Detecting Pathogenic Phytophthora Species Using Volatile Organic Compounds.

There are several highly damaging Phytophthora species pathogenic to forest trees, many of which have been spread beyond their native range by the international trade of live plants and infested materials. Such introductions can be reduced through the development of better tools capable of the early, rapid, and high-throughput detection of contaminated plants. This study utilized a volatilomics approach (solid-phase microextraction coupled to gas chromatography-mass spectrometry) to differentiate between several Phytophthora species in culture and discriminate between healthy and Phytophthora-inoculated European beech and pedunculate oak trees. We tentatively identified 14 compounds that could differentiate eight Phytophthora species from each other in vitro. All of the Phytophthora species examined, except Phytophthora cambivora, uniquely produced at least one compound not observed in the other species; however, most detected compounds were shared between multiple species. Phytophthora polonica had the most unique compounds and was the least similar of all the species examined. The inoculated seedlings had qualitatively different volatile profiles and could be distinguished from the healthy controls by the presence of isokaurene, anisole, and a mix of three unknown compounds. This study supports the notion that volatiles are suitable for screening plant material, detecting tree pathogens, and differentiating between healthy and diseased material.

Utilizing volatile organic compounds for early detection of Fusarium circinatum.

Fusarium circinatum, a fungal pathogen deadly to many Pinus species, can cause significant economic and ecological losses, especially if it were to become more widely established in Europe. Early detection tools with high-throughput capacity can increase our readiness to implement mitigation actions against new incursions. This study sought to develop a disease detection method based on volatile organic compound (VOC) emissions to detect F. circinatum on different Pinus species. The complete pipeline applied here, entailing gas chromatography-mass spectrometry of VOCs, automated data analysis and machine learning, distinguished diseased from healthy seedlings of Pinus sylvestris and Pinus radiata. In P. radiata, this distinction was possible even before the seedlings became visibly symptomatic, suggesting the possibility for this method to identify latently infected, yet healthy looking plants. Pinus pinea, which is known to be relatively resistant to F. circinatum, remained asymptomatic and showed no changes in VOCs over 28 days. In a separate analysis of in vitro VOCs collected from different species of Fusarium, we showed that even closely related Fusarium spp. can be readily distinguished based on their VOC profiles. The results further substantiate the potential for volatilomics to be used for early disease detection and diagnostic recognition.

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.

Water-deficit and fungal infection can differentially affect the production of different classes of defense compounds in two host pines of mountain pine beetle.

Bark beetles are important agents of tree mortality in conifer forests and their interaction with trees is influenced by host defense chemicals, such as monoterpenes and phenolics. Since mountain pine beetle (Dendroctonus ponderosae Hopkins) has expanded its host range from lodgepole pine (Pinus contorta Doug. ex Loud. (var. latifolia Engelm.))-dominated forests to the novel jack pine (Pinus banksiana Lamb.) forests in western Canada, studies investigating the jack pine suitability as a host for this beetle have exclusively focused on monoterpenes, and whether phenolics affect jack pine suitability to mountain pine beetle and its symbiotic fungus Grosmannia clavigera is unknown. We investigated the phenolic and monoterpene composition in phloem and foliage of jack and lodgepole pines, and their subsequent change in response to water deficit and G. clavigera inoculation treatments. In lodgepole pine phloem, water deficit treatment inhibited the accumulation of both the total and richness of phenolics, but had no effect on total monoterpene production or richness. Fungal infection also inhibited the total phenolic production and had no effect on phenolic or monoterpene richness, but increased total monoterpene synthesis by 71%. In jack pine phloem, water deficit treatment reduced phenolic production, but had no effect on phenolic or monoterpene richness or total monoterpenes. Fungal infection did not affect phenolic or monoterpene production. Lesions of both species contained lower phenolics but higher monoterpenes than non-infected phloem in the same tree. In both species, richness of monoterpenes and phenolics was greater in non-infected phloem than in lesions. We conclude that monoterpenes seem to be a critical component of induced defenses against G. clavigera in both jack and lodgepole pines; however, a lack of increased monoterpene response to fungal infection is an important evolutionary factor defining jack pine suitability to the mountain pine beetle invasion in western Canada.

Mechanisms of induced susceptibility to Diplodia tip blight in drought-stressed Austrian pine.

Plants experiencing drought stress are frequently more susceptible to pathogens, likely via alterations in physiology that create favorable conditions for pathogens. Common plant responses to drought include the production of reactive oxygen species (ROS) and the accumulation of free amino acids (AAs), particularly proline. These same phenomena also frequently occur during pathogenic attack. Therefore, drought-induced perturbations in AA and ROS metabolism could potentially contribute to the observed enhanced susceptibility. Furthermore, nitrogen (N) availability can influence AA accumulation and affect plant resistance, but its contributions to drought-induced susceptibility are largely unexplored. Here we show that drought induces accumulation of hydrogen peroxide (H2O2) in Austrian pine (Pinus nigra Arnold) shoots, but that shoot infection by the blight and canker pathogen Diplodia sapinea (Fr.) Fuckel leads to large reductions in H2O2 levels in droughted plants. In in vitro assays, H2O2 was toxic to D. sapinea, and the fungus responded to this oxidative stress by increasing catalase and peroxidase activities, resulting in substantial H2O2 degradation. Proline increased in response to drought and infection when examined independently, but unlike all other AAs, proline further increased in infected shoots of droughted trees. In the same tissues, the proline precursor, glutamate, decreased significantly. Proline was found to protect D. sapinea from H2O2 damage, while also serving as a preferred N source in vitro. Fertilization increased constitutive and drought-induced levels of some AAs, but did not affect plant resistance. A new model integrating interactions of proline and H2O2 metabolism with drought and fungal infection of plants is proposed.

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.

Austrian pine phenolics are likely contributors to systemic induced resistance against Diplodia pinea.

The molecular basis of the systemic induced resistance (SIR) phenotype known to occur in Austrian pine (Pinus nigra J.F. Arnold) in response to the tip blight and canker pathogen Diplodia pinea (Desm.) remains unclear. Specialized metabolites such as phenolics are considered to be an important component of plant defense, including SIR, but the antimicrobial activity of many of these putative defensive chemicals remains untested at realistic concentrations and in conjunction with each other. Here, we examined the anti-Diplodia activity of several previously identified Austrian pine phenolics associated with SIR by comparing the diameters of fungal colonies grown on media amended with ferulic acid, coumaric acid, taxifolin, pinosylvin, pinosylvin monomethyl ether and lignin. All of the compounds were tested both individually and as clusters (combinations) previously determined to occur in planta in a co-regulated fashion. Both the individual compounds and clusters were tested at constitutive concentrations and pathogen-induced concentrations linked to an SIR phenotype. Lignin possessed the strongest antifungal activity individually, and clusters at the SIR concentrations had the greatest antifungal effects, achieving fungistasis. This study exemplifies the value of evaluating potential biomarkers of resistance at in planta concentrations that are associated with the systemically resistant phenotype, and provides strong evidence that co-regulation of chemical defenses potentiates such a phenotype.

A novel type of glial cell in the retina is stimulated by insulin-like growth factor 1 and may exacerbate damage to neurons and Müller glia.

Recent studies have demonstrated that insulin can have profound affects on the survival of neurons within the retina. The purpose of this study was to determine how insulin-like growth factor 1 (IGF1) influences retinal cells; in particular, the glial cells. We identify a novel type of glial cell in the avian retina and provide evidence that these cells can respond to acute damage and IGF1. In normal retinas, we found a distinct cell-type, scattered across the ganglion cell and inner plexiform layers that express Sox2, Sox9, Nkx2.2, vimentin, and transitin, the avian homologue of mammalian nestin. These glial cells have a unique immunohistochemical profile, morphology, and distribution that are distinct among other known types of retinal glia, including microglia, oligodendrocytes, astrocytes, and Muller glia. We termed these cells nonastrocytic inner retinal glia-like (NIRG) cells. We found that the NIRG cells may express the IGF1 receptor and respond to IGF1 by proliferating, migrating distally into the retina, and upregulating transitin. In addition, IGF1 stimulated microglia to become reactive and upregulate lysosomal membrane glycoprotein and CD45. With microglia and NIRG cells stimulated by IGF1 there were elevated levels of cell death and numerous focal detachments across the retina in response to excitotoxic damage. Cell death was prominent within the areas of detachment coinciding with a stark loss of Müller glia and accumulation of NIRG cells. We conclude that NIRG cells are a novel type of retinal glia that is sensitive to IGF1 and whose activity may impact the survival of neurons and Müller glia.

Mitogen-activated protein kinase-signaling regulates the ability of Müller glia to proliferate and protect retinal neurons against excitotoxicity.

The purpose of this study was to investigate whether insulin, fibroblast growth factor (FGF), and mitogen-activated protein kinase (MAPK) pathways protect retinal neurons against excitotoxicity and regulate the proliferation of Müller glia. We found that intraocular injections of insulin or FGF2 had variable effects upon the phosphorylation of ERK1/2, p38 MAPK, and CREB, and the expression of immediate early genes, cFos and Egr1. Accumulations of pERK1/2, p38 MAPK, pCREB, cFos and Egr1 in response to insulin or FGF2 were confined to Müller glia, whereas retinal neurons did not seem to respond to growth factors. Unlike FGF2, insulin stimulated microglia-like cells to upregulate the intermediate filament transitin and lysosomal membrane glycoprotein (LMG). With microglia-like cells and Müller glia stimulated by insulin or FGF2 there were profound effects upon numbers of dying neurons in response to excitotoxic damage. Although FGF2 significantly reduced numbers of dying neurons, insulin significantly increased numbers of dying neurons. In addition to neuroprotective affects, FGF2 also "primed" the Müller glia to proliferate following retinal damage, whereas insulin had no effect upon glial proliferation. Further, we found that FGF receptor isoform 1 (FGFR1) and FGFR3 were prominently expressed in the retina, whereas the insulin receptor and FGFR2 are not expressed, or are expressed at very low levels. We conclude that MAPK-signaling through FGF receptors stimulates Müller glia to become more neuroprotective and progenitor-like, whereas insulin acting on Müller and microglia-like cells through unidentified receptors had the opposite effect.

Transient expression of LIM-domain transcription factors is coincident with delayed maturation of photoreceptors in the chicken retina.

In the retina of warm-blooded vertebrates, photoreceptors are specified many days before the onset of synaptogenesis and the expression of photopigments. The factors that regulate the maturation of photoreceptors in the developing retina remain unknown. We report here that photoreceptors transiently express LIM-domain transcription factors during the development of the chicken retina. We examined the differentiation of photoreceptors through the normal course of embryonic development and at the far periphery of the postnatal retina, where the differentiation of photoreceptors is slowed and persists across a spatial gradient. In the embryonic retina, we find visinin-positive photoreceptors that transiently express Islet2 and Lim3 starting at E8 and ending around E15, but persisting in far peripheral regions of the retina through the first 2 weeks of postnatal development. During early stages of photoreceptor maturation, there is coincident and transient expression of the LIM-domain factors with axonin1, a cell surface glycoprotein that is a member of the immunoglobulin superfamily. Coincident with the downregulation of Islet2 and Lim3, we find the upregulation of calbindin, red/green opsin, rhodopsin, and a synaptic marker in the outer plexiform layer (OPL; dystrophin). In the periphery of the postnatal retina, photoreceptors that express Islet2, Lim3, and axonin1 do not overlap with photoreceptors that express calbindin, red/green opsin, rhodopsin, and dystrophin. We propose that Islet2 and Lim3 may promote the expression of genes that are involved in the early stages of differentiation but may suppress the expression of genes that are required in the mature photoreceptors.