Microbial endophytes are well known to protect host plants against pathogens, thus representing a promising strategy for the control of xylem-colonizing pathogens. To date, the vast majority of microbial communities inhabiting the olive xylem are unknown; therefore, this work pursues the characterization of the xylem-limited microbiome and determines whether the culture isolation medium, olive genotype, and the plant material used to analyze it can have an effect on the bacterial populations retrieved. Macerated xylem tissue and xylem sap extracted with the Scholander chamber from olive branches obtained from two cultivated and a wild olive genotypes were analyzed using culture-dependent and -independent approaches. In the culture-dependent approach using four solid culture media, a total of 261 bacterial isolates were identified after performing Sanger sequencing of 16S rRNA. Culturable bacteria clustered into 34 genera, with some effect of culture media for bacterial isolation. The cultivated bacteria belonged to four phyla and the most abundant genera included Frigoribacterium (18.8%), Methylobacterium (16.4%), and Sphingomonas (14.6%). On the other hand, in the culture-independent approach conducted using Illumina MiSeq 16S rRNA amplicon sequencing [next-generation sequencing (NGS)] of the xylem extracts, we identified a total of 48 operational taxonomic units (OTUs) belonging to five phyla, being Sphingomonas (30.1%), Hymenobacter (24.1%) and Methylobacterium (22.4%) the most representative genera (>76% of reads). In addition, the results indicated significant differences in the bacterial communities detected in the xylem sap depending on the genotype of the olive tree studied and, to a minor extent, on the type of sap extraction method used. Among the total genera identified using NGS, 14 (41.2%) were recovered in the culture collection, whereas 20 (58.8%) in the culture collection were not captured by the NGS approach. Some of the xylem-inhabiting bacteria isolated are known biocontrol agents of plant pathogens, whereas for others little information is known and are first reported for olive. Consequently, the potential role of these bacteria in conferring olive tree protection against xylem pathogens should be explored in future research.

Current research on the influence of environmental and physicochemical factors in shaping the soil bacterial structure has seldom been approached from a pedological perspective. We studied the bacterial communities of eight soils selected along a pedogenic gradient at the local scale in a Mediterranean calcareous mountain (Sierra de María, SE Spain). The results showed that the relative abundance of Acidobacteria, Canditate division WPS-1, and Armatimonadetes decreased whereas that of Actinobacteria, Bacteroidetes, and Proteobacteria increased from the less-developed soils (Leptosol) to more-developed soils (Luvisol). This bacterial distribution pattern was also positively correlated with soil-quality parameters such as organic C, water-stable aggregates, porosity, moisture, and acidity. In addition, at a lower taxonomic level, the abundance of Acidobacteria Gp4, Armatimonadetes_gp4,Solirubrobacter,Microvirga,Terrimonas, andNocardioidesparalleled soil development and quality. Therefore, our work indicates that the composition of bacterial populations changes with pedogenesis, which could be considered a factor influencing the communities according to the environmental and physicochemical conditions during the soil formation.

Vascular pathogens are the causal agents of main diseases threatening the health and growth of olive crops worldwide. The use of endophytic microorganisms represents a challenging and promising strategy for management of vascular diseases in olive. Although current research has been focused on analyzing the structure and diversity of the endophytic microbial communities inhabiting the olive xylem, the characterization of this ecological niche has been overlooked and to date remain unexplored, despite that the characterization of the xylem sap composition is essential to unravel the nutritional requirements of xylem-limited microorganisms. In this study, branches from plantlets and adult olive trees of cultivars Picual and Arbequina were selected to characterize the chemical and microbial composition of olive xylem sap extracted using a Scholander pressure chamber. Metabolome and ionome analyses of xylem sap were performed by proton nuclear magnetic resonance (NMR) spectroscopy-based and by inductively coupled plasma with optical emission spectroscopy (ICP-OES), respectively. Olive xylem sap metabolites included a higher relative percentage of sugars (54.35%), followed by alcohols (28.85%), amino acids (8.01%), organic acids (7.68%), and osmolytes (1.12%). Within each of these groups, the main metabolites in the olive xylem sap were mannitol, ethanol, glutamine, acetic acid, and trigonelline, whereas K and Cl⁻ were the main element and inorganic anion, respectively. Metabolomic profile varied when comparing olive plant age and genotype. The levels of glucose, fructose, sucrose and mannitol, choline, B and PO₄³⁻ were significantly higher in adult trees than in plantlets for both olive genotypes, whereas NO³⁻ and Rb content showed the opposite behavior. On the other hand, levels of aspartic acid, phenylalanine, and Na were significantly higher in ‘Picual’ than in ‘Arbequina’, whereas Fe showed the opposite behavior, but only for adult trees. Microbiome composition identified Firmicutes (67%), Proteobacteria (22%) and Actinobacteriota (11%) as the main phyla, while at the genus level Anoxybacillus (52%), Cutibacterium (7%), Massilia (6%), and Pseudomonas (3%) were the most representative. Both non-supervised hierarchical clustering analysis and supervised PLS-DA analysis differentiated xylem sap chemical and microbial composition first, according to the age of the plant and then by the olive genotype. PLS-DA analysis revealed that B, ethanol, Fe, fructose, glucose, mannitol, sucrose, and Sr, and Anoxybacillus, Cutibacterium, and Bradyrhizobium were the most significant chemical compounds and bacterial genera, respectively, in the discrimination of adult olive trees and plantlets. Knowledge of the chemical composition of xylem sap will lead to a better understanding of the complex nutritional requirements of olive xylem-inhabiting microorganisms, including vascular pathogens and their potential antagonists, and may allow the better design of artificial growing media to improve the culturing of the olive microbiome.

Xylella fastidiosa represents a major threat to important crops worldwide including almond, citrus, grapevine, and olives. Nowadays, there are no efficient control measures for X. fastidiosa, and the use of preventive measures and host resistance represent the most practical disease management strategies. Research on vessel-associated microorganisms is gaining special interest as an innate natural defense of plants to cope against infection by xylem-inhabiting pathogens. The objective of this research has been to characterize, by next-generation sequencing (NGS) analysis, the microbial communities residing in the xylem sap of almond trees affected by almond leaf scorch disease (ALSD) in a recent X. fastidiosa outbreak occurring in Alicante province, Spain. We also determined community composition changes and network associations occurring between xylem-inhabiting microbial communities and X. fastidiosa. For that, a total of 91 trees with or without ALSD symptoms were selected from a total of eight representative orchards located in five municipalities within the X. fastidiosa-demarcated area. X. fastidiosa infection in each tree was verified by quantitative polymerase chain reaction (qPCR) analysis, with 54% of the trees being tested X. fastidiosa-positive. Globally, Xylella (27.4%), Sphingomonas (13.9%), and Hymenobacter (12.7%) were the most abundant bacterial genera, whereas Diplodia (30.18%), a member of the family Didymellaceae (10.7%), and Aureobasidium (9.9%) were the most predominant fungal taxa. Furthermore, principal coordinate analysis (PCoA) of Bray–Curtis and weighted UniFrac distances differentiated almond xylem bacterial communities mainly according to X. fastidiosa infection, in contrast to fungal community structure that was not closely related to the presence of the pathogen. Similar results were obtained when X. fastidiosa reads were removed from the bacterial data set although the effect was less pronounced. Co-occurrence network analysis revealed negative associations among four amplicon sequence variants (ASVs) assigned to X. fastidiosa with different bacterial ASVs belonging to 1174-901-12, Abditibacterium, Sphingomonas, Methylobacterium–Methylorubrum, Modestobacter, Xylophilus, and a non-identified member of the family Solirubrobacteraceae. Determination of the close-fitting associations between xylem-inhabiting microorganisms and X. fastidiosa may help to reveal specific microbial players associated with the suppression of ALSD under high X. fastidiosa inoculum pressure. These identified microorganisms would be good candidates to be tested in planta, to produce almond plants more resilient to X. fastidiosa infection when inoculated by endotherapy, contributing to suppress ALSD.

Understanding the unique and unexplored microbial environment of xylem sap is starting to be of relevant importance for plant health, as it could include microbes that may protect plants against xylem-limited pathogens, such as Verticillium dahliae and Xylella fastidiosa. In this study, we evaluated the effects that the method for extracting the xylem bacterial communities, the plant age and the PCR primers may have on characterizing the xylem-bacterial-community composition by using an NGS approach. Xylem sap was extracted from xylem vessels by using a Scholander pressure chamber, or by macerating wood shavings that were obtained from xylem tissues by using branches from 10-year-old olive trees, or the entire canopy of 1-year-old olive plantlets. Additionally, we compared four different PCR-primer pairs that target 16S rRNA for their efficacy to avoid the coamplification of mitochondria and chloroplast 16S rRNA, as this represents an important drawback in metabarcoding studies. The highest amplifications in the mitochondria and chloroplast reads were obtained when using xylem woody chips with the PCR1-799F/1062R (76.05%) and PCR3-967F/1391R (99.96%) primer pairs. To the contrary, the PCR2-799F/1115R and PCR4-799F/1193R primer pairs showed the lowest mitochondria 16S rRNA amplification (60% of reads) included Anoxybacillus, Cutibacterium, Pseudomonas, Spirosoma, Methylobacterium-Methylorubrum and Sphingomonas; however, their relative importance varied, depending on the matrix that was used for the DNA extraction and the primer pairs that were used, with the lowest effect due to plant age. These results will help to optimize the analysis of xylem-inhabiting bacteria, depending on whether whole xylematic tissue or xylem sap is used for the DNA extraction. More importantly, it will help to better understand the driving and modifying factors that shape the olive-xylem-bacterial-community composition.

Vascular pathogens are the causal agents of some of the most devastating plant diseases in the world, which can cause, under specific conditions, the destruction of entire crops. These plant pathogens activate a range of physiological and immune reactions in the host plant following infection, which may trigger the proliferation of a specific microbiome to combat them by, among others, inhibiting their growth and/or competing for space. Nowadays, it has been demonstrated that the plant microbiome can be modified by transplanting specific members of the microbiome, with exciting results for the control of plant diseases. However, its practical application in agriculture for the control of vascular plant pathogens is hampered by the limited knowledge of the plant endosphere, and, in particular, of the xylem niche. In this review, we present a comprehensive overview of how research on the plant microbiome has evolved during the last decades to unravel the factors and complex interactions that affect the associated microbial communities and their surrounding environment, focusing on the microbial communities inhabiting the xylem vessels of olive trees (Olea europaea subsp. europaea), the most ancient and important woody crop in the Mediterranean Basin. For that purpose, we have highlighted the role of xylem composition and its associated microorganisms in plants by describing the methodological approaches explored to study xylem microbiota, starting from the methods used to extract xylem microbial communities to their assessment by culture-dependent and next-generation sequencing approaches. Additionally, we have categorized some of the key biotic and abiotic factors, such as the host plant niche and genotype, the environment and the infection with vascular pathogens, that can be potential determinants to critically affect olive physiology and health status in a holobiont context (host and its associated organisms). Finally, we have outlined future directions and challenges for xylem microbiome studies based on the recent advances in molecular biology, focusing on metagenomics and culturomics, and bioinformatics network analysis. A better understanding of the xylem olive microbiome will contribute to facilitate the exploration and selection of specific keystone microorganisms that can live in close association with olives under a range of environmental/agronomic conditions. These microorganisms could be ideal targets for the design of microbial consortia that can be applied by endotherapy treatments to prevent or control diseases caused by vascular pathogens or modify the physiology and growth of olive trees.