Expansion of Ash Dieback towards the scattered Fraxinus excelsior range of the Italian peninsula

AbstractHymenoscyphus fraxineus, causal agent of Ash Dieback, has posed a threat to Fraxinus excelsior (common ash) in Europe since the 1990s. In south-western Europe, optimal climatic conditions for H. fraxineus become scattered and host density decreases, reducing disease spread rates. To date, the Ash Dieback agent has not been reported from southern and most of central Italy, where native F. excelsior is present as small fragmented populations. This study examines the expansion of Ash Dieback into central Italy, and it considers the consequences of further local spread with regards to the loss of F. excelsior genetic resource. Symptomatic F. excelsior were sampled from sixteen sites in northern and central Italy during 2020. Specimens were analyzed with a culturomics and a quantitative PCR approach. A bibliographic search of F. excelsior floristic reports was conducted for the creation of a detailed range map. The combined use of both techniques confirmed the presence of H. fraxineus in all the sites of central Italy where host plants were symptomatic. These new records represent the southern limit of the current known distribution of this pathogen in Italy, and together with Montenegro, in Europe. The characterization of the F. excelsior scattered range suggests that further spread of Ash Dieback across southern Italy is a realistic scenario. This presents a threat not just to the southern European proveniences of F. excelsior, but to the species as a whole, should Ash Dieback lead to the loss of warm climate adapted genetic material, which may become increasingly valuable under climate change

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

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

Figure S1. One of the most parsimonious trees from EF1-α sequence datasets. Data type: Alignment of genomic sequences. Explanation note: One of the most parsimonious trees from EF1-α gene sequence datasets is shown (length = 66, CI = 0.9999, RI = 0.9998, RC = 0.9988, HI = 0.9888). The MP and Bayesian posterior probability are indicated next to the branches. C. pseudotsugae and C. orientalis are used as outgroup. This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/).Figure S2. One of the most parsimonious trees from Bt1 sequence datasets. Data type: Alignment of genomic sequences. Explanation note: One of the most parsimonious trees from Bt1 sequence datasets is shown (CI = 0.9268, RI = 0.9840, RC = 0.936428, HI = 0.912039). The MP and Bayesian posterior probability are indicated next to the branches. C. pseudotsugae and C. orientalis are used as outgroup. This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/).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.Horizon 2020 Programme for Research & Innovationhttps://mycokeys.pensoft.netpm2020Forestry and Agricultural Biotechnology Institute (FABI

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.[Figure not available: see fulltext.]6n

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

Abstract 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