A LAMP Assay for Rapid Detection of the Pitch Canker Pathogen Fusarium circinatum.

The pine pitch canker pathogen Fusarium circinatum is endemic in the southeastern United States and Central America and represents an invasive threat globally. This ecologically adaptable fungus readily infects all parts of its pine hosts, leading to widespread mortality of nursery seedlings and decline in the health and productivity of forest stands. Because trees infected by F. circinatum can remain asymptomatic for long periods of time, accurate and rapid tools are needed for real-time diagnostics and surveillance at ports, in nurseries, and in plantations. To meet this need and to limit the spread and impact of the pathogen, we developed a molecular test using loop-mediated isothermal amplification (LAMP), a technology that allows for the rapid detection of pathogen DNA on portable, field-capable devices. LAMP primers were designed and validated to amplify a gene region unique to F. circinatum. Using a globally representative collection of F. circinatum isolates and other closely related species, we have demonstrated that the assay can be used to identify F. circinatum across its genetic diversity and that it is sensitive to as few as 10 cells from purified DNA extracts. The assay can also be used with a simple, pipette-free DNA extraction method and is compatible with testing symptomatic pine tissues in the field. This assay has the potential to facilitate diagnostic and surveillance efforts both in the laboratory and in the field and, thus, to reduce the spread and impact of pitch canker worldwide.

Rapid Detection of Raffaelea lauricola Directly from Host Plant and Beetle Vector Tissues Using Loop-Mediated Isothermal Amplification.

Since its introduction in 2002, laurel wilt disease has devastated indigenous lauraceous species in the southeastern United States. The causal agent is a fungal pathogen, Raffaelea lauricola, which, after being introduced into the xylem of trees by its vector beetle, Xyleborus glabratus, results in a fatal vascular wilt. Rapid detection and accurate diagnosis of infections is paramount to the successful implementation of disease management strategies. Current management operations to prevent the spread of laurel wilt disease are largely delayed by time-consuming laboratory procedures to confirm the diagnosis. In order to greatly speed up the operations, we developed a loop-mediated isothermal amplification (LAMP) species-specific assay that targets the ß-tubulin gene region of R. lauricola, and allows for the rapid detection of the pathogen directly from host plant and beetle tissues. The assay is capable of amplifying as little as 0.5 pg of fungal DNA and as few as 50 conidia. The assay is also capable of detecting R. lauricola directly from wood tissue of artificially inoculated redbay saplings as early as 10 and 12 days postinoculation, when testing high-quality and crude DNA extracts, respectively. Finally, crude DNA extracts of individual adult female X. glabratus beetles were assayed and the pathogen was detected from all specimens. This assay greatly reduces the time required to confirm a laurel wilt diagnosis and, because LAMP technology is well suited to provide point-of-care testing, it has the potential to expedite and facilitate implementation of management operations in response to disease outbreaks.

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.

Early Detection of Airborne Inoculum of Magnaporthe oryzae in Turfgrass Fields Using a Quantitative LAMP Assay.

Gray leaf spot (GLS) is a destructive disease of perennial ryegrass caused by a host specific pathotype of the ascomycete Magnaporthe oryzae. Early diagnosis is crucial for effective disease management and the implementation of Integrated Pest Management practices. However, a rapid protocol for the detection of low levels of airborne inoculum is still missing. We developed a pathogen-specific quantitative loop-mediated isothermal amplification (qLAMP) assay coupled with a spore trap system for rapid detection and quantification of airborne inoculum of the M. oryzae perennial ryegrass pathotype, and tested its suitability for implementation in GLS-infected turfgrass fields. In summer 2015, two perennial ryegrass plots were artificially inoculated with the pathogen, with four continuously running custom impaction spore traps placed in each plot. Sampling units were replaced daily and tested with the developed qLAMP assay, while plots were monitored for symptom development. Results confirmed that the qLAMP assay-trap system was able to detect as few as 10 conidia up to 12 days before symptoms developed in the field. LAMP technology is particularly appropriate for field implementation by nontechnical users, and has the potential to be a powerful decision support tool to guide timing of fungicide applications for GLS management.

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.

The Ophiostoma clavatum species complex: a newly defined group in the Ophiostomatales including three novel taxa.

Two species of blue-stain fungi with similar morphologies, Ophiostoma brunneo-ciliatum and Ophiostoma clavatum, are associates of bark beetles infesting Pinus spp. in Europe. This has raised questions whether they represent distinct taxa. Absence of herbarium specimens and contaminated or mistakenly identified cultures of O. brunneo-ciliatum and O. clavatum have accentuated the uncertainty regarding their correct identification. The aim of this study was to reconsider the identity of European isolates reported as O. brunneo-ciliatum and O. clavatum by applying DNA-based identification methods, and to provide appropriate type specimens for them. Phylogenetic analyses of the ITS, ßT, TEF-1α and CAL gene sequences revealed that the investigated isolates represent a complex of seven cryptic species. The study confirmed that ITS data is insufficient to delineate species in some Ophiostoma species clusters. Lectotypes and epitypes were designated for O. clavatum and O. brunneo-ciliatum, and three new species, Ophiostoma brunneolum, Ophiostoma macroclavatum and Ophiostoma pseudocatenulatum, are described in the newly defined O. clavatum-complex. The other two species included in the complex are Ophiostoma ainoae and Ophiostoma tapionis. The results suggest co-evolution of these fungi in association with specific bark beetles. The results also confirm the identity of the fungus associated with the pine bark beetle Ips acuminatus as O. clavatum, while O. brunneo-ciliatum appears to be mainly associated with another pine bark beetle, Ips sexdentatus.

The role of Manganese in tree defenses against pests and pathogens.

Manganese (Mn) deficiency is a widespread occurrence across different landscapes, including agricultural systems and managed forests, and causes interruptions in the normal metabolic functioning of plants. The microelement is well-characterized for its role in the oxygen-evolving complex in photosystem II and maintenance of photosynthetic structures. Mn is also required for a variety of enzymatic reactions in secondary metabolism, which play a crucial role in defense strategies for trees. Despite the strong relationship between Mn availability and the biosynthesis of defense-related compounds, there are few studies addressing how Mn deficiency can impact tree defense mechanisms and the ensuing ecological patterns and processes. Understanding this relationship and highlighting the potentially deleterious effects of Mn deficiency in trees can also inform silvicultural and management decisions to build more robust forests. In this review, we address this relationship, focusing on forest trees. We describe Mn availability in forest soils, characterize the known impacts of Mn deficiency in plant susceptibility, and discuss the relationship between Mn and defense-related compounds by secondary metabolite class. In our review, we find several lines of evidence that low Mn availability is linked with lowered or altered secondary metabolite activity. Additionally, we compile documented instances where Mn limitation has altered the defense capabilities of the host plant and propose potential ecological repercussions when studies are not available. Ultimately, this review aims to highlight the importance of untangling the effects of Mn limitation on the ecophysiology of plants, with a focus on forest trees in both managed and natural stands.

Winter diet of bats in working forests of the southeastern U.S. Coastal Plain.

Working forests comprise a large proportion of forested landscapes in the southeastern United States and are important to the conservation of bats, which rely on forests for roosting and foraging. While relationships between bat ecology and forest management are well studied during summer, winter bat ecology remains understudied. Hence, we aimed to identify the diet composition of overwintering bats, compare the composition of prey consumed by bat species, and determine the potential role of forest bats as pest controllers in working forest landscapes of the southeastern U.S. Coastal Plain. During January to March 2021-2022, we captured 264 bats of eight species. We used DNA metabarcoding to obtain diet composition from 126 individuals of seven bat species identifying 22 orders and 174 families of arthropod prey. Although Coleoptera, Diptera, and Lepidoptera were the most consumed orders, we found that bats had a generalist diet but with significant differences among some species. We also documented the consumption of multiple insect pests (e.g., Rhyacionia frustrana) and disease vectors (e.g., Culex spp). Our results provide important information regarding the winter diet of bats in the southeastern U.S. Coastal Plain and their potential role in controlling economically relevant pest species and disease vectors.

Use of loop-mediated isothermal amplification for detection of Ophiostoma clavatum, the primary blue stain fungus associated with Ips acuminatus.

Loop-mediated isothermal amplification (LAMP) is an alternative amplification technology which is highly sensitive and less time-consuming than conventional PCR-based methods. Three LAMP assays were developed, two for detection of species of symbiotic blue stain fungi associated with Ips acuminatus, a bark beetle infesting Scots pine (Pinus sylvestris), and an additional assay specific to I. acuminatus itself for use as a control. In common with most bark beetles, I. acuminatus is associated with phytopathogenic blue stain fungi involved in the process of exhausting tree defenses, which is a necessary step for the colonization of the plant by the insect. However, the identity of the main blue stain fungus vectored by I. acuminatus was still uncertain, as well as its frequency of association with I. acuminatus under outbreak and non-outbreak conditions. In this study, we employed LAMP technology to survey six populations of I. acuminatus sampled from the Southern Alps. Ophiostoma clavatum was detected at all sampling sites, while Ophiostoma brunneo-ciliatum, reported in part of the literature as the main blue stain fungus associated with I. acuminatus, was not detected on any of the samples. These results are consistent with the hypothesis that O. clavatum is the main blue stain fungus associated with I. acuminatus in the Southern Alps. The method developed in the course of this work provides a molecular tool by which it will be easy to screen populations and derive important data regarding the ecology of the species involved.

[Health surveillance for subjects with past occupational exposure to asbestos: the experience of Local Health Unit Messina 5 (Sicily)]. / La sorveglianza sanitaria dei soggetti con pregressa esposizione professionale ad amianto: l'esperienza dell'AUSL Messina 5.

OBJECTIVE: In the framework of the World Health Organization (WHO)-Istituto superiore di sanità (ISS) joint research project «Cohort studies in areas of high environmental risk in Sicily¼, since 2007 a working group ISS-Local Health Unit Messina 5 (AUSL ME5) assessed the issue emerged during the course of a health monitoring program launched in 2003, dedicated to former employees of an asbestos-cement factory, and their relatives (378 subjects, 119 ex employees and 259 relatives). METHODS: Communication about the health risks linked to previous exposure to asbestos, diagnostic tools for asbestos-related diseases, available preventive measures (stopping smoking tobacco, avoiding further exposure to respiratory irritants, prophylaxis of any intercurrent respiratory diseases) were focused. The working group discussed the whole structure of the activity in progress, and identified the difficulties emerged in previous years. RESULTS AND CONCLUSIONS: The authors developed a health plan that could be useful in similar Sicilian contexts with public health problems due to past activities with asbestos.

[Mortality from asbestos-related causes and incidence of pleural mesothelioma among former asbestos cement workers in San Filippo del Mela (Sicily)]. / Mortalità per cause asbesto-correlate e incidenza del mesotelioma fra i lavoratori del cemento-amianto di San Filippo del Mela (Messina).

OBJECTIVES: The present paper estimates the burden of asbestos-related disease among asbestos-cement production workers of the Sacelit plant that operated in San Filippo del Mela (Province of Messina) from 1958 through 1993. SETTING AND PARTICIPANTS: The cohort was enumerated by the local committee of formerly exposed workers, with whom a collaboration was set up. The cohort includes 198 subjects with complete individual anagraphic information, out of 231 previous workers identified by the committee. A record-linkage with the Sicilian centre of the National mesothelioma registry enabled estimation of mesothelioma incidence for the years 1998-2008. Standardised proportionate mortality (SPMR) for asbestos-related causes was computed for the years 1986-2009. Sicilian Region constituted the reference population. The rationale for using SPMR rather than standardized mortality ratio (SMR) was a consequence for the lack of company files from which to obtain dates of start and termination of employment, and thus to compute person-years of observation, following the guidelines of the international scientific literature. RESULTS: Standardised incidence ratio (SIR) for mesothelioma in the overall cohort was 251 (4 observed, 0.02 expected). Proportionate mortality analysis among male subjects showed significant increases for pneumoconiosis (SPMR 80.1, 5 observed), lung cancer (SPMR 2.81, 10 observed) and pleural neoplasms (SPMR 19.4, 2 observed). CONCLUSIONS: Notwithstanding limitations in cohort reconstruction, for which the proportion of eligible subjects was 87.5% of those detected by the local committee, and the lack of information on duration of employment, it was possible to estimate a significant increase of the incidence of pleural mesothelioma with respect to Sicilian population. Also mortality from asbestos-related causes was in excess with respect to the regional reference population.

A Review of the Ecology and Management of Black Turpentine Beetle (Coleoptera: Curculionidae).

The black turpentine beetle, Dendroctonus terebrans Olivier is the largest pine-infesting bark beetle native to the southern and eastern United States. It generally reproduces in fresh stumps and bases of trees weakened or killed by other biotic or abiotic agents, although it can also infest and sometimes kills apparently healthy trees. Its numbers can build when large amounts of host material become available (typically through a disturbance), and black turpentine beetle-caused mortality at a local scale can become considerable. Here, we provide a complete review of the literature on this species, including its taxonomy, host, life history, chemical ecology, arthropod and microbial associates, and management options. We also provide original data on numbers of instars, acoustic signals, and pheromone chirality in this species. Our survey of the existing literature revealed that key biological characteristics of black turpentine beetles are known, but interactions with closely associated organisms, economic and ecological impacts, and improvements to monitoring and management practices have been only partially investigated.

Inducibility of Plant Secondary Metabolites in the Stem Predicts Genetic Variation in Resistance Against a Key Insect Herbivore in Maritime Pine.

Resistance to herbivores and pathogens is considered a key plant trait with strong adaptive value in trees, usually involving high concentrations of a diverse array of plant secondary metabolites (PSM). Intraspecific genetic variation and plasticity of PSM are widely known. However, their ecology and evolution are unclear, and even the implication of PSM as traits that provide direct effective resistance against herbivores is currently questioned. We used control and methyl jasmonate (MJ) induced clonal copies of genotypes within families from ten populations of the main distribution range of maritime pine to exhaustively characterize the constitutive and induced profile and concentration of PSM in the stem phloem, and to measure insect herbivory damage as a proxy of resistance. Then, we explored whether genetic variation in resistance to herbivory may be predicted by the constitutive concentration of PSM, and the role of its inducibility to predict the increase in resistance once the plant is induced. We found large and structured genetic variation among populations but not between families within populations in resistance to herbivory. The MJ-induction treatment strongly increased resistance to the weevil in the species, and the genetic variation in the inducibility of resistance was significantly structured among populations, with greater inducibility in the Atlantic populations. Genetic variation in resistance was largely explained by the multivariate concentration and profile of PSM at the genotypic level, rather than by bivariate correlations with individual PSM, after accounting for genetic relatedness among genotypes. While the constitutive concentration of the PSM blend did not show a clear pattern of resistance to herbivory, specific changes in the chemical profile and the increase in concentration of the PSM blend after MJ induction were related to increased resistance. To date, this is the first example of a comprehensive and rigorous approach in which inducibility of PSM in trees and its implication in resistance was analyzed excluding spurious associations due to genetic relatedness, often overlooked in intraspecific studies. Here we provide evidences that multivariate analyses of PSM, rather than bivariate correlations, provide more realistic information about the potentially causal relationships between PSM and resistance to herbivory in pine trees.

Advanced spectroscopy-based phenotyping offers a potential solution to the ash dieback epidemic.

Natural and urban forests worldwide are increasingly threatened by global change resulting from human-mediated factors, including invasions by lethal exotic pathogens. Ash dieback (ADB), incited by the alien invasive fungus Hymenoscyphus fraxineus, has caused large-scale population decline of European ash (Fraxinus excelsior) across Europe, and is threatening to functionally extirpate this tree species. Genetically controlled host resistance is a key element to ensure European ash survival and to restore this keystone species where it has been decimated. We know that a low proportion of the natural population of European ash expresses heritable, quantitative resistance that is stable across environments. To exploit this resource for breeding and restoration efforts, tools that allow for effective and efficient, rapid identification and deployment of superior genotypes are now sorely needed. Here we show that Fourier-transform infrared (FT-IR) spectroscopy of phenolic extracts from uninfected bark tissue, coupled with a model based on soft independent modelling of class analogy (SIMCA), can robustly discriminate between ADB-resistant and susceptible European ash. The model was validated with populations of European ash grown across six European countries. Our work demonstrates that this approach can efficiently advance the effort to save such fundamental forest resource in Europe and elsewhere.

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.

Testing phenotypic trade-offs in the chemical defence strategy of Scots pine under growth-limiting field conditions.

Plants protect themselves from pathogens and herbivores through fine-tuned resource allocation, including trade-offs among resource investments to support constitutive and inducible defences. However, empirical research, especially concerning conifers growing under natural conditions, is still scarce. We investigated the complexity of constitutive and induced defences in a natural Scots pine (Pinus sylvestris L.) stand under growth-limiting conditions typical of alpine environments. Phenotypic trade-offs at three hierarchical levels were tested by investigating the behaviour of phenolic compounds and terpenoids of outer bark and phloem. We tested resource-derived phenotypic correlations between (i) constitutive and inducible defences vs tree ring growth, (ii) different constitutive defence metabolites and (iii) constitutive concentration and inducible variation of individual metabolites. Tree ring growth was positively correlated only with constitutive concentration of total terpenoids, and no overall phenotypic trade-offs between different constitutive defensive metabolites were found. At the lowest hierarchical level tested, i.e., at the level of relationship between constitutive and inducible variation of individual metabolites, we found that different compounds displayed different behaviours; we identified five different defensive metabolite response types, based on direction and strength of the response, regardless of tree age and growth rate. Therefore, under growth-limiting field conditions, Scots pine appears to utilize varied and complex outer bark and phloem defence chemistry, in which only part of the constitutive specialized metabolism is influenced by tree growth, and individual components do not appear to be expressed in a mutually exclusive manner in either constitutive or inducible metabolism.

Nutritional and pathogenic fungi associated with the pine engraver beetle trigger comparable defenses in Scots pine.

Conifer bark beetles are often associated with fungal complexes whose components have different ecological roles. Some associated species are nutritionally obligate fungi, serving as nourishment to the larvae, whereas others are pathogenic blue-stain fungi known to be involved in the interaction with host defenses. In this study we characterized the local and systemic defense responses of Scots pine (Pinus sylvestris L.) against Ophiostoma brunneo-ciliatum Math. (a blue-stain pathogen) and Hyalorhinocladiella macrospora (Franke-Grosm.) Harr. (a nutritional fungus). These fungi are the principal associates of the pine engraver beetle, Ips acuminatus (Gyll.). Host responses were studied following inoculation with the fungi, singly and as a fungal complex, and by identifying and quantifying terpenoids, phenolic compounds and lignin. Although the length of the necrotic lesions differed between control (wound) and fungal treatments, only two compounds (pinosylvin monomethyl ether and (+)-α-pinene) were significantly affected by the presence of the fungi, indicating that Scots pine has a generic, rather than specific, induced response. The fact that both nutritional and blue-stain fungi triggered comparable induced defense responses suggests that even a non-pathogenic fungus may participate in exhausting host plant defenses, indirectly assisting in the beetle establishment process. Our findings contribute to the further development of current theory on the role of associated fungal complexes in bark beetle ecology.