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In Apulia (southern Italy) since 2016 one of the largest monitoring campaign in EU is ongoing, with an average number of 100.000 samples tested per year in the framework of the official monitoring program for Xylella fastidiosa, in the demarcated and Xylella-free areas. More than 85% of the samples were from olive trees, the remaining from almond, oleander and other host plants. Before the promulgation of the EU Regulation 2020/1201, the diagnostic program was organized with two levels of checks. Samples were first screened in 4 regional laboratories by ELISA, with each laboratories processing 300-600 samples/day. Samples yielding positive or doubtful ELISA-results along with 3-5% of the negative samples, were re-tested by qPCR as confirmation tests and for verifying the lab performance for the serological tests. An average of 8.000 sample/year were re-tested by qPCR. Using this large dataset of samples (double tested by ELISA and qPCR) we determined the diagnostic specificity and sensitivity of the test used, and assessed the influence of other factors (period of sampling, origin of the samples). Overall, less than 0.03% of the ELISA-tested samples could not be assigned as negative/positive (i.e. undetermined) and thus re-tested by qPCR, yielding in the majority of the cases (>85%) negative qPCR-results. The diagnostic sensitivity and specificity calculated on the panel of ELISA-negative/positive samples re-tested in qPCR, approx. 26.000 samples, were 94% and 97%, respectively. These values did not vary significantly when the results were analyzed based on the period of sampling or location of the sampled trees. In conclusion, under the specific scenario characterizing the Apulian epidemics (olive predominant host plants, climatic conditions) this diagnostic workflow had allowed to screen with good diagnostic accuracy a relevant number of samples/year. Given the need to replace ELISA with qPCR tests, the capability of the laboratories to process the samples is now reduced (approx. half samples/day) and other possibilities will be consider for the implementation of the upcoming monitoring campaign (i.e testing pooled samples) to ensure testing the same amount of samples., IT; PDF; donato.boscia@ipsp.cnr.it

Up to date 35 different host species have been reported susceptible to Xylella fastidiosa, subsp. pauca, ST53 (Xfp), in the Apulia region (southern Italy). Xfp is genetically related to coffee strains of the subsp. pauca, particularly aggressive on olives, the predominant crop species in the area, and on other species (i.e. oleander, Acacia spp, Polygala myrtifolia). On the other hand, our previous investigations demonstrated that this genotype was not able to infect Vitis spp., several species of Citrus and the Prunus hybrid GF677. In the last 3 years, we extended the investigations by performing a series of artificial inoculations on different species, with the aim of identifying additional immune species. These included Pyrus communis, Juglans regia, Ceratonia siliqua, Mespilus germanica, Diospyros kaki, Citrus limon, Punica granatum, Persea americana. At least 10 plants for each species were needle-inoculated in 2017, along with susceptible controls (periwinkle and olive). Vector-mediated transmission was also used for P. americana, by caging the plants with Xfp-infected specimen of Philaenus spumarius. Inoculated plants were periodically inspected and sampled to assess bacterial multiplication and host colonization. Overall, the diagnostic tests performed during the 3 years post-inoculation did not reveal the presence of the bacterium in the tissues above (>10cm) the inoculation points of any of the inoculated plants. Detection occurred only when leaf petioles from the inoculation points were tested. Similarly, no positive detections occurred in the plants of P. americana exposed to infected insects. In addition, for C. limon, P. granatum, P. communis and C. siliqua surveys conducted in these last 3 years in the heavily infected area did not reveal the occurrence of natural infected plants. Overall, the data suggest that these species do not support the multiplication and the spread of the bacterium. However, even if for P. Americana needle-inoculations were complemented with vector transmissions, for the remaining species mechanical inoculations should be integrated by vector-mediated transmission tests for conclusive assessment of their immunity.

Inspections and controls for Xylella fastidiosa are mandatory on consignment and in place of productions for the most susceptible host plants listed in the EU Decision 2017/2352, as well as on the long list of “specified plants” when propagated in nurseries located in the infected, containment and buffer zones. The main constrains for testing samples collected from lots of plants are the large number of units to be sampled and the large amount of materials (n. of leaves or shoots/cuttings) to be processed at laboratory level. We carried out experiments by simulating composite samples containing different proportions of Xylella-infected plant tissues, to verify the diagnostic sensitivity of serological and molecular tests. Petioles recovered from infected leaves of Polygala myrtifolia, Nerium oleander and Olea europaea, and scraped xylem tissue from infected cuttings of Prunus avium were pooled at different ratio with healthy materials and processed by ELISA, LAMP and qPCR. Indeed, a protocol based on the extraction of large amount of tissues (40gr of plant material, 100-200 leaves) was also tested. The results provided preliminary important indications both for sampling and laboratory testing: (i) minimum n. of leaves to be collected from the single unit of the lot; (ii) the maximum n. of units that can be pooled and processed using either the standard extraction procedures or the protocol adapted for large tissue amount. The minimum number of the leaves to be collected from the individual plant of the sample unit, ranged from 2 to 6 according to the host species, allowing to pool up to 10 plants or more in relation to the extraction protocol used. Composite sampling is increasingly becoming an acceptable practice when a large number of samples have to be selected to satisfy sample size requirements, keeping the number of diagnostic tests affordable.

The recent global re-emergence of Xylella fastidiosa (Xf) has been associated to infections on several new host species and in particular to severe infections on olive trees, reported both in Europe and southern America. Although, initial report of Xf infections on olives, caused by strains of the subsp. multiplex, from California were associated to erratic appearance of olive leaf scorching or branch desiccation, severe phenomena of decline and extensive desiccations have been repeatedly reported associated to strains of the subspecies pauca in Italy, Brazil and Argentina. Furthermore, biological and molecular studies on olives belonging to different cultivars and infected with isolates of the subsp. pauca identified cultivars with differential susceptibility and traits of resistance. In this work, we used the strain CO33 taxonomically related to subsp. sandyi, to infect olive plants of the cultivar Cellina di Nardò and Leccino, previously characterized as susceptible and resistant, respectively. Twelve months after inoculation, Xf CO33 induced twig desiccations limitedly to the inoculated plants of the cv Cellina di Nardò. Indeed, the symptomless plants of the cv Leccino harbored lower bacterial populations than cv Cellina di Nardò. Transcriptome profiling of the xylem tissues showed that Xf is sensed by plants of both cultivars with high numbers of differentially expressed genes. Among transcripts showing the highest up regulation in cv Leccino are Leucine Rich Receptor like kinases, already identified in the transcriptomes of trees infected with Xf subsp. pauca. Conversely, tissues from symptomatic plants of Cellina di Nardò display a typical defense reaction with pathogenesis related proteins among major up regulated genes. Besides extending the data on the susceptibility of olives to Xf strains of a different subspecies, the data confirm the resistance of the cv Leccino to this pathogen, supporting evidence that different mechanisms of host-pathogen interactions exist in susceptible and resistant olive cultivars.

Genetic studies showed that the genotype of the olive-infecting strain of X. fastidiosa (Xf- De Donno) belongs to the sequence type ST53 within subspecies pauca, already reported to occur in Costa Rica. The analysis of single nucleotide polymorphisms (SNPs) and the study of the pan-genome of 27 available whole genomes were performed to determine the phylogenetic placement of Xf-De Donno. Maximum-parsimony and maximum likelihood trees constructed using the SNPs and the pangenome data distinguished the subsp. fastidiosa, multiplex, pauca, sandyi, and morus and groups the Italian and three Costa Rican ST53 isolates in a compact clade that diverges from the South American pauca isolates. These findings were supported by the characterization of a conjugative plasmid shared by the four ST53 isolates and by the identification of a gene encoding a putative histidine kinase-like ATPase, which is not present in isolates from the subsp. multiplex, sandyi and pauca, but was detected in the four ST53 and ST21 isolates of the subspecies fastidiosa from Costa Rica. These data support the common and recent origin of the ST53 isolates. The complete and annotated genome sequence of the strain De Donno of X. fastidiosa was obtained by a combined Illumina and PacBio sequencing. This strain, recovered from an olive tree affected by Olive Quick Decline Syndrome (OQDS), when mechanically inoculated in different olive cultivars, caused symptoms identical to those observed in contaminated olive groves.

While different sources of natural resistance to Xylella fastidiosa (Xf) have been described in grapevines and citrus, lack of consolidated information exists on the wide panel of cultivars characterizing the vast olive germplasm. Preliminary observations on few cultivars, support the evidence that differential cultivar responses to Xf infections may exist. To explore the response of a larger panel of cultivars, in April 2015, an experimental olive plot, located within the Xf-heavily affected olive groves, was established in the Apulia Region (Italy). Twenty-four trees for each of the ten different cultivars were planted in randomized blocks. Each tree was caged with 15-20 specimens of Philaenus spumarius collected from the neighboring infected olive groves. Upon removing the cages, the trees are then continuously exposed to the natural vector populations occurring in the area. Nine and 12-months after planting, the trees were sampled, tested for Xf and inspected for symptoms. The first data confirmed the infectivity of the vector populations occurring in the Apulian contaminated area and the Xf susceptibility of the olive cultivars tested. Almost 50% of the trees tested positive, with an infection incidence ranging from 25% (Leccino) to 78% (Koroneiki). Symptoms of shoot dieback started to appear 1-year after planting, limitedly on few replicates of Cellina di Nardò. In April 2016, the number of cultivars has been increased up to 30. Periodical surveys for symptoms and quantitative analyses to monitor the differential bacterial titer and expression of target genes involved in the host response, are underway.

Up to date 35 different host species have been reported susceptible to Xylella fastidiosa, subsp. pauca, ST53 (Xfp), in the Apulia region (southern Italy). Xfp is genetically related to coffee strains of the subsp. pauca, particularly aggressive on olives, the predominant crop species in the area, and on other species (i.e. oleander, Acacia spp, Polygala myrtifolia). On the other hand, our previous investigations demonstrated that this genotype was not able to infect Vitis spp., several species of Citrus and the Prunus hybrid GF677. In the last 3 years, we extended the investigations by performing a series of artificial inoculations on different species, with the aim of identifying additional immune species. These included Pyrus communis, Juglans regia, Ceratonia siliqua, Mespilus germanica, Diospyros kaki, Citrus limon, Punica granatum, Persea americana. At least 10 plants for each species were needle-inoculated in 2017, along with susceptible controls (periwinkle and olive). Vector-mediated transmission was also used for P. americana, by caging the plants with Xfp-infected specimen of Philaenus spumarius. Inoculated plants were periodically inspected and sampled to assess bacterial multiplication and host colonization. Overall, the diagnostic tests performed during the 3 years post-inoculation did not reveal the presence of the bacterium in the tissues above (>10cm) the inoculation points of any of the inoculated plants. Detection occurred only when leaf petioles from the inoculation points were tested. Similarly, no positive detections occurred in the plants of P. americana exposed to infected insects. In addition, for C. limon, P. granatum, P. communis and C. siliqua surveys conducted in these last 3 years in the heavily infected area did not reveal the occurrence of natural infected plants. Overall, the data suggest that these species do not support the multiplication and the spread of the bacterium. However, even if for P. Americana needle-inoculations were complemented with vector transmissions, for the remaining species mechanical inoculations should be integrated by vector-mediated transmission tests for conclusive assessment of their immunity., IT; PPT; leonardo.susca@uniba.it

Up to date 35 different host species have been reported susceptible to Xylella fastidiosa, subsp. pauca, ST53 (Xfp), in the Apulia region (southern Italy). Xfp is genetically related to coffee strains of the subsp. pauca, particularly aggressive on olives, the predominant crop species in the area, and on other species (i.e. oleander, Acacia spp, Polygala myrtifolia). On the other hand, our previous investigations demonstrated that this genotype was not able to infect Vitis spp., several species of Citrus and the Prunus hybrid GF677. In the last 3 years, we extended the investigations by performing a series of artificial inoculations on different species, with the aim of identifying additional immune species. These included Pyrus communis, Juglans regia, Ceratonia siliqua, Mespilus germanica, Diospyros kaki, Citrus limon, Punica granatum, Persea americana. At least 10 plants for each species were needle-inoculated in 2017, along with susceptible controls (periwinkle and olive). Vector-mediated transmission was also used for P. americana, by caging the plants with Xfp-infected specimen of Philaenus spumarius. Inoculated plants were periodically inspected and sampled to assess bacterial multiplication and host colonization. Overall, the diagnostic tests performed during the 3 years post-inoculation did not reveal the presence of the bacterium in the tissues above (>10cm) the inoculation points of any of the inoculated plants. Detection occurred only when leaf petioles from the inoculation points were tested. Similarly, no positive detections occurred in the plants of P. americana exposed to infected insects. In addition, for C. limon, P. granatum, P. communis and C. siliqua surveys conducted in these last 3 years in the heavily infected area did not reveal the occurrence of natural infected plants. Overall, the data suggest that these species do not support the multiplication and the spread of the bacterium. However, even if for P. Americana needle-inoculations were complemented with vector transmissions, for the remaining species mechanical inoculations should be integrated by vector-mediated transmission tests for conclusive assessment of their immunity. &nbsp