Evaluation of Established Methods for DNA Extraction and Primer Pairs Targeting 16S rRNA Gene for Bacterial Microbiota Profiling of Olive Xylem Sap.

Carmen Haro,Madis Metsis,Manuel Anguita-Maeso,Blanca B Landa,Juan A Navas-Cortés

doi:10.3389/fpls.2021.640829

Carmen Haro, Madis Metsis + Show 3 more

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https://doi.org/10.3389/fpls.2021.640829

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Abstract

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.

Highlights

Olive (Olea europaea subsp. europaea) is a primary element in the agricultural economy of most countries in the Mediterranean Basin, where about 5 million hectares of olive orchards are grown only in European countries
The number of plant organellar rRNA gene sequences amplified varied according to the DNA extraction kit, with the NorgenMicrobiomeV2 and PureLink DNA extraction kits showing the highest proportion of organellar reads (≥70%) and both NucleoSpin and the PowerLyzer DNA extraction kits showing the lowest proportion (≤4%), independently of the reference database used for taxonomic
Hierarchical clustering analysis and principal coordinate analysis (PCoA) of Bray–Curtis index and weighted UniFrac distances using operational taxonomic units (OTUs) frequencies differentiated xylem bacterial communities in four clusters according to the DNA extraction method regardless of the database used for taxonomy assignment (Figure 2A)

Summary

Introduction

Olive (Olea europaea subsp. europaea) is a primary element in the agricultural economy of most countries in the Mediterranean Basin, where about 5 million hectares of olive orchards are grown only in European countries. The health of the olive groves is being seriously threatened, as a consequence of a notable increase, both in extent and in severity, of diseases caused by diverse pathogens, which are capable of adversely affecting its growth and production. Among olive diseases, those caused by the vascular plant pathogenic bacterium Xylella fastidiosa and the soilborne fungus Verticillium dahliae are, without a doubt, the two major global threats to olive production worldwide (Jiménez-Díaz et al, 2011; Saponari et al, 2018; Landa et al, 2019; Anguita-Maeso et al, 2020). Information on the nature and role of the xylem microbiome and its contribution to plant health and crop productivity is still scarce (Anguita-Maeso et al, 2020)

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