Evaluation of established methods for DNA extraction and primer pairs targeting 16S rRNA gene for bacterial microbiome profiling of olive xylem sap

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 nonbiased 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, and 16 or 21 families, and 12 or 14 genera when using the Greengenes or Silva database for taxonomic assignation, 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 assignation. 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 within the xylem sap of olives or other woody crop species.

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

Frontiers in Plant Science ◽

10.3389/fpls.2021.640829 ◽

2021 ◽

Vol 12 ◽

Author(s):

Carmen Haro ◽

Manuel Anguita-Maeso ◽

Madis Metsis ◽

Juan A. Navas-Cortés ◽

Blanca B. Landa

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Dna Extraction ◽

Bacterial Communities ◽

Xylem Sap ◽

Pcr Primers ◽

Rrna Gene ◽

Community Assessment ◽

Taxonomic Assignment ◽

Woody Crop

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.

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Molecular characterization of the bacterial communities present in sheep's milk and cheese produced in South Brazilian Region via 16S rRNA gene metabarcoding sequencing

LWT ◽

10.1016/j.lwt.2021.111579 ◽

2021 ◽

Vol 147 ◽

pp. 111579

Author(s):

Creciana M. Endres ◽

Ícaro Maia S. Castro ◽

Laura D. Trevisol ◽

Juliana M. Severo ◽

Michele B. Mann ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Molecular Characterization ◽

Bacterial Communities ◽

Rrna Gene

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Sponge-Associated Bacteria Are Strictly Maintained in Two Closely Related but Geographically Distant Sponge Hosts

Applied and Environmental Microbiology ◽

10.1128/aem.05285-11 ◽

2011 ◽

Vol 77 (20) ◽

pp. 7207-7216 ◽

Cited By ~ 76

Author(s):

Naomi F. Montalvo ◽

Russell T. Hill

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Bacterial Communities ◽

Sponge Species ◽

Rrna Gene ◽

Gene Sequences ◽

Associated Bacteria ◽

Content Type ◽

Key Species ◽

Sponge Associated Bacteria

ABSTRACTThe giant barrel spongesXestospongiamutaandXestospongiatestudinariaare ubiquitous in tropical reefs of the Atlantic and Pacific Oceans, respectively. They are key species in their respective environments and are hosts to diverse assemblages of bacteria. These two closely related sponges from different oceans provide a unique opportunity to examine the evolution of sponge-associated bacterial communities. Mitochondrial cytochrome oxidase subunit I gene sequences fromX.mutaandX.testudinariashowed little divergence between the two species. A detailed analysis of the bacterial communities associated with these sponges, comprising over 900 full-length 16S rRNA gene sequences, revealed remarkable similarity in the bacterial communities of the two species. Both sponge-associated communities include sequences found only in the twoXestospongiaspecies, as well as sequences found also in other sponge species and are dominated by three bacterial groups,Chloroflexi,Acidobacteria, andActinobacteria. While these groups consistently dominate the bacterial communities revealed by 16S rRNA gene-based analysis of sponge-associated bacteria, the depth of sequencing undertaken in this study revealed clades of bacteria specifically associated with each of the twoXestospongiaspecies, and also with the genusXestospongia, that have not been found associated with other sponge species or other ecosystems. This study, comparing the bacterial communities associated with closely related but geographically distant sponge hosts, gives new insight into the intimate relationships between marine sponges and some of their bacterial symbionts.

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Diversity and Structure of the Endophytic Bacterial Communities Associated With Three Terrestrial Orchid Species as Revealed by 16S rRNA Gene Metabarcoding

Frontiers in Microbiology ◽

10.3389/fmicb.2020.604964 ◽

2020 ◽

Vol 11 ◽

Author(s):

Pasquale Alibrandi ◽

Sylvia Schnell ◽

Silvia Perotto ◽

Massimiliano Cardinale

Keyword(s):

Bacterial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

Host Plant ◽

Bacterial Communities ◽

Reproductive Organs ◽

Distribution Model ◽

Rrna Gene ◽

Orchid Species ◽

Core Microbiota

The endophytic microbiota can establish mutualistic or commensalistic interactions within the host plant tissues. We investigated the bacterial endophytic microbiota in three species of Mediterranean orchids (Neottia ovata, Serapias vomeracea, and Spiranthes spiralis) by metabarcoding of the 16S rRNA gene. We examined whether the different orchid species and organs, both underground and aboveground, influenced the endophytic bacterial communities. A total of 1,930 operational taxonomic units (OTUs) were obtained, mainly Proteobacteria and Actinobacteria, whose distribution model indicated that the plant organ was the main determinant of the bacterial community structure. The co-occurrence network was not modular, suggesting a relative homogeneity of the microbiota between both plant species and organs. Moreover, the decrease in species richness and diversity in the aerial vegetative organs may indicate a filtering effect by the host plant. We identified four hub OTUs, three of them already reported as plant-associated taxa (Pseudoxanthomonas, Rhizobium, and Mitsuaria), whereas Thermus was an unusual member of the plant microbiota. Core microbiota analysis revealed a selective and systemic ascent of bacterial communities from the vegetative to the reproductive organs. The core microbiota was also maintained in the S. spiralis seeds, suggesting a potential vertical transfer of the microbiota. Surprisingly, some S. spiralis seed samples displayed a very rich endophytic microbiota, with a large number of OTUs shared with the roots, a situation that may lead to a putative restoring process of the root-associated microbiota in the progeny. Our results indicate that the bacterial community has adapted to colonize the orchid organs selectively and systemically, suggesting an active involvement in the orchid holobiont.

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