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.
Needle blights are serious needle fungal diseases affecting pines both in natural and productive forests. Among needle blight agents, the ascomycetes Lecanosticta acicola, Dothistroma pini and D. septosporum are of particular concern. These pathogens need specific, fast and accurate diagnostics since they are regulated species in many countries and may require differential management measures. Due to the similarities in fungal morphology and the symptoms they elicit, these species are hard to distinguish using morphological characteristics. The symptoms can also be confused with those caused by insects or abiotic agents. DNA-based detection is therefore recommended. However, the specific PCR assays that have been produced to date for the differential diagnosis of these pathogens can be applied only in a well-furnished laboratory and the procedure takes a relatively long execution time. Surveillance and forest protection would benefit from a faster diagnostic method, such as a loop-mediated isothermal amplification (LAMP) assay, which requires less sophisticated equipment and can also be deployed directly on-site using portable devices. LAMP assays for the rapid and early detection of L. acicola, D. pini and D. septosporum were developed in this work. Species-specific LAMP primers and fluorescent assimilating probes were designed for each assay, targeting the beta tubulin (β-tub2) gene for the two Dothistroma species and the elongation factor (EF-1α) region for L. acicola. Each reaction detected its respective pathogen rapidly and with high specificity and sensitivity in DNA extracts from both pure fungal cultures and directly from infected pine needles. These qualities and the compatibility with inexpensive portable instrumentation position these LAMP assays as an effective method for routine phytosanitary control of plant material in real time, and they could profitably assist the management of L. acicola, D. pini and D. septosporum.
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.
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