairmaire; Coleoptera: Buprestidae), is an exotic wood-boring beetle that has been threatening North American ash (Fraxinus spp.) resources since its discovery in Michigan and Ontario in 2002. In this study, we investigated the phytochemical responses of the three most common North American ash species (black, green, and white ash) in northeastern USA to EAB adult feeding. Black ash was the least responsive to EAB adult feeding in terms of the induction of volatile compounds, and levels of only two (indole and benzyl cyanide) of the 11 compounds studied increased. In green ash, levels of two [(E)-ß-ocimene and indole] of the 11 volatile compounds studied were elevated, while the levels of two green leaf volatiles [hexanal and (E)-2-hexenal] decreased. White ash showed the greatest response with an increase in levels of seven of the 11 compounds studied. Qualitative differences among ash species were detected. Among the phenolic compounds detected, ligustroside was the only one detected in all three species. Oleuropein aglycone and 2 unidentified compounds were found only in black ash; coumaroylquinic acid and feruloylquinic acid were detected only in green ash; and verbascoside hexoside was detected only in white ash. EAB adult feeding did not elicit or decrease concentrations of any selected individual phenolic compounds. However, although levels of total phenolics from black and green ash foliage were not affected by EAB adult feeding, they decreased significantly in white ash. EAB adult feeding elevated chymotrypsin inhibitors in black ash. The possible ecological implications of these findings are discussed.