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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.

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