Broth media cultivation of xylem microbiome from cultivated olive trees

Nowadays there is no culture media described for axenic growth of olive xylem microbiome which can be essential to understand the ecological interactions of xylem-inhabiting pathogens. This work focused on the study of culture media of defined composition to sustain growth of xylem-inhabiting microorganisms. Xylem sap extracted with a Scholander pressure chamber from two olive cultivars were inoculated in SMX, XVM2, XF26, PD3, 3G10R and XDM2 liquid culture media for two weeks at 28°C. The increase in absorbance was measured daily and aliquots were sampled every two days to analyze changes in bacterial composition by Illumina MiSeq metagenomic analysis. Globally, the most abundant genus of culturable bacteria was Massilia (21.25%), followed by Sphingomonas (20.54%), Curtobacterium (9.80%), Pseudomonas (7.51%) and Frigoribacterium (7.12%), although there were substantial differences in microbial communities over time and among culture media. Thus, SMX and XF26 displayed the highest bacterial OTU richness variation on time (33-9 and 30-6, respectively), followed by XVM2 (29-11), XDM2 (25-9), 3G10R (19-9) and PD3 (9-6). Currently, we have evaluated the possibility to modify the xylem microbiome composition of olive seedlings by transplanting a microbiome artificially cultured in liquid media by an endotherapy treatment and monitored the stability of this modification over time. Results indicated that consortia of xylem microorganisms can be cultivated in vitro using predefined culture media, and that those can artificially introduced in xylem vessels of seedling plants modifying their native xylem microbiome. This technique, when optimized, can settle the basis to inoculate ad-hoc selected microorganisms with the aim to control diseases caused by xylem inhabiting pathogens such as Xylella fastidiosa or modify olive plant physiology and growth.