Search Results (13)

The quarantine pathogen Phytophthora ramorum has a high potential for dispersal due to its airborne inoculum, its wide range of hosts, and its ability to spread through the trade of nursery plants. For these reasons, it represents a serious threat to ornamental nursery production and, consequently, to urban, natural and semi-natural ecosystems. This oomycete pathogen (EU1 lineage, A1 mating type) has been detected on Viburnum tinus in a commercial nursery located in the Pistoia nursery district (PND) (Tuscany, central Italy), one of the main nursery areas for the production of ornamentals in Europe. Artificial inoculations were carried out in the laboratory under controlled conditions, following a standard detached-leaf assay protocol, on leaves of 16 ornamental shrub species commonly marketed by the PND. Disease severity was assessed, and susceptibility categories (high, moderate, low, and non-susceptible) were defined based on data collected at 7 and 14 days post-inoculation and validated through statistical analysis. Inoculated species exhibited variable levels of disease severity. The results confirmed the pathogen’s high virulence on Viburnum tinus and Rhododendron hybrid ‘Madame Masson’. The following species were also found to be highly susceptible: Ilex aquifolium, Loropetalum chinense, Magnolia stellata, Osmanthus fragrans, and Trachelospermum jasminoides. Camellia japonica, Nerium oleander, Osmanthus heterophyllus, Prunus laurocerasus, and Rhododendron obtusum showed moderate susceptibility. Arbutus unedo, Laurus nobilis, Photinia fraseri and Syringa vulgaris exhibited low susceptibility. At the end of the trial, no infected species fell into the non-susceptible categories. The oomycete proved particularly aggressive on Ilex aquifolium, the most susceptible host among those tested. This high susceptibility is a new finding that could have significant epidemiological implications. Our findings emphasize the need for rigorous phytosanitary surveillance in nursery systems, based on constant monitoring and the adoption of high-throughput diagnostic protocols, in order to implement effective and rapid control measures. Full article

International timber trade has accelerated the global spread of the invasive red-haired pine bark beetle H. ligniperda, posing persistent challenges to phytosanitary inspection and border biosecurity. Rapid isothermal amplification assays are increasingly deployed in frontline quarantine settings to support timely regulatory decisions. However, their performance under the heterogeneous biological backgrounds typical of traded timber remains insufficiently evaluated, particularly with respect to the practical implications of low-level false-positive signals. We re-evaluated a previously reported isothermal assay for H. ligniperda using conditions that simulate timber transport and routine customs workflows. Fifty non-target arthropod species (predominantly insects), selected from quarantine interception records, were included to represent taxa likely to co-occur in operational contexts. Material from Lema decempunctata consistently generated weak but reproducible amplification signals across replicates. Sanger sequencing excluded contamination, confirming low-level non-target amplification in complex biological matrices. Although the signals were faint, ambiguous results in quarantine settings may trigger shipment detention, confirmatory laboratory testing, or temporary trade restrictions, thereby increasing inspection workload, delaying clearance, and generating avoidable compliance costs. These findings indicate that trade-mediated species assemblages can compromise assay performance beyond expectations derived from conventional taxonomy-based specificity testing. To reduce interpretive uncertainty and associated regulatory burden, we propose a tiered diagnostic workflow combining rapid on-site isothermal screening with specificity-oriented SYBR Green qPCR confirmation. This strategy enhances diagnostic reliability while preserving operational efficiency in applied biosecurity surveillance. Full article

Ips typographus is one of the most destructive bark beetles affecting conifer forests in Europe, where climatic disturbances and the movement of infested wood can rapidly shift populations from endemic levels to severe outbreaks. Early detection through border inspections and forest monitoring is essential to prevent new introductions and limit the spread of established populations. Here, we developed and validated a probe-based TaqMan qPCR assay, targeting the mitochondrial COI barcode region, for the rapid and species-specific detection of I. typographus from both insect material and environmental DNA recovered from frass and exit-hole wood chips. Validation followed EPPO PM7/98(5) guidelines, assessing analytical specificity, sensitivity, repeatability, reproducibility, and inter-laboratory transferability. High analytical specificity was demonstrated against a broad panel of non-target species, and reliable amplification was obtained across different tested matrices. The method showed strong analytical sensitivity, with limits of detection of 0.32 pg/µL for adult-derived DNA and 1.6 pg/µL for artificial frass. Repeatability, reproducibility, and inter-laboratory blind testing further confirmed the diagnostic reliability of the method. This validated qPCR assay provides a rapid and sensitive molecular tool for the early detection of I. typographus, supporting border inspection and phytosanitary diagnostic laboratories in forest biosecurity activities. Full article

Colletotrichum species are major plant pathogens and emerging opportunistic human pathogens. Due to their vast genetic diversity, existing diagnostic tools often suffer from narrow specificity or labor-intensive workflows. In this study, we developed a rapid, universal, and highly sensitive genus-specific real-time PCR assay utilizing a TaqMan MGB probe targeting the conserved 28S rDNA region. The assay demonstrated exceptional specificity, with no cross-reactivity against closely related fungal taxa or common co-occurring pathogens. The method exhibited high sensitivity, achieving a limit of detection (LOD) of 680 fg of genomic DNA. Furthermore, the assay was successfully validated using simulated environmental samples, where it accurately identified Colletotrichum within complex fungal communities. By providing a robust platform for genus-level screening, this methodology significantly enhances the efficiency of phytosanitary inspections and clinical diagnostics, facilitating timely biosecurity interventions and therapeutic decisions. Full article

Woody tissue diseases of avocado (Persea americana Mill. var. Hass) pose a major phytosanitary threat due to their chronic progression, late symptom expression, and severe impact on tree stability and productivity. Although white rot has traditionally been attributed to saprobic basidiomycetes, increasing evidence suggests corticioid fungi may act as facultative pathogens in agricultural systems. This study examined corticioid basidiomycetes associated with white rot in stems and branches of avocado in Michoacán, Mexico. Field surveys revealed consistent symptoms of structural weakening, branch dieback, and wood decay. Fungal isolates obtained from symptomatic tissues and sporomes were characterized morphologically and identified through ITS-based phylogenetic analyses. Representative isolates of Grammothele spp. and Dentocorticium portoricense were evaluated in pathogenicity assays under controlled conditions. All isolates reproduced field symptoms, confirming pathogenicity, though aggressiveness varied. D. portoricense exhibited the highest incidence, severity, and AUDPC values, indicating greater virulence, while Grammothele isolates showed slower, moderate progression. Phylogenetic analyses provided robust support for D. portoricense, whereas Grammothele was resolved at genus level. Integration of field, pathogenicity, and molecular data demonstrates corticioid fungi are not merely secondary saprotrophs but relevant pathogens in avocado white rot. These findings highlight the need to include corticioid fungi in diagnostic, monitoring, and management strategies for trunk and branch diseases. Full article

Sweet cherry (Prunus avium L.) cultivation in China covers an estimated area of 25,600 hectares, representing more than one-third of the global total. Viral diseases present a serious challenge to cherry production worldwide; however, the phytosanitary status of sweet cherry in China has remained poorly understood. In this study, 191 sweet cherry samples were collected from major growing regions and screened using RT-PCR combined with DNA sequencing for the presence of 14 viruses previously reported in China. Results revealed that 80.1% of the tested samples were infected with at least one virus, with mixed infections detected in 51.3% of the samples. Prevalent viruses included cherry virus A (CVA, 53.4%), prunus necrotic ringspot virus (PNRSV, 35.1%), cherry green ring mottle virus (CGRMV, 32.5%), plum bark necrosis stem pitting-associated virus (PBNSPaV, 31.4%), and prune dwarf virus (PDV, 10.5%). Cherry necrotic rusty mottle virus (CNRMV) was found at a very low frequency (0.5%), and the remaining eight viruses were not detected in any sample. Based on these findings, we developed multiplex RT-PCR assays for simultaneous detection of CVA, PNRSV, CGRMV, PBNSPaV, and PDV. Several dual and triplex RT-PCR systems were successfully established, including combinations such as PBNSPaV/PNRSV, CVA/PDV, CVA/CGRMV, PBNSPaV/PDV/CGRMV, and PBNSPaV/PNRSV/PDV. This study identifies CVA, PNRSV, CGRMV, PBNSPaV, and PDV as the prevalent viruses in the investigated Chinese sweet cherry orchards. Accordingly, multiplex RT-PCR assays were developed for their simultaneous detection. Our work advances the understanding of sweet cherry viral diseases in China and provides a valuable complementary tool for the existing diagnostic toolkit. Full article

Setophoma terrestris is an important phytopathogenic fungus listed by China as a harmful fungus subject to phytosanitary import control. This pathogen is a threat to a wide range of plants, particularly as the causal agent of onion pink root rot, one of the most severe diseases of onions. In order to provide rapid identification and early warning of S. terrestris and prevent its spread, we have developed a rapid, accurate, and visually intuitive diagnostic assay for this pathogen, by utilizing recombinase polymerase amplification (RPA), coupled with CRISPR/Cas12a cleavage and fluorescence-based detection systems or paper-based lateral flow strips. The developed RPA-CRISPR/Cas12a assay exhibited remarkable specificity for the detection of S. terrestris. Moreover, this protocol can detect the pathogen at a sensitivity level of 0.01 pg/μL, which significantly outperforms the 1 pg/μL sensitivity achieved by the existing qPCR-based detection method. The entire diagnostic procedure, including DNA extraction, the RPA reaction, the Cas12a cleavage, and the result interpretation, can be accomplished in 40 min. Furthermore, the successful application of the assay in infected plant samples highlighted its potential for rapid and accurate pathogen detection in agricultural settings. In summary, this RPA-CRISPR/Cas12a diagnostic method offers a potentially valuable technological solution for quarantine and disease management. Full article

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. Full article

Rapid and safe access to new plant genetic stocks is crucial for primary plant industries to remain profitable, sustainable, and internationally competitive. Imported plant species may spend several years in Post Entry Quarantine (PEQ) facilities, undergoing pathogen testing which can impact the ability of plant industries to quickly adapt to new global market opportunities by accessing new varieties. Advances in high throughput sequencing (HTS) technologies provide new opportunities for a broad range of fields, including phytosanitary diagnostics. In this study, we compare the performance of two HTS methods (RNA-Seq and sRNA-Seq) with that of existing PEQ molecular assays in detecting and identifying viruses and viroids from various plant commodities. To analyze the data, we tested several bioinformatics tools which rely on different approaches, including direct-read, de novo, and reference-guided assembly. We implemented VirusReport, a new portable, scalable, and reproducible nextflow pipeline that analyses sRNA datasets to detect and identify viruses and viroids. We raise awareness of the need to evaluate cross-sample contamination when analyzing HTS data routinely and of using methods to mitigate index cross-talk. Overall, our results suggest that sRNA analyzed using VirReport provides opportunities to improve quarantine testing at PEQ by detecting all regulated exotic viruses from imported plants in a single assay. Full article

The walnut twig beetle Pityophthorus juglandis is a phloem-boring bark beetle responsible, in association with the ascomycete Geosmithia morbida, for the Thousand Cankers Disease (TCD) of walnut trees. The recent finding of TCD in Europe prompted the development of effective diagnostic protocols for the early detection of members of this insect/fungus complex. Here we report the development of a highly efficient, low-cost, and rapid method for detecting the beetle, or even just its biological traces, from environmental samples: the loop-mediated isothermal amplification (LAMP) assay. The method, designed on the 28S ribosomal RNA gene, showed high specificity and sensitivity, with no cross reactivity to other bark beetles and wood-boring insects. The test was successful even with very small amounts of the target insect’s nucleic acid, with limit values of 0.64 pg/µL and 3.2 pg/µL for WTB adults and frass, respectively. A comparison of the method (both in real time and visual) with conventional PCR did not display significant differences in terms of LoD. This LAMP protocol will enable quick, low-cost, and early detection of P. juglandis in areas with new infestations and for phytosanitary inspections at vulnerable sites (e.g., seaports, airports, loading stations, storage facilities, and wood processing companies). Full article

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. Full article

Xanthomonas gardneri is one of the causal agents of bacterial spot (BS), an economically important bacterial disease of tomato and pepper. Field-deployable and portable loop-mediated isothermal amplification (LAMP)-based instruments provide rapid and sensitive detection of plant pathogens. In order to rapidly and accurately identify and differentiate X. gardneri from other BS-causing Xanthomonas spp., we optimized a new real-time monitoring LAMP-based method targeting the X. gardneri-specific hrpB gene. Specificity and sensitivity of real-time and colorimetric LAMP assays were tested on the complex of bacterial strains pathogenic to tomato and pepper and on plants infected by the pathogen. The assay detection limit was 1 pg/μL of genomic DNA with an assay duration of only 30 min. The use of portable and handheld instruments allows for fast analysis, reducing the diagnosis time, and can contribute to proper disease management and control of X. gardneri. Due to the high efficiency of this method, we suggest its use as a standard diagnostic tool during phytosanitary controls. Full article

Venturia inaequalis, an agent of apple scab, is the most important pathogen of Malus x domestica. Control measures against this pathogen rely on intensive phytosanitary programs based on predictive models to identify the meteorological conditions conducive to the primary infection. The detection of the pathogen in field, both in naturally infected symptomatic and asymptomatic leaves, is desirable. Loop-mediated isothermal amplification (LAMP) assays are profitable molecular diagnostic tools for the direct detection of pathogens in field. A LAMP assay for V. inaequalis has been designed on the elongation factor 1-alpha sequence. The validation of the LAMP assay was carried out following the international EPPO standard PM 7/98 in terms of specificity, sensitivity, repeatability and reproducibility. Specificity testing was performed using target and non-target species, such as phylogenetically related Venturia species and other pathogens commonly found in apple, resulting in positive amplification only for the target with a time to positive ranging from 20 to 30 min. Sensitivity testing was performed with serial dilutions of DNA of the target and by artificial inoculation of young apple leaves. The reliability of the LAMP assay as an early-detection tool and its user-friendly application make it suitable for the diagnosis of apple scab in the field. Full article