Branches (ca. 35 cm long, 2 years old) from 8-year-old cultivated olive (O. europaea var. europaea) trees of “Picual” and “Arbequina” cultivars were sampled from an experimental rainfed field located at the Institute for Sustainable Agriculture from the Spanish National Research Council (IAS-CSIC) in Córdoba (Southern Spain). Trees of both varieties have been submitted to the same agricultural practices throughout the years. Cv. Picual has been found to be highly susceptible to Defoliating (D) and susceptible to Nondefoliating (ND) V. dahliae isolates, respectively, while cv. Arbequina has been shown to be susceptible to D and moderately resistant to ND V. dahliae (Calderon et al., 2014). In addition, clones of a wild olive tree of O. europaea var. sylvestris “Acebuche” known to be moderately resistant to D V. dahliae infection from a collection of wild-olive genotypes at IAS-CSIC was also sampled. Three 35-cm-long terminal branches (one per tree) from independent “Picual” and “Arbequina” cultivated olive trees and from clone trees of “Acebuche” were sampled in December 2018 to perform xylem sap extraction with the Scholander chamber. Xylem sap was extracted at the end of Autumn, a season of the year when the olive stem water potential is more stable (Iniesta et al., 2009) and lower than 40 bar of pressure, the maximum pressure allowed by the Scholander chamber device that was used to extract the xylem sap (see below). Likewise, for wood chips collection, a similar number of branches (one branch per tree, with three trees in total per olive genotype) were sampled, and 6-cm-long stem portions were selected from each branch (three pieces per branch). All pruned branches were placed in sterile plastic bags, sprayed with distilled water and kept in a cold room at 4°C to avoid desiccation until later processing in the same day.

The three 6-cm-long pieces of branches from each sample were debarked with a sterile scalpel. Bark tissue or xylem chips were obtained by scraping the debarked woody pieces with a sterile scalpel. A total of 0.5 g of xylem chips was weighted by mixing the chips obtained from all pieces sampled from same branch and tree and placed in a Bioreba bag containing 5 ml of sterile phosphate-buffered saline (PBS). Bioreba bags were closed with a thermal sealer and the content was homogenized with a hand homogenizer (BIOREBA, Reinach, Switzerland). Extracts were stored at 4°C until plating onto culture media, and then at -80°C, until DNA extraction. A total of three replicates per olive tree within each genotype was processed. All the processes described above took place under sterile conditions within a flow hood chamber.

Xylem sap extraction from olive branches was performed with a Scholander pressure chamber connected to a nitrogen cylinder following the Bollard process described by Alexou and Peuke (2013). An external port allowed switching from the internal Scholander chamber to an external 60-cm-long super chamber admitting a maximum of 40 bar of pressure. After inserting the branch in the super chamber, 5 cm of the main stem protruded to the exterior of the lid. At this point, it was important to preserve the branch with no cuts or loose leaves that could compromise the pressure within the chamber. To avoid microbial contamination of the xylem sap from bark and phloem, 2 cm of the main stem was debarked and disinfested. The edge between bark and xylem tissue was covered with parafilm to avoid leaks, and the pressure was increased gradually until xylem sap drops were observed to a maximum of 35 bars. The first drops of xylem sap were discarded to avoid external contamination. Xylem sap was collected within a 15 ml sterile falcon tube placed on ice. An average of 10 ml of xylem sap per genotype and branch was obtained and was preserved at 4°C until plating onto culture media, and then kept at -80°C until DNA extraction. All the processes described above took place under sterile conditions within a flow hood chamber.

For the culture-dependent approach, four solid culture media were evaluated: BCYE (Wells et al., 1981), PD2 (Davis et al., 1981), R2A (Reasoner and Geldreich, 1985), and Nutrient agar (NA) (CONDALAB, Madrid, Spain). R2A and NA are general culture media of low- or high-nutrient contents, respectively. BCYE and PD2 are media specifically designed for isolation of the fastidious bacterium X. fastidiosa from xylem tissues.

Aliquots or 1/10 dilutions (100 µl each) of xylem extracts obtained by each of the two techniques indicated above were plated directly onto three plates of each medium and incubated at 28°C in the dark for 2 weeks. After incubation, all bacterial colonies were counted and a representative number of colonies from each medium and genotype was selected based on abundance and colony feature morphological criteria. Selected colonies were purified by triple serial colony isolation in the same medium. Purified isolates were grown at 28°C in the dark for 1 to 2 weeks depending on their rate of growth prior to DNA extraction.

DNA was extracted from a total of 261 bacterial isolates using the DNeasy kit (QIAGEN, Madrid, Spain). The near-complete 16S rDNA gene was amplified using primers 8f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-ACGGCTACCTTGTTACGACTT-3′) (Weisburg et al., 1991) as described in Aranda et al. (2011). Amplicons were purified with ExoSAP-IT (Thermo Fisher Scientific, Madrid, Spain), and directly sequenced in both directions using primers 8f and 1492r (STABVIDA, Caparica, Portugal). Sequences were assembled and manually corrected using DNASTAR software version 15.3.0.66 (Madison, WI, USA). Isolates were identified to genus/species level by the nearest neighbor in the GenBank “nt” database after alignment with reference 16S rRNA gene sequences using the BLAST algorithm according to Altschul et al. (1997).

Aliquots of xylem sap samples (0.5 ml) obtained from macerated xylem chips were placed in PowerBead tubes (DNeasy PowerSoil Kit, QIAGEN) and homogenized 7 min at 50 pulses s^-1 with the Tissuelyser LT (QIAGEN). Sap extracts were incubated in the lysis buffer for 1 h at 60°C to increase cell lysis, and then processed following the DNeasy PowerSoil Kit manufacturer's instructions.

Aliquots (8 ml) of xylem sap samples extracted with the Scholander chamber were filtered through a 0.22-µm pore MF-Millipore™ filter (Merck Millipore, Madrid, Spain). Then, filters were placed into 1.5-ml Eppendorf tubes and the filtrate resuspended by vortexing for 5 min in the DNeasy PowerSoil Kit lysis buffer, incubated 1 h at 60°C and processed as described before. DNA obtained was quantified and outsourced to the Integrated Microbiome Resource (IMR) at Dalhousie University (Canada) to perform V5-V6 amplicon library sequencing with primers 799F (5'-AACMGGATTAGATACCCKG-3') and 1115R (5'-AGGGTTGCGCTCGTTG-3') and paired-end sequenced by using Illumina MiSeq sequencing platform (V3; PE 2x 300 bp). The ZymoBIOMICS microbial standard (Zymo Research Corp., Irvine, CA, USA) and water (no template DNA) were used as internal positive and negative controls, respectively, for library construction and sequencing. Raw sequence data were deposited in the Sequence Read Archive (SRA) database at the NCBI under BioProject accession number PRJNA574439.

To determine the effects of the olive genotype, type of xylem sap extraction procedure, and culture media, data of culturable bacterial populations obtained in the culture-depended approach were subjected to analysis of variance (ANOVA) using the GLM (General Linear Models) procedure in Statistical Analysis System v. 9.4 (SAS Institute Inc.). Data of culturable bacterial population were log-transformed to fulfill ANOVA assumption. The experiment had a completely randomized design, with olive genotype, type of xylem sap, and culture media as factors with three replications (plates) per experimental unit. Data fulfilled the assumptions for ANOVA according to proper statistics. Orthogonal single-degree-of-freedom contrasts were computed to test the effect of selected experimental treatment combinations.

The 16S rRNA sequences obtained were analyzed using the Quantitative Insights into Microbial Ecology bioinformatics pipeline, QIIME2 (version 2018.11; https://view.qiime2.org/) (Caporaso et al., 2010; Bolyen et al., 2018) with default parameters unless otherwise noted. DADA2 pipeline was used for denoising raw fastq paired-end sequences and filtering chimeras. Operational taxonomic units (OTUs) were obtained at 1% of dissimilarity and were taxonomically classified using RDP Bayesian classifier (Wang et al., 2007) against Silva SSU v.132 reference database. Singletons were discarded for taxonomy and statistical analyses.

Differences among bacterial communities derived from the culture-independent approach were calculated in QIIME2 using rarefaction curves of alpha-diversity indexes (including Shannon, Simpson, Faith_PD, and Richness) at the genus level. Alpha and beta diversity as well as alpha rarefaction curves were conducted rarefying all samples to the minimum number of reads found. The Kruskal-Wallis test (P <0.05) with FDR correction (Benjamini and Hochberg, 1995) was used to find differences in alpha diversity indexes among the studied factors. Venn diagrams were generated using the “Venn diagram” online tool (http://bioinformatics.psb.ugent.be/webtools/Venn/) and were used to identify shared (core microbiome) or unique taxa according to the type of xylem sap extract and olive genotypes studied, and to compare the culture-dependent and culture-independent approaches. We filtered bacterial taxa and retained those occurring in at least 50% of the samples in a given category. A heat tree summarizing main results was created using Metacoder package in R software (Foster et al., 2017). We unified the taxonomic affiliation derived from BLAST analysis (culture-dependent approach) and Silva SSU v.132 reference database (culture-independent approach) using the NCBI Taxonomy Browser (https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Root). Taxonomic abundances within each identified Phylum to genus level were visualized using Krona hierarchical data browser (Ondov et al., 2011).

A non-supervised principal component analysis (PCA) and multivariate hierarchical clustering analysis (using Pearson's correlation to measure distance and the Ward clustering algorithm) were performed using the OTU frequency matrixes at the genus level derived from the culture-dependent and culture-independent approach with the online tool MetaboAnalyst 4.0 (http://www.metaboanalyst.ca; Chong et al., 2018).

In the culture-dependent approach, bacterial population densities in xylem sap extracted from woody chips ranged from 40 to 1,920 colony forming units (cfu)/ml, whereas those obtained from xylem sap samples obtained with the Scholander pressure chamber ranged from 10 to 610 cfu/ml. Only two factors in the study were found to be significant [olive genotype (F=12.6, P < 0.0001) and type of xylem sap extraction method (F = 22.4, P < 0.0001), representing 16.6% and 29.7% of the mean square error (MSE) in the model, respectively]. Only the interaction type of xylem sap extraction method by olive genotype was significant (F = 35.67, P < 0.0001) representing 47.2% of the MSE in the model.

A significantly higher (F > 32.09, P < 0.0001) bacterial population density was estimated on xylem samples extracted from woody chips compared to xylem sap extracted with the Scholander chamber for “Acebuche” and “Picual,” while the opposite occurred for “Arbequina” (F = 17.13, P = 0.0001) for both xylem extraction procedures ( Figure 1 ). “Arbequina” showed a significantly higher (F > 10.45, P < 0.0021) bacterial population than “Picual” and “Acebuche” when xylem sap was obtained with the Scholander chamber, whereas “Acebuche” showed a significantly higher (F > 9.24, P < 0.0037) bacterial population than “Arbequina” and “Picual” when the xylem sap was extracted from woody chips macerates ( Figure 1 ).

Maximal recoveries of bacterial population were observed with R2A medium (478.9 ± 131.9 cfu/ml), and decreased with BCYE (352.2 ± 101.3 cfu/ml), PD2 (335.0 ± 97.6 cfu/ml), and NA (270.0 ± 82.0 cfu/ml), in that order, although no significant differences were found among culture media (F = 1.98, P = 0.1298) (data not shown).

A total of 261 bacterial isolated were selected for further study from xylem sap and xylem chips extracts after cultivation in NA, PD2, R2A, or BCYE culture media ( Figure 1 ; Table S1 ). Out of those, 137 bacterial isolates were from xylem sap extracted with the Scholander chamber while 124 were selected from xylem chips extracts. From the 261 isolates, 51 and 117 bacterial isolates were recovered from cultivated olives “Picual” and “Arbequina,” respectively, and 93 bacterial isolates from the wild olive genotype “Acebuche” ( Figure 1 ).

In the culture-independent approach, Illumina MiSeq sequencing analysis resulted in a total of 21,411 good quality reads with an average of 334 bp after removal of chimeras, unassigned or mitochondrial reads. No chloroplasts reads were detected in our samples. A total of 58 OTUs were identified for all treatments, out of which 48 OTUs were retained after rarefying all data to 1,234 sequences (the minimum number of reads obtained in one of the samples) and singleton removal.

Rarefaction curves of observed OTUs (Richness) displayed significantly higher (H = 12.79; P < 0.0003) values in xylem sap extracted with the Scholander chamber as compared to the woody chips maceration method. Interestingly, although “Picual” was the olive cultivar showing the lowest number of culturable bacteria ( Figure 1 ), it presented a significantly higher number of OTUs for both extraction methods (H = 20.19, P < 0.0001), followed by “Acebuche” and “Arbequina” ( Figure S1 ). Maximum values of Good's coverage of 1.0 were obtained for all samples (data not shown).

Alpha diversity indices (Richness, Shannon, Faith_PD and Simpson) did not show global significant differences between xylem sap extraction methods (H < 3.185, P > 0.074), among olive genotypes (H < 1.977, P > 0.372) or their interaction (H < 6.591, P > 0.253) (data not shown).

In the culture-dependent approach, a total of 4 phyla, 7 classes, 14 orders, 22 families, and 34 genera were identified ( Figure 2 ). Significant differences (P < 0.05) were found in the number of genera isolated according to the culture media ( Figure S2 ). Out of the total of 34 bacterial genera identified, 11 genera were detected in AN, 17 in BCYE and PD2, and 24 in R2A. Seven of the genera (Bacillus, Brachybacterium, Curtobacterium, Dermacoccus, Frigoribacterium, Methylobacterium, and Sphingomonas) were isolated in all culture media, whereas seven, four and four, and one genera were unique to R2A, PD2 and AN, and BCYE, respectively. Those unique genera corresponded normally to single isolates ( Figure S2 ; Table S1 ).

Regarding olive cultivars, 13 genera were identified in “Picual,” 22 in “Arbequina,” and 21 in “Acebuche.” Only four bacterial genera (11.8% of the total identified) were shared among the three olive genotypes and were present in more than 50% of all samples (Core bacterial genera: Bacillus, Frigoribacterium, Methylobacterium, Sphingomonas), six were shared between the two cultivated olive genotypes (Brevibacillus, Dermacoccus, Kineococcus, Marmoricola, Micrococcus, Staphylococcus), seven were shared between the wild olive genotype “Acebuche” and “Arbequina” (Amnibacterium, Curtobacterium, Frondihabitans, Microbacterium, Modestobacter, Nocardioides, Paenibacillus), and only one (Variovorax) between “Acebuche” and “Picual” ( Figure 3 ). “Acebuche” showed the highest number of unique genera (nine in total). Finally, regarding xylem sap extraction method, 13 (38.2%) of the culturable bacterial genera (Bacillus, Brevibacillus, Curtobacterium, Dermacoccus, Frigoribacterium, Methylobacterium, Micrococcus, Modestobacter, Nocardioides, Paenibacillus, Sphingomonas, Staphylococcus and Variovorax) were isolated by both extraction methods, whereas 11 (32.4%) and 10 (29.4%) of bacterial genera were unique to the Scholander chamber extraction method (Acinetobacter, Amnibacterium, Clavibacter, Corynebacterium, Frondihabitans, Kocuria, Marmoricola, Novosphingobium, Patulibacter, Quadrisphaera, Roseomonas) or the woody chips extraction method (Brachybacterium, Cellulomonas, Enterococcus, Friedmanniella, Kineococcus, Microbacterium, Rhodococcus, Risungbinella, Spirosoma, Terribacillus), respectively ( Figure 4 ).

When using the culture-independent approach, a total of 5 phyla, 8 classes, 17 orders, 23 families, and 31 genera were identified ( Figure 2 ). Twenty-one of the genera were identified in “Picual,” 14 in “Arbequina,” and 16 in “Acebuche” ( Figure 3 ). Only seven bacterial genera (22.6% of the total identified) were shared among the three olive genotypes (Core bacterial genera: Curtobacterium, Friedmanniella, Geodermatophilus, Hymenobacter, Kineococcus, Methylobacterium, Sphingomonas), two were shared between the two cultivated olive genotypes (Cutibacterium and Pseudomonas), two were shared between the wild olive genotype “Acebuche” and “Arbequina” (Amnibacterium and Mucilaginibacter), and two (Exiguobacterium and Novosphingobium) between “Acebuche” and “Picual.” “Picual” showed the highest number of unique genera (10 in total) ( Figure 3 ). Regarding xylem sap extraction method 14 (45.2%) of the bacterial genera identified were isolated by both extraction methods (Acidibacter, Curtobacterium, Cutibacterium, Exiguobacterium, Friedmanniella, Geodermatophilus, Hymenobacter, Kineococcus, Marmoricola, Methylobacterium, Pseudomonas, Roseomonas, Sphingomonas, Spirosoma), whereas 14 (45.2%) and 3 (0.10%) of bacterial genera were unique to the Scholander chamber extraction method (1174-901-12, Acidiphilium, Amnibacterium, Arthrobacter, Brevibacterium, Deinococcus, Frigoribacterium, Mucilaginibacter, Nocardioides, Novosphingobium, Quadrisphaera, Rathayibacter, Streptococcus, Variovorax) or the woody chips extraction method (Bradyrhizobium, Pectobacterium, Rubellimicrobium), respectively ( Figure 4 ).

Four of the phyla, Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria, were obtained both by the culture-dependent and culture-independent approaches. Only the phylum Deinococcus-Thermus emerged exclusively when using the culture-independent approach ( Figure 2 ). At the Class level Actinobacteria, Bacilli, Alphaproteobacteria Cytophagia, Betaproteobacteria, and Gammaproteobacteria were identified following both approaches. Thermophilia class was detected only in the culture-dependent approach while Sphingobacteriia and Deinococci classes were identified only when using the NGS procedure ( Figure 2 ). At the genus level, a total of 51 genera were identified combining both culture-dependent and culture-independent approaches; 20 and 17 genera were exclusive of the culture-dependent and culture-independent approaches, respectively, and 14 bacterial genera were shared by both methodologies ( Figure 2 ).

Actinobacteria phylum presented the highest relative abundance in the culture-dependent approach (46.37%) followed by Proteobacteria (34.09%), Firmicutes (19.15%), and Bacteroidetes (0.38%) ( Figure S3 ). Differently, Proteobacteria showed more than half of the total bacterial genera in the culture-independent approach (56.25%), followed by Bacteroidetes (24.76%), Actinobacteria (13.44%), Firmicutes (0.77%), and Deinococcus-Thermus (0.21%) ( Figure S4 ). At the family level, the most abundant bacterial families were Microbacteriaceae (34.48%), Methylobacteriaceae (16.86%), Sphingomonadaceae (31.44%), and Bacillaceae (13.03%) when using the culture-dependent approach, whereas Sphingomonadaceae (31.44%), Hymenobacteraceae (24.11%), and Methylobacteriaceae (22.36%) were the most abundant when using the culture-independent approach. Focusing on the culture-dependent methodology, the most abundant genera were Frigoribacterium (18.77%), Methylobacterium (16.36%), Sphingomonas (14.56%), Bacillus (12.64%), and Curtobacterium (11.88%), while when we used the NGS approach, the most abundant genera identified were Sphingomonas (30.10%), Hymenobacter (24.11%), and Methylobacterium (22.36%) ( Figures S3 and S4 ).

Hierarchical clustering analysis and PCA using OTU frequencies at the genus level differentiated xylem bacterial communities according to the sap extraction method or the olive genotype irrespective of the culture approach used ( Figure 5 ; Figure S5 ). However, these differences were more noticeable when using the culture-independent approach, for which there was a clear trend to group the bacterial communities first by olive genotype and then by extraction method with only one exception, and with “Acebuche” showing the most distant bacterial communities as compared to the cultivated olive genotypes.