Apulian olive sapwood endophytes to cope with Xylella fastidiosa: community analysis, antagonists screening, colonization efficiency, and genes expression

This dissertation comprises a series of studies aimed at exploring the cultivable endophytic communities indigenous to the sapwood of Apulian olive cultivars, identifying potential antagonists against Xylella fastidiosa, pauca ST53, and demonstrating their colonization efficiency and associated effects on olive defence-related genes. This work starts with an extensive analysis of cultivable sapwood endophytes from resistant and susceptible cultivars distributed across phytosanitary zones in Apulia. After multiple isolation of endophyte colonies, the results were translated into quantitative indicators. It was found that the resistant cultivar 'Leccino' showed high stability and diversity of the endophyte unit compared to the susceptible cultivar 'Ogliarola salentina', whether infected or not by Xf. Moreover, this variation was observed at the genus level, where several bacterial (such as Bacillus and Pantoea) and fungal (Pithomyces and Paraconiothyrium) genera with commonly known antagonistic potency were isolated more frequently from the resistant cultivar. Later, several isolates of bacterial and fungal species were subjected to in vitro screening for antagonistic activity against Xf ST53. Isolates of Bacillus subtilis, Bacillus pumilus and Pantoea agglomerans exhibited significant potency of growth inhibition of Xf. A similar effect was shown by the fungal isolates of Paraconiothyrium brasiliense. Meanwhile, SYBR-Green real-time primers with high specificity and sensitivity to the target species of direct antagonists (B. subtilis, P. agglomerans and P. brasiliense), indirect antagonists (Curotbacterium flaccumfaciens) and symbionts (Methylobacterium mesophilicum) were developed to follow the establishment of the inoculum in resistant and susceptible olive cultivars. After inoculation of the endophytes, the regulation of defence-related genes in the above-ground tissues of the olive was studied. Both real-time PCR and plate counting revealed the successful establishment of the endophytic inoculum in the cultivars 'Leccino' and 'Cima di Mola'. Among the bacterial direct antagonists, B. sublitis showed high stability and persistence within the internal tissues of both olive cultivars. Similarly, the fungal isolate of P. brasiliense was consistently recovered with a high colonization rate in the stem segment of both cultivars. Induction of defence-related gene expression was found almost exclusively in olives inoculated with B. sublitis. Overall, our results demonstrate the efficacy and suitability of direct/indirect antagonists such as B. sublitis as a potential control strategy of Xf ST53 in Apulian olives.