Evaluation of a sampling method for Xylella fastidiosa detection in olive trees

To assess the presence of the xylem-limited bacterium Xylella fastidiosa subsp. pauca strain CoDiRO in olive trees, a specific sampling method was evaluated. Symptomatic and symptomless plants were randomly selected in four olive orchards located in the province of Lecce (Southern Italy). The crown of each plant was subdivided into a lower and an upper portion; four samples were collected from each layer in the main four cardinal directions. A total of eight samples per plant, composed of one- or two-year-old asymptomatic twigs, were collected next to branches showing leafscorch symptoms. In this preliminary study, the null hypothesis was tested. i.e. there is no difference between the lower and the upper portions of the tree canopy and across the four cardinal directions. Samples (472), collected from 60 plants belonging to 11 different olive cultivars, were tested by qPCR. Out of 236 samples taken from the upper and lower parts of the canopy only 38.1% of lower samples, in contrast to 56.8% taken from the upper crown layer, were positive to the bacterium,. The McNemar test determined that there is a statistically significant difference in the proportion of positive samples between the upper and lower crown (p < 0.001). The Cochran’s Q test was performed to evaluate differences in the four cardinal directions. The null hypothesis suggesting there is no difference across cardinal directions was confirmed (p = 0.097). Based on these preliminary results, it appears that sampling should be directed to the upper part of the canopy. However, further studies are needed to improve the efficiency of the sampling technique

First international proficiency testing for laboratory performance on Xylella fastidiosa detection

A proficiency test (PT) to evaluate the performance of laboratories involved in molecular and serological detection of X. fastidiosa was carried out in early 2017; 35 laboratories from EU/non- EU Countries tested 4 different methods to purify DNA, conventional and qPCR assays, and 2 ELISA tests. The number of resultant positive agreement/negative agreement/positive deviation/negative deviation was used to determine the laboratory performance (i.e. accuracy 100%). The overall results showed that all laboratories were able to correctly diagnose X. fastidiosa in the blind samples containing the highest X. fastidiosa concentrations, whereas the performance of several laboratories was negatively affected by the lack of detection in the samples with the lowest concentrations, both through molecular and serological tests. Accuracy level of 100% (laboratory conformed to the PT) was successfully recovered in the majority of the laboratories performing qPCR and PCR assays on DNA purified using at least 2 of the 4 tested protocols. The use of automated platform ensured higher laboratory performance. As expected, results of the ELISA tests generated lower performance values in the majority of the laboratories, due to the lack of detection of positive samples containing the lowest the bacterial concentration. This study provides a good overview on the laboratory performance for the diagnostics currently used in the EPPO countries and indicate useful improvements that laboratories can adopt to achieve a better performance

SURVEY FOR THE PRESENCE OF XYLELLA FASTIDIOSA subsp. PAUCA STRAIN CoDiRO IN THE NATIVE FLORA OF THE SALENTO PENINSULA

Xylella fastidiosa subsp. pauca strain CoDiRO was identified as associated with the “Olive Quick Decline Syndrome”, a devastating disease first observed in October 2013 in the southeastern Apulia. At least 350 plant species belonging to 75 families are reported as hosts of X. fastidiosa. These provide a source of inoculum for the vectors (xylem sap-feeding leafhoppers), thus playing a major epidemiological role and facilitating the entrenchment of the pathogen in the affected area. To investigate the CoDiRO strain host range in Salento, monthly samplings of the native flora of two heavily infected olive groves and of the side of adjacent roads were conducted from January 2014 onwards. One of the groves was grass-covered, whereas periodic tillage was performed in the other. Overall, more than 200 species of 50 families were sampled, observed for the presence of symptoms, photographed and identified. In the spring, Philaenus spumarius the main vector of the Salentinian X. fastidiosa strain was abundantly present on the herbaceous flora and shrubs at all sites. All samples, in pools of no less that 3 to 5 plants, were tested by DAS-ELISA and uncertain/positive results were verified by conventional and real time PCR. Bacterial isolates were obtained in axenic culture from some positive species. In a two-year survey, only Euphorbia terracina proved to be Xylella-positive among the herbaceous hosts, whereas some shrubs and subshrubs i.e. Asparagus acutifolius, Cistus creticus, Myrtus communis, Phillyrea latifolia, Rhamnus alaternus and Rosmarinus officinalis were infected. These results provide as strong indication that, rather than weeds, are the perennial shrubs that play a major role in the epidemiology of the X. fastidiosa in this area

Phytosanitary Rules for the Movement of Olive (<i>Olea europaea</i> L.) Propagation Material into the European Union (EU)

Phytosanitary legislation involves government laws that are essential to minimize the risk of the introduction and diffusion of pests, especially invasive non-native species, as a consequence of the international exchange of plant material, thus allowing us to safeguard agricultural production and biodiversity of a territory. These measures ensure compliance with adequate requirements relating to the absence of pests, especially of harmful quarantine organisms through inspections and diagnosis tests of the consignments to ascertain the presence of the pests concerned. They also regulate the eradication and containment measures that are implemented in the eventuality of an unintentional introduction of these organisms. In the present contribution, the current plant protection legislation for the exchange of plants or propagation material within the European Union or for export to foreign countries, represented by Regulation (EU) 2016/2031, has been reviewed, with a particular focus on the olive tree (Olea europaea L.). Furthermore, a brief summary of the main olive tree pests transmissible with the propagation material is also reported, indicating their current categorization with respect to the relative quarantine status

Phytosanitary Rules for the Movement of Olive (Olea europaea L.) Propagation Material into the European Union (EU)

Phytosanitary legislation involves government laws that are essential to minimize the risk of the introduction and diffusion of pests, especially invasive non-native species, as a consequence of the international exchange of plant material, thus allowing us to safeguard agricultural production and biodiversity of a territory. These measures ensure compliance with adequate requirements relating to the absence of pests, especially of harmful quarantine organisms through inspections and diagnosis tests of the consignments to ascertain the presence of the pests concerned. They also regulate the eradication and containment measures that are implemented in the eventuality of an unintentional introduction of these organisms. In the present contribution, the current plant protection legislation for the exchange of plants or propagation material within the European Union or for export to foreign countries, represented by Regulation (EU) 2016/2031, has been reviewed, with a particular focus on the olive tree (Olea europaea L.). Furthermore, a brief summary of the main olive tree pests transmissible with the propagation material is also reported, indicating their current categorization with respect to the relative quarantine status

Embryo Culture, In Vitro Propagation, and Molecular Identification for Advanced Olive Breeding Programs

The high biodiversity of the olive tree is an important opportunity to develop sustainable plans to control Xylella fastidiosa (Xf) through breeding programs. Olive tree breeding activities have been limited due to various features of this species including the long time required for seed germination caused by the inhibition effect of the woody endocarp, the seed integument, and the endosperm. Starting from F1 seeds by cross-breeding, the embryo culture was compared with traditional seed germination, evaluating the effectiveness of in vitro multiplication of the plantlets for large-scale production. The isolated embryos were established on a new medium based on Rugini ‘84 macroelements, Murashige & Skoog ‘62 microelements, with Nitsch J. P. & Nitsch C. ‘69 vitamine and subcultured on Leva MSM modified. The results obtained confirmed that in vitro culture of olive embryos is a valid tool for increasing the percentage and speed of germination, helping to reduce the time of the olive breeding programs, offering the possibility to effectively propagate plantlets for further experiments

Isolation of a Xylella fastidiosa strain infecting olive and oleander in Apulia, Italy

The isolation in pure culture of the Xylella fastidiosa strain associated with the quick decline syndrome of olive, recently observed in Apulia (Salento peninsula, southern Italy) was attempted from symptomatic, naturally infected olive and oleander plants, and a periwinkle seedling that had been exposed to, and was infected by Xylella-positive spittlebugs. Prior to isolation, the presence of Xylella was ascertained in all donor hosts by PCR, indirect immunofluorescence and electron microscopy. solations from olive failed because of the heavy contamination by bacteria other than Xylella. By contrast, pure bacterial cultures were obtained from oleander and periwinkle extracts plated in periwinkle wilt gelrite (PWG) and buffered cysteine-yeast extract (BCYE) media. In both media, colonies were slow-growing, small-sized (less than 1 mm 25 days from plating), non pigmented, opalescent and exhibited the same morphology, except for the margin that was entire in BCYE and somewhat irregular in PWG. Bacterial cells were rod-shaped with rounded ends, had a thick and rippled cell wall, an average width of 0.35 μm, and a maximum length of ca. 5 μm. They gave a positive reaction in immunofluorence assays and were clearly decorated by colloidal gold in immunogold labelling tests. Sequenced PCR products amplified from periwinkle and oleander colonies shared 97-99% sequence identity with known X. fastidiosa strains from database and were 100% identical to one another and to comparable sequences obtained from infected olive trees. These sequences grouped in a distinct cluster of a branch comprising X. fastidiosa isolates belonging to the subspecies pauca

Apulian Autochthonous Olive Germplasm: A Promising Resource to Restore Cultivation in <i>Xylella fastidiosa</i>-Infected Areas

The olive tree (Olea europaea subsp. europaea var. europaea) represents the cornerstone crop of Apulian agriculture, which is based on the production of oil and table olives. The high genetic variability of the Apulian olive germplasm is at risk of genetic erosion due to social, economic, and climatic changes. Furthermore, since 2013, the spread of the Gram-negative bacterium Xylella fastidiosa subsp. pauca responsible for the olive quick decline syndrome (OQDS) has been threatening olive biodiversity in Apulia, damaging the regional economy and landscape heritage. The aim of this study was to investigate the differential response to X. fastidiosa infection in a collection of 100 autochthonous Apulian olive genotypes, including minor varieties, F1 genotypes, and reference cultivars. They were genotyped using 10 SSR markers and grown for 5 years in an experimental field; then, they were inoculated with the bacterium. Symptom assessments and the quantification of bacterium using a qPCR assay and colony forming units (CFUs) were carried out three and five years after inoculation. The study allowed the identification of nine putatively resistant genotypes that represent a first panel of olive germplasm resources that are useful both for studying the mechanisms of response to the pathogen and as a reserve for replanting in infected areas

Venous thromboembolism secondary to hospitalization for COVID-19: patient management and long-term outcomes

Background: Venous thromboembolism (VTE) is a complication of COVID-19 in hospitalized patients. Little information is available on long-term outcomes of VTE in this population.Objectives: We aimed to compare the characteristics, management strategies, and long-term clinical outcomes between patients with COVID-19-associated VTE and patients with VTE provoked by hospitalization for other acute medical illnesses.Methods: This is an observational cohort study, with a prospective cohort of 278 patients with COVID-19-associated VTE enrolled between 2020 and 2021 and a comparison cohort of 300 patients without COVID-19 enrolled in the ongoing START2-Register between 2018 and 2020. Exclusion criteria included age &lt;18 years, other indications to anticoagulant treatment, active cancer, recent (&lt;3 months) major surgery, trauma, pregnancy, and participation in interventional studies. All patients were followed up for a minimum of 12 months after treatment discontinuation. Primary end point was the occurrence of venous and arterial thrombotic events.Results: Patients with VTE secondary to COVID-19 had more frequent pulmonary embolism without deep vein thrombosis than controls (83.1% vs 46.2%, P &lt;.001), lower prevalence of chronic inflammatory disease (1.4% and 16.3%, P &lt;.001), and history of VTE (5.0% and 19.0%, P &lt;.001). The median duration of anticoagulant treatment (194 and 225 days, P = 0.9) and the proportion of patients who discontinued anticoagulation (78.0% and 75.0%, P = 0.4) were similar between the 2 groups. Thrombotic event rates after discontinuation were 1.5 and 2.6 per 100 patient-years, respectively (P = 0.4). Conclusion: The risk of recurrent thrombotic events in patients with COVID-19- associated VTE is low and similar to the risk observed in patients with VTE secondary to hospitalization for other medical diseases