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Trabajo presentado en la 3rd European Conference on Xylella fastidiosa (Building knowledge, protecting plant health), celebrada online el 29 y 30 de abril de 2021., The plant-pathogenic bacterium Xylella fastidiosa, widely distributed in the Americas and with several recent outbreaks in EU, emerged as a serious threat to olive crop and the typical Mediterranean landscape. This xylem-limited plant pathogen spreads by xylem sap-feeding insects, causes symptoms of marginal leaf scorch, leaf chlorosis, defoliation, and a general decay in olive trees. Research on plant-associated microorganisms has gained importance in the last decade as a key component in plant health. However, the role of the xylem microbiome and its contribution to plant health is still scarce. Therefore, this work is focused on mapping X. fastidiosa infection and deciphering the changes in the composition of microbial communities on symptomatic and asymptomatic olive tree branches. Three olives trees of cultivar Grappolo growing at Sao Paulo state (Brazil) were selected and within each tree three main branches were sampled showing different severity of symptoms development; one branch presenting typical X. fastidiosa symptoms of pathogen infection in contrast to the other two branches that were asymptomatic. X. fastidiosa infection was verified by qPCR. Leaves and stem samples were collected in a gradual scale above and below the symptomatic branches. Chips extracts of xylem tissue from its stems and leaf veins and petioles were used for DNA extraction to undergo metagenomic analysis (NGS) for microbial composition estimation and to diagnose the presence of the pathogen by real-time PCR, respectively. These results will help to understand the occurrence of step-wise changes in olive xylem microbiome as a consequence of X. fastidiosa infection and determine which microorganisms respond faster or can be associated to a symptom progression on infected trees., Study supported by Projects 727987 XF-ACTORS (EU-H2020) and AGL2016-75606-R (MEIC Spain and FEDER-EU) and SEGIB – Carolina Foundation.

Trabajo presentado en la 3rd European Conference on Xylella fastidiosa (Building knowledge, protecting plant health), celebrada online el 29 y 30 de abril de 2021., The xylem-inhabiting plant pathogenic bacterium Xylella fastidiosa (Xf) represents one of the major phytopathological threats to olive crop worldwide, due to its devastating effects on agricultural yields losses and high tree mortality that causes profound socioeconomic and environmental impacts. Endophytes play an essential role on plant growth and its physiological status, but they can also act as an innate natural defense to cope against infection by xylem-inhabiting pathogenic organisms. Today, vast majority of microorganisms residing in olive xylem are unknown; therefore this work pursues the characterization of the olive microbiome through culture-dependent and independent (NGS) techniques as a tool for identifying potential biological control agents for this pathogen. Hence, four cultivated olive genotypes (Arbequina, Arbosana, Koroneiki and Grappolo) located in Sao Paulo state (Brazil) showing visual Xf symptoms or asymptomatic-non-infected were selected. Xf infection was verified by qPCR. For the culture-dependent approach, chips extracts of xylem tissue from branches and roots were plated in two solid media (R2A and R2A supplemented with plant extract). For culture independent approach, total DNA extracted from xylem tissue was analyzed by metagenomic analysis of 16S and ITS region to characterize the xylem-inhabiting bacterial and fungal communities. Preliminary culture results indicated differences in the frequency of microbial communities depending on the olive genotype and the type of plant material analyzed, as well as, the presence or absence of Xf symptoms on the sampled trees that correlated with Xf infection. These results will help to expand our knowledge on the olive xylem microbiome community composition and understand its driving factors when Xf infection occurs and more importantly to identify xylem-inhabiting microorganisms with potential to combat this harmful bacterium., Study supported by Projects 727987 XF-ACTORS (EU-H2020) and AGL2016-75606-R (MEIC Spain and FEDER-EU) and SEGIB – Carolina Foundation.

Trabajo presentado en la 3rd European Conference on Xylella fastidiosa (Building knowledge, protecting plant health), celebrada online el 29 y 30 de abril de 2021., 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., Research financially supported by Projects XF-ACTORS 727987 (EU-H2020) and AGL2016-75606-R (MICINN Spain and FEDER-EU).

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. Research financially supported by Projects XF-ACTORS 727987 (EU-H2020) and AGL2016-75606-R (MICINN Spain and FEDER-EU).