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

First report and molecular characterization of citrus dwarfing viroid (CDVd) and citrus bark cracking viroid (CBCVd) on Citrus volkameriana in Egypt

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Antagonism and Antimicrobial Capacity of Epiphytic and Endophytic Bacteria against the Phytopathogen Xylella fastidiosa

Olive quick decline syndrome (OQDS), which is caused by Xylella fastidiosa, poses a severe threat to the agriculture of Mediterranean countries and causes severe damage to the olive trees in Italy. Since no effective control measures are currently available, the objective of this study was the screening of antagonistic bacteria that are potentially deployable as biocontrol agents against X. fastidiosa. Therefore, two approaches were used, i.e., the evaluation of the antagonistic activity of (i) endophytic bacteria isolated from two different cultivars of olive trees (Leccino and Ogliarola salentina) and (ii) epiphytic bacteria isolated from the phyllospheres of different host plant species of X. fastidiosa. In vitro dual culture tests showed that 12 out of 200 isolates inhibited X. fastidiosa growth, with appearances of clear zones between 4.0 and 38.6 mm. 16S rRNA gene sequencing revealed different species of the genera Paenibacillus, Bacillus, Pantoea, Microbacterium, Stenotrophomonas, Delftia, and Pseudomonas. Furthermore, an investigation for antimicrobial activity identified 5 out of the 12 antagonistic bacteria, Paenibacillus rigui, Bacillus subtilis, Bacillus pumilus, Microbacterium oxydans, and Stenotrophomonas rhizophila, that were able to produce culture filtrates with inhibitory activities. Our results are promising for further investigation to develop an eco-sustainable strategy to control X. fastidiosa using biocontrol agents or their secreted metabolites

Community Analysis of Culturable Sapwood Endophytes from Apulian Olive Varieties with Different Susceptibility to Xylella fastidiosa

Endophytes are symptomless fungal and/or bacterial microorganisms found in almost all living plant species. The symbiotic association with their host plants by colonizing the internal tissues has endowed them as a valuable tool to suppress diseases, stimulate growth, and promote stress resistance. In this context, the study of culturable endophytes residing the sapwood of Apulian olives might be a promising control strategy for xylem colonizing pathogens as Xylella fastidiosa. To date, olive sapwood cultivable endophytes are still under exploration; therefore, this work pursues a study of cultivable endophytes occurrence variation in the sapwood of different olive varieties under the effect of seasonality, geographical coordinates, and X. fastidiosa infection status. Our study confirms the stability of sapwood endophytic culturable communities in the resistant olive variety and presents the seasonal and geographical fluctuation of olive trees’ sapwood endophytes. It also describes the diversity and occurrence frequency of fungal and bacterial genera, and finally retrieves some of the sapwood-inhabiting fungal and bacterial isolates, known as biocontrol agents of plant pathogens. Thus, the potential role of these bacterial and fungal isolates in conferring olive tree protection against X. fastidiosa should be further investigated

Community Analysis of Culturable Sapwood Endophytes from Apulian Olive Varieties with Different Susceptibility to <i>Xylella fastidiosa</i>

Endophytes are symptomless fungal and/or bacterial microorganisms found in almost all living plant species. The symbiotic association with their host plants by colonizing the internal tissues has endowed them as a valuable tool to suppress diseases, stimulate growth, and promote stress resistance. In this context, the study of culturable endophytes residing the sapwood of Apulian olives might be a promising control strategy for xylem colonizing pathogens as Xylella fastidiosa. To date, olive sapwood cultivable endophytes are still under exploration; therefore, this work pursues a study of cultivable endophytes occurrence variation in the sapwood of different olive varieties under the effect of seasonality, geographical coordinates, and X. fastidiosa infection status. Our study confirms the stability of sapwood endophytic culturable communities in the resistant olive variety and presents the seasonal and geographical fluctuation of olive trees’ sapwood endophytes. It also describes the diversity and occurrence frequency of fungal and bacterial genera, and finally retrieves some of the sapwood-inhabiting fungal and bacterial isolates, known as biocontrol agents of plant pathogens. Thus, the potential role of these bacterial and fungal isolates in conferring olive tree protection against X. fastidiosa should be further investigated