Xylella fastidiosa subsp. pauca ST53 (XFP), the causal agent of olive quick decline syndrome (OQDS), was thoroughly investigated after a 2013 outbreak in the Salento region of Southern Italy. Some trees from Ogliarola Salentina and Cellina di Nardò, susceptible cultivars in the Gallipoli area, the first XFP infection hotspot in Italy, have resprouted crowns and are starting to flower and yield fruits. Satellite imagery and Normalized Difference Vegetation Index analyses revealed a significant improvement in vegetation health and productivity from 2018 to 2022 of these trees. Lipid molecules have long been recognized as plant defense modulators, and recently, we investigated their role in XFP-positive hosts and in XFP-resistant as well as in XFP-susceptible cultivars of olive trees. Here, we present a case study regarding 36 olive trees (12 XFP-positive resprouting, 12 XFP-positive OQDS-symptomatic, and 12 XFP-negative trees) harvested in 2022 within the area where XFP struck first, killing millions of trees in a decade. These trees were analyzed for some free fatty acid, oxylipin, and plant hormones, in particular jasmonic and salicylic acid, by targeted LC-MS/MS. Multivariate analysis revealed that lipid markers of resistance (e.g., 13-HpOTrE), along with jasmonic and salicylic acid, were accumulated differently in the XFP-positive resprouting trees from both cultivars with respect to XFP-positive OQDS symptomatic and XFP-negative trees, suggesting a correlation of lipid metabolism with the resprouting, which can be an indication of the resiliency of these trees to OQDS. This is the first report concerning the resprouting of OQDS-infected olive trees in the Salento area.

In 2022, a test performance study (TPS) assessing the influence of different master mixes on the performance of the tetraplex real-time PCR (TqPCR) assay was organized. TqPCR allows for the specific detection and identification of Xylella fastidiosa (Xf) subspecies in a single reaction. Eighteen official laboratories of the Italian National Plant Protection Organization received a panel of 12 blind samples, controls, primers, probes, and different master mixes to participate in the TPS. Furthermore, the Research Centre for Plant Protection and Certification of the Council for Agricultural Research and Economics performed an intra-laboratory study (ITS) on spiked plant matrices to evaluate the analytical sensitivity of TqPCR employing the selected master mixes with the best performance. Naturally infected samples were analyzed for subspecies identification via TqPCR compared with the official multilocus-sequence-typing (MLST) method. The best results in this comparative study were obtained using Fast Universal PCR Master Mix (Applied Biosystems) and Brilliant multiplex QPCR Master Mix (Agilent), and they confirmed that the TqPCR test is reliable, offering the advantage of identifying this subspecies at the same time, thus saving time and resources. The TqPCR assay is suggested among the tests to be used by laboratories performing the official diagnosis of Xf to support the activities of official monitoring.

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.