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.

Introduced species alter established trophic interactions and molecular analysis can resolve changes in community structure and associated foraging links. Joro spiders (Trichonephila clavata) were recently introduced to the United States and their range is rapidly expanding across the east coast. Here, we used DNA metabarcoding of fecal samples, prey remains from webs, and dissected guts to compare diet composition of female Joro spiders in the southeastern United States. We amplified DNA from three material types using arthropod-targeted COI primers and sequenced with IIlumina MiSeq. Prey remains from webs had the highest diversity, richness, as well as the highest proportion of prey reads relative to Joro spider reads. Recovery of prey reads from fecal samples and dissected gut content was low and both were overwhelmed by Joro spider DNA. Although fecal samples and gut content had high proportions of Joro spider reads, fecal samples had higher prey diversity and richness. Moreover, we detected prey DNA from fecal samples several days after capture from the field, which reveals initial gut retention time estimates for fecal samples collected from web-building spiders. Combined, our results offer a first glimpse at the complexity of trophic associations for an introduced web-building spider and identify a viable material, prey remains from webs, as a source of prey DNA for estimates of biodiversity associated with web-building spiders.

The pine pitch canker pathogen Fusarium circinatum is endemic in the Southeast 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 ten 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.