Culture-Dependent and Culture-Independent Characterization of the Olive Xylem Microbiota: Effect of Sap Extraction Methods

Abstract

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.

Keywords: culture; microbiome; next-generation sequencing; vascular pathogens; xylem.

Figure 1

Culturable bacterial population densities [colony forming units (cfu)/ml] present in xylem sap of different olive genotypes extracted with the Scholander chamber or from woody chips macerates from three independent trees per genotype, irrespective of the culture medium used. The number of bacterial isolates selected for 16S taxonomic identification for each treatment is indicated between brackets. For each extraction method, bars with the same letter do not differ significantly among olive genotypes. The “*” indicate for each genotype the existence of significant differences at P≤ 0.05 between both xylem sap extraction methods.

Figure 2

Heat tree of the abundance of bacterial taxa at different ranks present in olive xylem sap and determined using culture-dependent and culture-independent approaches and extracted with the Scholander chamber (SCh) or from woody chips (WC) macerates. The size and color of nodes and edges are correlated with the abundance of taxa. The central nodes are the total of all the other nodes in the tree for each phylum.

Figure 3

Prevalence Venn diagram showing the unique and shared bacterial genera obtained using culture-dependent approaches (upper panel) or culture-independent approach (lower panel) in olive xylem sap samples when compared by olive genotype (“Acebuche,” “Arbequina,” and “Picual”).

Figure 4

Prevalence Venn diagram showing the unique and shared bacterial genera obtained using culture-dependent approaches (upper panel) or culture-independent approach (lower panel) in olive xylem sap samples when compared by xylem sap extraction method [Scholander chamber (SCh) or wood chips (WC) maceration].

Figure 5

Principal component analysis of the relative abundance of bacterial genera obtained using culture-dependent approaches (left panel) or culture-independent approach (right panel) in olive xylem sap samples when compared by olive genotype (Ace: “Acebuche,” Arb: “Arbequina,” and Pi: “Picual”) or by xylem sap extraction method [Scholander chamber (SCh) or wood chips (WC) maceration].