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 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.

Differences in the Endophytic Microbiome of Olive Cultivars Infected by Xylella fastidiosa across Seasons

“…A good correlation in the classification of the bacterial taxa was obtained between the two sequencing approaches at higher taxonomical levels (phylum, order, and class), while it decreased when lower levels were considered, likely due to the different depth of the data. These four dominant phyla were indeed found to be predominant in the microbiomes recovered from Xylella -infected Leccino and Cellina di Nardò trees [ 46 ], as well as in the endophytic microbiome of healthy olive trees [ 41 ]. The dominant fungal phyla were Ascomycota and Basidiomycota , confirming previous ITS microbiome analyses [ 42 , 44 , 46 , 54 ], but the agreement between WMSS and ITS rRNA gene approaches was not maintained as different taxa abundances were classified at all taxonomic levels.…”

“…Among the season-associated Bacteria, WMSS identified the nitrogen-fixing Bradyrhizobium genus [ 77 ], already reported in olive by [ 41 ], the anaerobes Peptoniphilus [ 78 ], which include human pathogenic species, and the Streptomyces genus, whose members are known as bioremediators and plant-growth-promoters [ 79 ]. The only WMSS and 16S/ITS rRNA gene shared genus was Malassezia that was positively associated to plants with high Xylella abundance.…”

“…However, differently from Müller et al [ 39 ], in their 16S rRNA gene analysis, no traces of Archaea were found in the xylem sap while these microorganisms were present with low abundances in leaves and soil fractions. A comparison between cultivation-dependent and independent approaches to study the xylem microbiome of olive cultivars Picual, Arbequina, and Acebuche [ 41 ] showed that the main factor shaping the xylem-inhabiting microbiome was the olive genotype. Interestingly minor variations in the microbiome composition were detected between the xylem-sap (recovered using the Scholander pressure chamber) and the whole homogenized xylem tissue.…”

Verticillium dahliaeinoculation andin vitropropagation modify the xylem microbiome and disease reaction to Verticillium wilt in a wild olive genotype

“…In our study, 10 keystone bacterial genera could be considered the core microbiome being transmitted from generation to generation in olive, since they were detected in all samples regardless plant propagation procedure ( in vitro vs nursery) or inoculation with the pathogen; with Cutibacterium, Pseudomonas, Anoxybacillus, Staphylococcus, Methylobacterium-Methylorubrum , and Bradyrhizobium being the most abundant, in that order. These bacterial taxa have also been identified in olive xylem in other works in which olive trees of different ages, belonging to different cultivars or growing under different environments were evaluated (Müller et al 2015; Fausto et al, 2018; Sofo et al, 2019; Anguita-Maeso et al, 2020; Giampetruzzi et al, 2020) which strength the hypothesis that those genera may represent keystone olive xylem bacteria. More interestingly, some of these genera have been already reported or used as plant growth promoting bacteria (Otieno et al, 2015; Subramanian et al, 2015) or as biological control agents against V. dahliae (Berg et al, 2002, 2006).…”

“…Possibly, technical difficulties for isolation of xylem microbiome have difficulted its study, and only a few have focused on microorganisms inhabiting the olive xylem. However, different methodological approaches, including culture-dependent procedures complemented with next-generation sequencing (NGS) technologies, have recently made possible to characterize microbial communities associated with olive xylem tissue (Hardoim et al, 2015; Fausto et al, 2018; Anguita-Maeso et al, 2020; Zicca et al, 2020).…”