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 culturedependent 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, and Nocardioides paralleled 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.

Host resistance is the most practical, long-term, and economically efficient disease control measure for Verticillium wilt in olive caused by the xylem-invading fungus Verticillium dahliae (Vd), and it is at the core of the integrated disease management. Plant’s microbiome at the site of infection may have an influence on the host reaction to pathogens; however, the role of xylem microbial communities in the olive resistance to Vd has been overlooked and remains unexplored to date. This research was focused on elucidating whether in vitro olive propagation may alter the diversity and composition of the xylem-inhabiting microbiome and if those changes may modify the resistance response that a wild olive clone shows to the highly virulent defoliating (D) pathotype of Vd. Results indicated that although there were differences in microbial communities among the different propagation methodologies, most substantial changes occurred when plants were inoculated with Vd, regardless of whether the infection process took place, with a significant increase in the diversity of bacterial communities when the pathogen was present in the soil. Furthermore, it was noticeable that olive plants multiplied under in vitro conditions developed a susceptible reaction to D Vd, characterized by severe wilting symptoms and 100% vascular infection. Moreover, those in vitro propagated plants showed an altered xylem microbiome with a decrease in total OTU numbers as compared to that of plants multiplied under non-aseptic conditions. Overall, 10 keystone bacterial genera were detected in olive xylem regardless of infection by Vd and the propagation procedure of plants (in vitro vs nursery), with Cutibacterium (36.85%), Pseudomonas (20.93%), Anoxybacillus (6.28%), Staphylococcus (4.95%), Methylobacterium-Methylorubrum (3.91%), and Bradyrhizobium (3.54%) being the most abundant. Pseudomonas spp. appeared as the most predominant bacterial group in micropropagated plants and Anoxybacillus appeared as a keystone bacterium in Vd-inoculated plants irrespective of their propagation process. Our results are the first to show a breakdown of resistance to Vd in a wild olive that potentially may be related to a modification of its xylem microbiome and will help to expand our knowledge of the role of indigenous xylem microbiome on host resistance, which can be of use to fight against main vascular diseases of olive.

Next-generation sequencing has revolutionized our ability to investigate the microbiota composition of diverse and complex environments. However, a number of factors can affect the accuracy of microbial community assessment, such as the DNA extraction method, the hypervariable region of 16S rRNA gene targeted, or the PCR primers used for amplification. The aim of this study was to assess the influence of commercially available DNA extraction kits and different primer pairs to provide a non-biased vision of the composition of bacterial communities present in olive xylem sap. For that purpose, branches from “Picual” and “Arbequina” olive cultivars were used for xylem sap extraction using a Scholander chamber device. The DNA extraction protocol significantly affected xylem sap bacterial community assessment. That resulted in significant differences in alpha (Richness) and beta diversity (UniFrac distances) metrics among DNA extraction protocols, with the 12 DNA extraction kits evaluated being clustered in four groups behaving differently. Although the core number of taxa detected by all DNA extraction kits included four phyla, seven classes, 12 orders, 16 or 21 families, and 12 or 14 genera when using the Greengenes or Silva database for taxonomic assignment, respectively, some taxa, particularly those identified at low frequency, were detected by some DNA extraction kits only. The most accurate depiction of a bacterial mock community artificially inoculated on sap samples was generated when using the PowerPlant DNA extraction kit, the combination of 799F/1193R primers amplifying the hypervariable V5–V7 region, and the Silva 132 database for taxonomic assignment. The DESeq2 analysis displayed significant differences among genera abundance between the different PCR primer pairs tested. Thus, Enterobacter, Granulicatella, Prevotella, and Brevibacterium presented a significant higher abundance in all PCR protocols when compared with primer pair 799F/1193R, while the opposite was true for Pseudomonas and Pectobacterium. The methodological approach followed in this study can be useful to optimize plant-associated microbiome analysis, especially when exploring new plant niches. Some of the DNA extraction kits and PCR primers selected in this study will contribute to better characterize bacterial communities inhabiting the xylem sap of olives or other woody crop species.

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 (<27.48%), no chloroplast sequences and the highest numbers of bacterial OTUs identified (i.e., 254 and 266, respectively). Interestingly, only 73 out of 172 and 46 out of 181 genera were shared between the xylem sap and woody chips after amplification with PCR2 or PCR4 primers, respectively, which indicates a strong bias of the bacterial-community description, depending on the primers used. Globally, the most abundant bacterial genera (>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.

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.

In Brazil, olive trees (Olea europaea) are grown in the southern state of Rio Grande do Sul and in the south-eastern states of Minas Gerais, São Paulo, Rio de Janeiro and Espírito Santo on a landscape known as Serra da Mantiqueira, characterized by mountains with altitudes above 1000 m (Belarmino et al., 2020). The cultivated area is restricted to 7000 and 3000 ha in the southern and south-eastern regions, respectively. However, the national demand for olive oil was over 100 million tonnes in 2020 (https://www.inter natio nalol iveoil.org/wp-conte nt/uploa ds/2021/02/IOC-Impor t-profi les-Brazi l-2019-20-rev0.html). In the south-eastern region, olive oil production is carried out by small-scale producers who, in most cases, use their own extractor plant and require intensive use of local labour as a consequence of the landscape shape. Therefore, olive oil production has a significant economic and social importance as it keeps