Domenico Rizzo

Arbutus unedo L. is an ecologically and economically important Mediterranean evergreen shrub or small tree, increasingly used in restoration and ornamental plantings. Despite its significance, its diseases and associated pathogens remain poorly studied. Phytophthora cinnamomi, one of the world’s most destructive plant pathogens, represents an emerging threat to Mediterranean ecosystems but has not previously been reported on A. unedo in Italy. In recent years, dieback has been observed in natural populations near Monticiano, Tuscany, while similar symptoms, associated with extensive mortality, have occurred in commercial nurseries in the Pistoia district. Woodland plants showed reduced vigour and hook-shaped shoot tips, whereas nursery plants exhibited root rot, collar necrosis, leaf chlorosis, crown dieback, wilting, and death. A total of 184 oomycete isolates were consistently recovered from symptomatic roots and collars and from rhizosphere soil. Morphological characterization and multilocus molecular analyses identified the isolates as P. cinnamomi. The pathogenicity of the oomycete was demonstrated by artificially inoculating healthy A. unedo seedlings, which displayed symptoms very similar to those observed under field conditions. Both representative isolates caused severe disease, resulting in 100% mortality of inoculated plants within 30 days, whereas no mortality occurred in control plants. Re-isolation of the pathogen from inoculated tissues fulfilled Koch’s postulates. These findings constitute the first report of P. cinnamomi on A. unedo in Italy and raise concern about its possible spread in natural ecosystems and through nursery production and trade.

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.

Species of the genus Phytophthora are among the most detrimental plant pathogens globally, representing a significant threat to global agriculture, horticulture, and forestry. These zoosporic oomycetes have historically caused devastating outbreaks, including, just to mention a few, late blight of potato in Ireland; jarrah dieback of eucalyptus in Western Australia; ink disease of chestnut in Europe; sudden oak death and sudden larch death of coast live oak and tanoak in the Western US, and of Japanese larch in the UK. The environmental and ecological impacts of the diseases they cause result in significant economic costs that often have social repercussions. With the acceleration of globalization, enhancing the movement of plant material, in particular with the global live plant trade, the spread of Phytophthora to new, uncontaminated territories has intensified. Nurseries play a key role in the movement of these pathogens, the trade of contaminated stocks representing their major dissemination route. However valuable, conventional detection techniques, including baiting and direct isolation, are too slow and labour-intensive to meet current diagnostic requirements, particularly given the huge volumes of plants traded globally. This problem becomes even more acute when large volumes of potentially infectious plant material need to be processed in a short time frame, as it is often necessary to provide accurate and timely responses to interested parties. Early and precise detection is thus vital to avert outbreaks and mitigate long-term consequences. This review evaluates and contrasts the efficacy of novel detection methods against traditional approaches, emphasizing their significance in managing the escalating threat posed by Phytophthora spp. worldwide. Despite technological advances, critical challenges remain that limit the reliability and large-scale adoption of new diagnostic methods. Research still needs to bridge the gap between the laboratory and the field in terms of accuracy, sensitivity and diagnostic costs. Recent innovations focus on sensor technology and point-of-care (POC) devices for faster, more sensitive, and low-cost specific detection of Phytophthora spp. in plant matrices, water and soil. Enhancing diagnostic capabilities through these tools is crucial for protecting agricultural productivity, local economies, and natural ecosystems.

To date, Philaenus spumarius (Linnaeus, 1758), Philaenus italosignus Drosopoulos & Remane, 2000, and Neophilaenus campestris (Fallén, 1805) are proven vectors of the phytopathogenic bacterium Xylella fastidiosa Wells et al., 1987 in Europe. Currently, the identification of these three species relies on the well-documented status of morphological and taxonomical characters, making the discrimination of vector adult males possible by genitalia comparison. This study updates the biomolecular diagnostic tests with a rapid identification tool for P. italosignus, using locked nucleic acid (LNA) probe technology. The test also overcomes the difficulties associated with the morphological identification of females and juveniles. The morphological α-taxonomic identification of the male, achieved through comparison with the type of the species, retains its primary role in specimen identification for probe building. Later, the proposed assay can contribute to the rapid identification of P. italosignus by the secondary (molecular) identification step. The new LNA qPCR test offers high reliability and reproducibility in the identification of P. italosignus instars, thus improving targeted surveys of X. fastidiosa vector populations and allowing discrimination between species collected in the field. The accurate identification and census of vector individuals, regardless of their gender and instar, enhances the efficacy of Xylella IPM-DSS (Integrated Pest Management Decision Support System) strategies.

The invasive sap-feeding pest Toumeyella parvicornis (pine tortoise scale) is rapidly spreading across Europe, threatening pine ecosystems, particularly in forest–urban areas of Italy. In this scenario, early detection and monitoring strategies are critical to prevent new outbreaks and mitigate impacts in infested regions. Current surveillance is challenged by the lack of rapid, sensitive tools for indirect detection of this cryptic, canopy-dwelling pest, despite advancements in molecular diagnostics and environmental DNA (eDNA). Here, we established a highly specific qPCR assay using LNA probe chemistry for detecting T. parvicornis DNA from both adult insects and their excreted honeydew. DNA was successfully isolated/quantified from all tested matrices. We recorded average Cq values of 20.9 for insect specimens and 30.3 for collected honeydew samples. Targeting the COI barcoding region, the assay demonstrated excellent specificity in both in silico and in vitro tests, showing no cross-reactivity to other pine-associated taxa. The limit of detection for DNA isolated from insect was 64 fg/µL. This is the first diagnostic protocol to use honeydew as a matrix for indirect detection of T. parvicornis. Optimized for routine application by Plant Health Services, this eDNA-based tool offers a valuable approach for future monitoring of sap-sucking hemipterans in multiple environments.

English walnut (Juglans regia) is a species that is highly valued for the quality of its wood and the nutritional and nutraceutical properties of its fruit. A severe dieback of J. regia trees was observed recently in orchards located in three geographically distinct areas of Tuscany, central Italy. Symptoms included root and collar rot, necrosis of the under-bark tissue, bleeding cankers, stunted growth, and crown dieback. Four Phytophthora species were obtained from 239 isolates found on symptomatic J. regia individuals. They were identified, on the basis of macro-morphological (colony shape and texture), micro-morphometric (shape and size of oogonia, antheridia, oospores, sporangia, and chlamydospores) and molecular (ITS sequencing) characters, as P. gonapodyides, P. cactorum, P. citricola, and P. plurivora. Among these species, P. plurivora was the species isolated with overwhelming frequency from symptomatic tissue and rhizosphere soil, suggesting it to be the putative etiological agent. Pathogenicity assays were conducted on 20 cm long detached J. regia branches for a definitive establishment of disease causation. Severe symptoms (extended necroses) were exhibited by branches infected with P. plurivora, proving its pathogenicity and high virulence on this host. The other Phytophtora species produced negligible necroses around the inoculation site. P. plurivora was recovered from all the investigated orchards, providing evidence that it is quite widespread. This study highlights the growing threat posed by the polyphagous P. plurivora to walnut cultivation and the sustainable business it fuels in Europe, underscoring the need for integrated management strategies to mitigate its economic and ecological impacts.

Pantoea stewartii subsp. stewartii (Pss) is a Gram-negative bacterium causing Stewart wilt, a severe disease in maize. Native to North America, it has spread globally through the maize seed trade. Resistant maize varieties and insecticides are crucial to mitigate the disease’s economic impact. Pss is a quarantine pest, requiring phytosanitary certification for the seed trade in European countries. Accurate diagnostic tests, including real-time PCR, are fundamental to detect Pss and distinguish it from other bacteria, like Pantoea stewartii subsp. indologenes (Psi), a non-quarantine bacteria associated with maize seeds. Population genetics is a valuable tool for studying adaptation, speciation, population structure, diversity, and evolution in plant bacterial pathogens. In this study, the key activities of interlaboratory comparisons are reported to assess diagnostic sensitivity (DSE), diagnostic specificity (DSP) and accuracy (ACC) for different real-time PCR able to detect Pss in seeds. The results of complete sequencing of Italian bacterial isolates are presented. This study enhances our understanding of molecular methods for diagnosing and identifying pathogens in maize seeds, improving knowledge of Pss genomes to prevent their spread and trace possible entry routes from endemic to non-endemic areas.

Aleurocanthus spiniferus (Quaintance) (Hemiptera: Aleyrodidae) has rapidly spread, mainly in the central and eastern Mediterranean coastal area, infesting various new host plants alongside known ones. This invasive species poses a significant threat to agricultural ecosystems, necessitating urgent action to monitor and control outbreaks in previously pest-free areas. While entomological and morphological recognitions are crucial for initial detection, challenges often arise in quickly identifying different developmental stages or genus-level distinctions, particularly in surveys conducted by personnel with limited entomological skills. Due to these challenges, a qPCR probe protocol was developed to enhance the diagnostic capacity of laboratories responsible for the territorial control of pests. This biomolecular tool integrates morphological surveys, enabling prompt and reliable proof of A. spiniferus presence in free areas, delimited territories, or during phytosanitary import inspections. The protocol’s high analytical specificity, inclusivity, and exclusivity ensure accurate identification of A. spiniferus, while its low limit of detection and high repeatability and reproducibility reinforce its utility as a standardized diagnostic method. By facilitating prompt and targeted control efforts, this innovative approach strengthens the resilience of agricultural systems against the widespread threat of A. spiniferus infestations.

This study presents a qPCR-based molecular diagnostic method utilizing a TaqMan probe to ac-curately identify Agrilus auroguttatus Schäffer (Coleoptera: Buprestidae) from adult insects, ex-creta samples, and frass residues. The method demonstrated excellent DNA amplification across all sample types, highlighting strong analytical specificity. The protocol exhibited high repeata-bility and reproducibility, with inter-run and intra-run variability standard deviations consist-ently at or below 0.5. This assay distinguished A. auroguttatus samples from non-target species with 100% sequence correspondence. Analytical sensitivity (LoD) for adult and frass samples was determined at 8 pg/µL and at a 1:5 dilution from an initial concentration of 5 ng/µL, yield-ing mean Cq values of 35.14 ± 0.71 and 37.07 ± 0.59, respectively. The indirect diagnostic capabil-ity of this method is particularly valuable for detecting the presence of wood-boring insects, po-tentially aiding in the early detection of A. auroguttatus in new environments or in managing cur-rent outbreak areas. This diagnostic tool could thus play a critical role in biosecurity efforts to limit the spread of this invasive species.

Monitoring of emerging plant diseases in the Apennine mountains (central Italy) identified Cryptostroma corticale as the cause of the disease sooty bark of maple trees. The identified sites were located in rural or forested areas, next to buildings, in villages or suburbs, and one site was in a forest. Samples of symptomatic tissues were taken from Acer pseudoplatanus and/or A. campestre, as well as asymptomatic samples from A. campestre and Aesculus hippocastanum. All samples tested positive to a species-specific qPCR assay for the presence of C. corticale, indicating wide incidence of the disease in the northern Apennines of Italy, after attempted eradication of the first small group of infected plants were found in 2012.