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.

Complexities underlying the breeding and deployment of Dutch elm disease resistant elms.

Dutch elm disease (DED) is a vascular wilt disease caused by the pathogens Ophiostoma ulmi and Ophiostoma novo-ulmi with multiple ecological phases including pathogenic (xylem), saprotrophic (bark) and vector (beetle flight and beetle feeding wound) phases. Due to the two DED pandemics during the twentieth century the use of elms in landscape and forest restoration has declined significantly. However new initiatives for elm breeding and restoration are now underway in Europe and North America. Here we discuss complexities in the DED 'system' that can lead to unintended consequences during elm breeding and some of the wider options for obtaining durability or 'field resistance' in released material, including (1) the phenotypic plasticity of disease levels in resistant cultivars infected by O. novo-ulmi; (2) shortcomings in test methods when selecting for resistance; (3) the implications of rapid evolutionary changes in current O. novo-ulmi populations for the choice of pathogen inoculum when screening; (4) the possibility of using active resistance to the pathogen in the beetle feeding wound, and low attractiveness of elm cultivars to feeding beetles, in addition to resistance in the xylem; (5) the risk that genes from susceptible and exotic elms be introgressed into resistant cultivars; (6) risks posed by unintentional changes in the host microbiome; and (7) the biosecurity risks posed by resistant elm deployment. In addition, attention needs to be paid to the disease pressures within which resistant elms will be released. In the future, biotechnology may further enhance our understanding of the various resistance processes in elms and our potential to deploy trees with highly durable resistance in elm restoration. Hopefully the different elm resistance processes will prove to be largely under durable, additive, multigenic control. Elm breeding programmes cannot afford to get into the host-pathogen arms races that characterise some agricultural host-pathogen systems.

Real-Time Portable LAMP Assay for a Rapid Detection of Xylella fastidiosa In-Field.

Early diagnosis is part of a decision-making process which in the case of plant diseases may prevent the spread of invasive plant pathogens and assist in their eradication. Significant advantages could be obtained from moving testing technology closer to the sampling site, thereby reducing the detection time. This chapter describes a portable real-time LAMP assay for a specific detection of Xylella fastidiosa in-field. The LAMP assay, including DNA extraction, allows a complete and specific in-field analysis in just 40 minutes, enabling the detection of pathogen DNA in host tissues.

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.

Invasive Alien Plant Pathogens: The Need of New Detection Methods.

Invasive alien species are a major threat to natural and anthropogenic ecosystems and to the economy. Many invasive fungal species have severely impacted ecology and human lifestyle in the past. Most of them express a pathogenic lifestyle following introduction into a new region and hosts. They are usually cryptic during the introduction phase and hard to be identified, classified, and monitored.The increasing number of new alien pests coincide with the rapid increase in the volume and diversity of intercontinental trade in plants for planting, underlying the need to reduce the risk of their introduction with the development of molecular-based, inexpensive, rapid, accurate, and reliable methods that can identify and intercept plant pathogens even before symptoms occur in the new environment of diffusion. Applicative aerobiology, for instance, represents a challenging research line for the implementation of pest detection protocols during the early stage of fungal introduction, being capable to target aerial dispersed propagules.In addition to this, new metabarcoding protocols based on an innovative multigene approach, although not yet tested on fungi, are able to provide an output with very high taxonomic resolution and are likely to be considered in the next-future biosurveillance of invasive fungal pathogens.

Loop-Mediated Isothermal Amplification (LAMP) and SYBR Green qPCR for Fast and Reliable Detection of Geosmithia morbida (Kolarik) in Infected Walnut.

Walnut species (Juglans spp.) are multipurpose trees, widely employed in plantation forestry for high-quality timber and nut production, as well as in urban greening as ornamental plants. These species are currently threatened by the thousand cankers disease (TCD) complex, an insect-fungus association which involves the ascomycete Geosmithia morbida (GM) and its vector, the bark beetle Pityophthorus juglandis. While TCD has been studied extensively where it originated in North America, little research has been carried out in Europe, where it was more recently introduced. A key step in research to cope with this new phytosanitary emergency is the development of effective molecular detection tools. In this work, we report two accurate molecular methods for the diagnosis of GM, based on LAMP (real-time and visual) and SYBR Green qPCR, which are complimentary to and integrated with similar recently developed assays. Our protocols detected GM DNA from pure mycelium and from infected woody tissue with high accuracy, sensitivity, and specificity, without cross-reactivity to a large panel of taxonomically related species. The precision and robustness of our tests guarantee high diagnostic standards and could be used to support field diagnostic end-users in TCD monitoring and surveillance campaigns.

Novel Insights Into Refugia at the Southern Margin of the Distribution Range of the Endangered Species Ulmus laevis.

Riparian ecosystems, in long-time developed regions, are among the most heavily impacted by human activities; therefore, the distribution of tree riparian species, such as Ulmus laevis, is highly affected. This phenomenon is particularly relevant at the margins of the natural habitat of the species, where populations are small and rare. In these cases, it is difficult to distinguish between relics or introductions, but it is relevant for the restoration of natural habitats and conservation strategies. The aim of this study was to study the phylogeography of the southern distribution of the species. We sequenced the entire chloroplast (cp) genomes of 54 individuals from five sampled populations across different European regions to highlight polymorphisms and analyze their distribution. Thirty-two haplotypes were identified. All the sampled populations showed private haplotypes that can be considered an indicator of long-term residency, given the low mutation rate of organellar DNA. The network of all haplotypes showed a star-like topology, and Serbian haplotypes were present in all branches. The Balkan population showed the highest level of nucleotide and genetic diversity. Low genetic differentiation between populations was observed but we found a significant differentiation among Serbia vs. other provenances. Our estimates of divergent time of U. laevis samples highlight the early split of above all Serbian individuals from other populations, emphasizing the reservoir role of white elm genetic diversity of Serbian population.

Rapid diagnostics for Gnomoniopsis smithogilvyi (syn. Gnomoniopsis castaneae) in chestnut nuts: new challenges by using LAMP and real-time PCR methods.

Nuts of the sweet chestnut (Castanea sativa) are a widely appreciated traditional food in Europe. In recent years producers and consumers reported a drop of nut quality due to the presence of rot diseases caused by Gnomoniopsis smithogilvyi. Early detection of this pathogen is fundamental to the economic viability of the chestnut industry. In the present study, we developed three molecular methods based on real-time portable LAMP, visual LAMP and qPCR assays for G. smithogilvyi. The molecular assays were specific for G. smithogilvyi and did not amplify the other 11 Gnomoniopsis species and 11 other fungal species commonly associated with chestnuts. The detection limit of both the qPCR and real-time portable LAMP (P-LAMP) assays was 0.128 pg/µL, while the visual LAMP (V-LAMP) assay enabled the detection up to 0.64 pg/µL. By using these newly developed molecular tools, the pathogen was detected in symptomatic and asymptomatic nuts, but not in leaves. The reliability of these molecular methods, including the P-LAMP assay, was particularly useful in detecting G. smithogilvyi of harvested nuts in field, even in the absence of rot symptoms.

Optimising the assignment of swabs and reagent for PCR testing during a viral epidemic.

Early large-scale swab testing is a fundamental tool for health authorities to assess the prevalence of a virus and enact appropriate mitigation measures during an epidemic. The COVID-19 pandemic has shown that the availability of chemical reagent required to carry out the tests is often a bottleneck in increasing a country's testing capacity. Further, demand is unevenly spread between more affected regions (which require more tests they can perform) and less affected ones (which have spare capacity). These issues hint at the opportunity of increasing test capacity via the optimal allocation of swabs and reagent to laboratories. We prove that this is the case, proposing an Integer Programming formulation to maximise the number of tests a country can perform and validating our approach on both real-life data from Italy and synthetic instances. Our results show that increased inter-regional collaboration and a steadier supply of reagent (i.e., coming from local production sites rather than international shipments) can dramatically increase testing capacity. Accordingly, we propose short-term and long-term recommendations for policy makers and health authorities.

Data for a meta-analysis of the adaptive layer in adaptive large neighborhood search.

Meta-analysis, a systematic statistical examination that combines the results of several independent studies, has the potential of obtaining problem- and implementation-independent knowledge and understanding of metaheuristic algorithms, but has not yet been applied in the domain of operations research. To illustrate the procedure, we carried out a meta-analysis of the adaptive layer in adaptive large neighborhood search (ALNS). Although ALNS has been widely used to solve a broad range of problems, it has not yet been established whether or not adaptiveness actually contributes to the performance of an ALNS algorithm. A total of 134 studies were identified through Google Scholar or personal e-mail correspondence with researchers in the domain, 63 of which fit a set of predefined eligibility criteria. The results for 25 different implementations of ALNS solving a variety of problems were collected and analyzed using a random effects model. This dataset contains a detailed comparison of ALNS with the non-adaptive variant per study and per instance, together with the meta-analysis summary results. The data enable to replicate the analysis, to evaluate the algorithms using other metrics, to revisit the importance of ALNS adaptive layer if results from more studies become available, or to simply consult the ready-to-use formulas in the summary file to carry out a meta-analysis of any research question. The individual studies, the meta-analysis and its results are described and interpreted in detail in Renata Turkes, Kenneth Sörensen, Lars Magnus Hvattum, Meta-analysis of Metaheuristics: Quantifying the Effect of Adaptiveness in Adaptive Large Neighborhood Search, in the European Journal of Operational Research.

Caliciopsis moriondi, a new species for a fungus long confused with the pine pathogen C. pinea.

The genus Caliciopsis (Eurotiomycetes, Coryneliales) includes saprobic and plant pathogenic species. Caliciopsis canker is caused by Caliciopsis pinea Peck, a species first reported in the 19th century in North America. In recent years, increasing numbers of outbreaks of Caliciopsis canker have been reported on different Pinus spp. in the eastern USA. In Europe, the disease has only occasionally been reported causing cankers, mostly on Pinus radiata in stressed plantations. The aim of this study was to clarify the taxonomy of Caliciopsis specimens collected from infected Pinus spp. in Europe and North America using an integrative approach, combining morphology and phylogenetic analyses of three loci. The pathogenicity of the fungus was also considered. Two distinct groups were evident, based on morphology and multilocus phylogenetic analyses. These represent the known pathogen Caliciopsis pinea that occurs in North America and a morphologically similar, but phylogenetically distinct, species described here as Caliciopsis moriondi sp. nov., found in Europe and at least one location in eastern North America. Caliciopsis moriondi differs from C. pinea in various morphological features including the length of the ascomata, as well as their distribution on the stromata.

Real-time loop-mediated isothermal amplification assay for rapid detection of Fusarium circinatum.

Fusarium circinatum is the causal agent of pitch canker, a lethal disease of pine and other conifers. Since F. circinatum is a quarantine organism, its timely detection could efficiently prevent its introduction into new areas or facilitate spread management in already infected sites. In this study, we developed a sequence-specific probe loop-mediated isothermal amplification (LAMP) assay for F. circinatum using a field-deployable portable instrument. The assay was able to recognize the pathogen in host tissues in just 30 min, and the sensitivity of the assay made it possible to detect even small amounts of F. circinatum DNA (as low as 0.5 pg/µl). The high efficiency of this method suggests its use as a standard diagnostic tool during phytosanitary controls.

Fast and reliable molecular methods to detect fungal pathogens in woody plants.

Plant diseases caused by pathogenic microorganisms represent a serious threat to plant productivity, food security, and natural ecosystems. An effective framework for early warning and rapid response is a crucial element to mitigate or prevent the impacts of biological invasions of plant pathogens. For these reasons, detection tools play an important role in monitoring plant health, surveillance, and quantitative pathogen risk assessment, thus improving best practices to mitigate and prevent microbial threats. The need to reduce the time of diagnosis has prompted plant pathologists to move towards more sensitive and rapid methods such as molecular techniques. Considering prevention to be the best strategy to protect plants from diseases, this review focuses on fast and reliable molecular methods to detect the presence of woody plant pathogens at early stage of disease development before symptoms occur in the host. A harmonized pool of novel technical, methodological, and conceptual solutions is needed to prevent entry and establishment of new diseases in a country and mitigate the impact of both invasive and indigenous organisms to agricultural and forest ecosystem biodiversity and productivity.

Early Detection of Fungal Plant Pathogens by Real-Time Quantitative PCR: The Case of Diplodia sapinea on Pine.

This chapter reports the use of real-time quantitative PCR to detect Diplodia sapinea, a fungal plant pathogen that causes shoot tip dieback and tree mortality on pine trees. This molecular approach represents a reliable and sensitive tool to detect fungal pathogens in DNA extracted from plant tissues and its use can be also recommended to study fungal behavior in host tissues by quantifying fungal growth in the latent phase, when symptoms in the host are not present yet.

Life-threatening Misdiagnosis of Bulbar Type Onset Myasthenia Gravis as a Subacute Thyroiditis.

Myasthenia gravis (MG) is an autoimmune disease that can mimic a variety of symptoms leading to a delay in diagnosis and treatment. We report the case of 48 year-old woman, in whom initial presentation of MG with predominance of bulbar symptoms was mistaken for thyroid disease complications. Subsequently, correct diagnosis and optimal management resulted on significant improvement of her functional state. We discuss the importance of considering MG as one of the potential differential diagnoses among cases of new onset or recurrent unexplained bulbar symptoms.

Complex Insect-Pathogen Interactions in Tree Pandemics.

Tree pandemics are a major cause of economic and ecological loss in forest and urban ecosystems. They often depend on the introduction of a non-native pathogen, which is occupying the niche of a native, non-aggressive organism. Complex interactions with native insects carrying fungi and nematodes can be established based on the proximity of the aggressive pathogenic agents. Here we review three major pandemics of forest and urban trees in temperate ecosystems at world scale, i.e., the Dutch elm disease, the cypress canker, and the pine wilt disease. For each system, the relationships between aggressive and non-aggressive fungi and nematodes with the native insect vectors are presented. Hidden players such as insects, microorganisms or plants, which may have the role of facilitating or contrasting the performance of the agents, are also considered. Results suggest that pandemics rely on the introduction of a non-native pathogen that exploits well-developed interactions between native non-aggressive organisms and insects associated with trees. The success of the invaders depends on the morpho-physiological proximity of the players and on the mutual benefits resulting from the associations. Deciphering such interactions in native systems may help to predict the outcome of the introduction of new pathogens and the development of new tree pandemics.

Real-time loop-mediated isothermal amplification: an early-warning tool for quarantine plant pathogen detection.

An effective framework for early warning and rapid response is a crucial element to prevent or mitigate the impact of biological invasions of plant pathogens, especially at ports of entry. Molecular detection of pathogens by using PCR-based methods usually requires a well-equipped laboratory. Rapid detection tools that can be applied as point-of-care diagnostics are highly desirable, especially to intercept quarantine plant pathogens such as Xylella fastidiosa, Ceratocystis platani and Phytophthora ramorum, three of the most devastating pathogens of trees and ornamental plants in Europe and North America. To this aim, in this study we developed three different loop mediated isothermal amplification (LAMP) assays able to detect each target pathogen both in DNA extracted from axenic culture and in infected plant tissues. By using the portable instrument Genie® II, the LAMP assay was able to recognize X. fastidiosa, C. platani and P. ramorum DNA within 30 min of isothermal amplification reaction, with high levels of specificity and sensitivity (up to 0.02 pg µL-1 of DNA). These new LAMP-based tools, allowing an on-site rapid detection of pathogens, are especially suited for being used at ports of entry, but they can be also profitably used to monitor and prevent the possible spread of invasive pathogens in natural ecosystems.

Comparative transcriptional and metabolic responses of Pinus pinea to a native and a non-native Heterobasidion species.

Heterobasidion irregulare is a causal agent of root and butt-rot disease in conifers, and is native to North America. In 1944 it was introduced in central Italy in a Pinus pinea stand, where it shares the same niche with the native species Heterobasidion annosum. The introduction of a non-native pathogen may have significant negative effects on a naïve host tree and the ecosystem in which it resides, requiring a better understanding of the system. We compared the spatio-temporal phenotypic, transcriptional and metabolic host responses to inoculation with the two Heterobasidion species in a large experiment with P. pinea seedlings. Differences in length of lesions at the inoculation site (IS), expression of host genes involved in lignin pathway and in cell rescue and defence, and analysis of terpenes at both IS and 12 cm above the IS (distal site, DS), were assessed at 3, 14 and 35 days post inoculation (dpi). Results clearly showed that both species elicit similar physiological and biochemical responses in P. pinea seedlings. The analysis of host transcripts and total terpenes showed differences between inoculation sites and between pathogen and mock inoculated plants. Both pathogen and mock inoculations induced antimicrobial peptide and phenylalanine ammonia-lyase overexpression at IS beginning at 3 dpi; while at DS all the analysed genes, except for peroxidase, were overexpressed at 14 dpi. A significantly higher accumulation of terpenoids was observed at 14 dpi at IS, and at 35 dpi at DS. The terpene blend at IS showed significant variation among treatments and sampling times, while no significant differences were ever observed in DS tissues. Based on our results, H. irregulare does not seem to have competitive advantages over the native species H. annosum in terms of pathogenicity towards P. pinea trees; this may explain why the non-native species has not widely spread over the 73 years since its putative year of introduction into central Italy.

Duplex real-time PCR assay for the simultaneous detection of Caliciopsis pinea and Fusarium circinatum in pine samples.

Fusarium circinatum and Caliciopsis pinea are the causal agents of Pitch canker and Caliciopsis canker, respectively. These diseases affect pines and other conifers both in Europe and North America. The two pathogens cause similar bleeding cankers, especially at the early stage of colonization. Symptoms closely resembling those due to F. circinatum can be instead associated with C. pinea. Since F. circinatum is a quarantine organism, subjected to provisional emergency measures, its report immediately causes serious economic implications, while C. pinea, even if now emerging, is not regulated in the EU nor in the USA. For this reason, a reliable and accurate diagnostic tool able to distinguish between the two organisms was considered a priority. In this study, we developed and standardized a duplex real-time PCR assay allowing the simultaneous recognition of C. pinea and F. circinatum DNA in pine tissue in a reasonably short time and for amounts as small as 0.06 pg/µl. The molecular assay is, therefore, able to detect the infection even before symptoms have fully developed. The test was challenged with a very large set of strains (110 different isolates) collected in different regions of the world and host trees, and gave reliable results. The high efficiency of this method suggests its use as a standard diagnostic tool during phytosanitary controls. In addition, the duplex real-time PCR assay presented here is the first DNA-based method designed to detect C. pinea, which is becoming an increasing threat to pine stands both in North America and in Europe.