Eco-sustainable strategies to control Xylella fastidiosa subsp. pauca Strain De Donno by novel biological control agents: screening and mode of action in Apulian olive cultivars and Oleander plants
Author(s)
Mourou, Marwa
Date Issued
July 26, 2022
Type
Doctoral Thesis
Abstract
This dissertation comprises a series of studies aimed at exploring the cultivable bacterial communities inhabiting leaf surfaces of the main host species of Xylella fastidiosa ST53 present in the Apulia region, screening of potential antagonists, revealing their mechanism of action and identifying possible antimicrobial compounds secreted. Furthermore, evaluating their capability to colonize Apulian olive cultivars and Nerium. oleander host. This work started with the isolation of potential biological control agents (BCAs) from leaves of Olea europaea, Polygala myrtifolia, Rosmarinus officinalis, Nerium oleander, Laurus nobilis, Myrtus communis, Prunus dulcis, Prunus avium and Cistus creticus hosts present in various X. fastidiosa outbreaks occurred in the demarcated area of Apulia region. Later, several isolates were obtained and screened in vitro for antagonistic activity against X. fastidiosa ST53 and were subjected to morphological, biochemical, and molecular characterization. In vitro dual culture tests showed that 12 out of 89 isolates inhibited X. fastidiosa growth with an appearance of clear inhibition zones between 4.0 and 34 mm. Sequencing the 16S rRNA gene revealed different species belonged to the genera Pantoea, Microbacterium, Stenotrophomonas, Delftia, Sphingomonas, Curtobacterium and Pseudomonas. In the framework of the thesis, several tests were carried out to elucidate and understand the mode of action of different antagonists and their ability to produce culture filtrates (CFs) with antimicrobial compounds. Moreover, five bacterial endophytic strains natively obtained from Apulian olive cultivars were also included in the experiment of antimicrobial activity against X. fastidiosa ST53. Results showed that Pseudomonas. graminis BA2 and Pantoea. agglomerans BA69 isolates produced siderophores. However, Stenotrophomonas. rhizophila BA102 and Microbacterium. oxydans BA104 isolates produced protease and HCN respectively. Importantly, our results showed that Bacillus subtilis, Bacillus pumilus, Peanbacillus rigui, S. rhizophila and M. oxydans had the highest antagonistic activity in vitro with the production of culture filtrates which inhibit the growth of the pathogen likely secreting active substances in the liquid medium. Bacterial isolates demonstrating the strongest antagonistic activity (P. agglomerans, Curotbacterium flaccumfaciens, B. subtilis, M. oxydans and S. rhizophila) were inoculated in Apulian olive cultivars and N. oleander hosts. The establishment of bacterial inoculums were monitored by Real-time PCR, conventional PCR and plat counting. The results obtained showed that the different isolates tested were able to colonize the stem of the two olive cultivars and N. oleander plant during the assessment period of about one month. In detail, P. agglomerans BA69 was recovered with relatively high population size (106 CFU/ml) from N. Oleander plant and was less efficient in establishing in the two olive cultivars. Both isolates of B. subtilis L39 and C. flaccumfaciens BA54 were found to be stable in the stem segments of both olive cultivars and N. oleander plant. They were able to reach approximately 106 CFU/ml population size in N. oleander and over 104 CFU/ml population in olive cultivars. Endophytic colonization of M. oxydans BA104 and S. rhizophila BA102 were found to require more time compared with other isolates tested based on the plate counting approach. On the other hand, preliminary promising results were obtained from HPLC-HRMS analysis in which we succeed to annotate 10 metabolites that were mainly antibiotics from CFS of B. subtilis and M.oxydans. Particularly, 3'-Deoxydihydrostreptomycin metabolite that was detected in CFS of M. oxydans BA104 seems to be the most interesting since we revealed also its fragmentation pattern compared to other metabolites that did not show enough high abundance to be fragmented. Overall, our results demonstrate the efficacy and suitability of several antagonists to be exploited in control strategy against X. fastidiosa ST53. Nevertheless, HPLC-HRMS results needs to be carried on to clearly identify the metabolites responsible for the antibacterial activity against X. fastidiosa ST53.
Additional information
Dottorato di ricerca in Scienze delle produzioni vegetali e animali
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