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This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Plant Science
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In this study, we investigated the impact of different land use intensities (LUI) on the root-associated microbiome of
Numerous studies have shown that healthy plants can host up to a few thousand different microbial species in their different organs, including roots, stems and leaves (
The various plant compartments colonized by microbes do not provide constant environments, but change with plant developmental stage or as a result of environmental factors including temperature, water content, or nutrient availability (
Land use intensity strongly influences a large number of environmental factors including the amount and quality of nutrients, the soil structure and general biodiversity pattern (
This study was performed within the framework of the German Biodiversity Exploratories, a project investigating large-scale and long-term relationships of biodiversity and land use in Central European grasslands (
Nine plants of
Dissolved carbon and nitrogen content in soil was measured after extraction of 5.0 g fresh soil using 0.01 M calcium chloride after shaking on a horizontal shaker for 45 min followed by centrifugation (2 min at 4500 x
Above-ground plant material was dried at 65°C for 2 days prior to pulverization in a Tissue LyserII (Qiagen GmbH, Germany). About 1.5 mg of the pulverized material was weighted into 3.5 mm × 5 mm tin capsules (HEKAtech GmbH, Wegberg, Germany). Total carbon and nitrogen contents were determined using an Elemental-Analysator ‘Euro-EA’ (Eurovector, Milano, Italy).
After rhizosphere removal, roots were submerged in sterile 1% Tween 20 for 2 min, washed in sterile water, incubated for 2 min in 70% ethanol and subsequently rinsed three times with sterile distilled water. Afterward, roots were surface sterilized using 5% sodium hypochlorite for 10 min, washed eight times with sterile distilled water, frozen in liquid nitrogen and stored at -80°C. The disinfection efficiency was confirmed by the absence of PCR products after amplification of the 16S rRNA genes using DNA extracted from the final wash water as a template (data not shown). Additionally, no colonies were obtained when 200 μl of the final wash water were plated onto NB-agar plates and incubated for 10 days on 28°C (data not shown).
DNA was extracted using a phenol-chloroform-based method according to
Next generation sequencing was performed using the Illumina MiSeq platform (Illumina Inc., San Diego, CA, United States). Library preparation was accomplished according to the “16S Metagenomic Sequencing Library Preparation” protocol proposed by Illumina Inc., San Diego, CA, United States. Briefly, polymerase chain reaction (PCR) of the 16S rRNA region was performed in triplicates using the primers 335Fc (5′-CADACTCCTACGGGAGGC-3′) and 769Rc (5′-ATCCTGTTTGMTMCCCVCRC-3′) published by
Sequences were analyzed using QIIME (Quantitative Insights into Microbial Ecology) software package version 1.9.1 (
Water extractable organic carbon values were evenly distributed among different LUI levels. In contrast, WEON, nitrate, and ammonium concentrations changed with LUI levels. WEON and nitrate decreased from intensively to extensively managed sites. While WEON and nitrate levels were significantly higher in soil samples from the high LUI sites (Supplementary Table
A total of 3,883,552 raw reads were obtained. After quality filtering and chimera check with DeconSeq 3,853,784 remained, which could be clustered into a total of 3,241 OTUs at 97% sequence similarity. Afterward, plant derived 16S sequences (displayed by
As expected, α-diversity (
Faith’s phylogenetic diversity of all compartments under low and high land use intensity, respectively. The distribution of phylogenetic diversity is shown at 97% sequence similarity. The boxplot indicates the first and third quartile. The median is indicated as horizontal line and whiskers indicate minimum or maximum, respectively. Significant differences among land use intensities are indicated with an asterisk (
Phylogenetic classification of the obtained OTUs revealed 18 phyla, highlighting
Analysis of α-diversity revealed a significant impact of LUI on bacterial diversity in bulk soil as well as in roots (
To identify changes in bacterial communities as a consequence of different LUIs in the three compartments, ternary plots were prepared (
Ternary plots of community structure in roots, rhizosphere and bulk soil. The colors represent the classes to which corresponding OTUs were assigned.
To gain deeper insights into the shared communities in all compartments (OTUs present across 95% of the samples) under low or high LUI, respectively, an analysis of the core microbiome was performed. Consistent with the ternary plot, more shared OTUs were found under low LUI (38 OTUs) compared to high LUI (17 OTUs). Among these, a shared core of 12 OTUs could be defined which was independent of compartment and LUI (Supplementary Figure
Significant differences with respect to LUI in the abundance of assigned families in roots could only be detected for four taxa (Supplementary Table
To better understand the driving factors of the differences in bacterial community structure in the three compartments, we performed a canonical analysis of principal coordinates. Including the soil characteristics in the ordination revealed that LUI is best represented by nitrate (high LUI) and ammonium (low LUI). The soil characteristics used explained 36% of the variation of microbial community structure in the roots (
Canonical analysis of principal coordinates of Bray–Curtis dissimilarities in roots. Crosses mark centroids of low or high LUI, respectively. Low LUI is represented in green, high LUI is represented in red color.
The phylogenetic classification of plant-associated bacterial communities of
Moreover, our results revealed that
In this study, the main aim was to identify the impact of LUI on the plant-associated microbiome of
Nevertheless, results of diversity analysis corroborate our findings on the significant differences of the abundant bacterial families in roots. Most abundant taxa in the endosphere were not affected by LUI. It has been proposed that host tissues represent unique niches for associated bacteria, leading to little variation in dominant communities (
Together with the analysis of microbial patterns between the compartments (
Previous studies had indicated the strong influence of plants on the microbial colonization in the rhizosphere through exudation. However, it remained unclear whether soil type or selection by the host plant was the major determinant of root microbiome composition (
Our results indicate that roots of reproducing
JE designed the experiment, carried out the field work, laboratory experiments, data analysis and wrote the manuscript. BS and PS contributed to the design of the experiment. PH contributed to the field work and the laboratory experiments. GV provided the pipelines for sequencing data analysis and advised data analysis. BS, PS, GV, JJ, MR, and MS advised the studies and critically revised the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank the managers of the three Exploratories, Kirsten Reichel-Jung, Katrin Lorenzen, Martin Gorke and all former managers for their work in maintaining the plot and project infrastructure; Christiane Fischer for giving support through the central office, Michael Owonibi for managing the central data base, and Markus Fischer, Eduard Linsenmair, Dominik Hessenmöller, Jens Nieschulze, Daniel Prati, Ingo Schöning, François Buscot, Ernst-Detlef Schulze, Wolfgang W. Weisser and the late Elisabeth Kalko for their role in setting up the Biodiversity Exploratories project. Furthermore we want to thank Dr. Anne Schöler and Christoph Schmid for their help in R and Dr. Stefanie Schulz for a her advisory help.
The Supplementary Material for this article can be found online at: