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.

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.

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: 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.

Effects of a three-year exposure to ambient ozone on biomass allocation in poplar using ethylenediurea.

We examined the effect of ambient ozone on visible foliar injury, growth and biomass in field-grown poplar cuttings of an Oxford clone sensitive to ozone (Populus maximoviczii Henry × berolinensis Dippel) irrigated with ethylenediurea (EDU) or water for three years. EDU is used as an ozone protectant for plants. Protective effects of EDU on ozone visible injury were found. As a result, poplar trees grown under EDU treatment increased leaves, lateral branches and root density in the third year, although no significant enhancement of stem height and diameter was found. Ambient ozone (AOT40, 24.6 ppm h; diurnal hourly average, 40.3 ppb) may finally reduce carbon gain by reducing the number of branches, and thus sites for leaf formation, in ozone-sensitive poplar trees under not-limiting conditions.