Long-Term Warming Shifts the Composition of Bacterial Communities in the Phyllosphere of Galium album in a Permanent Grassland Field-Experiment

Abstract

Global warming is currently a much discussed topic with as yet largely unexplored consequences for agro-ecosystems. Little is known about the warming effect on the bacterial microbiota inhabiting the plant surface (phyllosphere), which can have a strong impact on plant growth and health, as well as on plant diseases and colonization by human pathogens. The aim of this study was to investigate the effect of moderate surface warming on the diversity and composition of the bacterial leaf microbiota of the herbaceous plant Galium album. Leaves were collected from four control and four surface warmed (+2°C) plots located at the field site of the Environmental Monitoring and Climate Impact Research Station Linden in Germany over a 6-year period. Warming had no effect on the concentration of total number of cells attached to the leaf surface as counted by Sybr Green I staining after detachment, but changes in the diversity and phylogenetic composition of the bacterial leaf microbiota analyzed by bacterial 16S rRNA gene Illumina amplicon sequencing were observed. The bacterial phyllosphere microbiota were dominated by Proteobacteria, Bacteroidetes, and Actinobacteria. Warming caused a significant higher relative abundance of members of the Gammaproteobacteria, Actinobacteria, and Firmicutes, and a lower relative abundance of members of the Alphaproteobacteria and Bacteroidetes. Plant beneficial bacteria like Sphingomonas spp. and Rhizobium spp. occurred in significantly lower relative abundance in leaf samples of warmed plots. In contrast, several members of the Enterobacteriaceae, especially Enterobacter and Erwinia, and other potential plant or human pathogenic genera such as Acinetobacter and insect-associated Buchnera and Wolbachia spp. occurred in higher relative abundances in the phyllosphere samples from warmed plots. This study showed for the first time the long-term impact of moderate (+2°C) surface warming on the phyllosphere microbiota on plants. A reduction of beneficial bacteria and an enhancement of potential pathogenic bacteria in the phyllosphere of plants may indicate that this aspect of the ecosystem which has been largely neglected up till now, can be a potential risk for pathogen transmission in agro-ecosystems in the near future.

Keywords: Buchnera; Enterobacteriaceae; Sphingomonas; global climate change; grassland; phyllosphere; warming experiment.

FIGURE 1

Box-plots of total cell number counts per g G. album leaf of C and T plots. C = ambient temperature (C), T = elevated temperature (T). Squares = four C plots, triangles = four T plots, box-plots were performed in SigmaPLOT (Applied Maths).

FIGURE 2

Phylogenetic composition of phyllosphere inhabiting bacteria of Galium album leaves from plots with ambient temperature (C 1–4) and +2°C surface temperature elevated plots (T 1–4) resolved at the level of Bacteria phyla. (A) Relative abundance of different bacterial phyla on G. album leaves from C and T plots. (B) Principal component analysis (PCA) of relative abundance pattern of respective bacterial communities based on the phyla assignment. Eigenvalues for the compared principal components are given in brackets (%) at the respective axes of the graphs. The contribution of different taxonomic groups to the placement of the samples in the PCA plots are indicated as biplots in the graph, and (C) variation of the relative abundances of the most abundant phyla among G. album leaf samples from C and T plots. Asterisks are representing statistical significance: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. G = Galium album; C = ambient temperature; T = elevated temperature; 1–4 = biological replicates.

FIGURE 3

Phylogenetic composition of phyllosphere inhabiting bacteria of Galium album leaves from plots with ambient temperature (C 1–4) and +2°C surface temperature elevated plots (T 1–4) with a resolution at the level of Bacteria taxa. (A) Non-metric multidimensional scaling (nMDS) analysis of relative abundance pattern of respective bacterial communities based on the taxa assignment, calculated with Bray–Curtis similarity matrix, and (B) Rank differences of the leaf microbiota from C and T plots analyzed at the level of bacterial taxa. Analysis was performed in PAST3 and based on a Bray–Curtis similarity matrix calculation. Box-plots were calculated with the interpolated quartile method. Outliers were not determined. Significant differences were determined by one-way ANOSIM. Asterisk is representing statistical significance: ^∗p < 0.05.

FIGURE 4

Box-plots of alpha diversity indices Chao 1 (A), evenness (B), dominance (C) and Shannon index (D) and rarefaction curves (E) for the phyllosphere community of Galium album leaves from C and T plots. C = ambient temperature (C), T = elevated temperature (T). Triangles = four C plots, squares = four T plots. (E) Rarefaction curves (specimens versus taxa) based on Illumina 16S rRNA gene amplicon sequencing of microbial communities from all C (C 1–4) and T (T 1–4) plots.

FIGURE 5

(A) Principal component analysis (PCA) of relative abundance pattern of respective bacterial communities from Galium album leaves based on the taxa assignment. Eigenvalues for the compared principal components were given in brackets (%) at the respective axes of the graphs. The contribution of different taxonomic groups to the placement of the samples in the PCA plots are indicated as biplots in the graph. Different taxa were numbered with T1-T111 in accordance with their contribution to the differences between leaf microbiota from C and T plots; T1 represents the taxa with the highest contribution. (B) Relative abundance of the most abundant 10 taxa present on Galium album leaves from C and T plots. Asterisks are representing statistical significance: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

FIGURE 6

Relative abundance pattern of bacterial taxa present on Galium album leaves grown in control (C) and warming (T) plots. Analysis were performed at the level of taxonomic paths (resolved up to the genus level). Community pattern were compared by cluster analysis in PAST3 using UPGMA as clustering algorithm and Bray–Curtis similarity calculation. Taxa were sorted by their contribution to the differentiation between leaf microbiota from C and T plots by the SIMPER analysis using PAST. Data are shown for taxa with ≥0.1% contribution.