The effect of elevated atmospheric CO2 concentrations on the abundance and community composition of heterotrophic and methylotrophic bacteria in the phyllosphere of abundant plant species of the permanent grassland ecosystem

Abstract

The consequences of global climate change are currently a major problem. Elevated CO2 concentration in the atmosphere can affect plants, bacterial communities in the phyllosphere and plant-bacteria interactions. Phyllosphere bacteria play an important role in plant health and growth. In this study, it was hypothesized that long-term elevated CO2 concentration (17 years) affects the abundance and composition of oligo/heterotrophic and methylotrophic bacterial communities of the phyllosphere of abundant plant species (A. elatius, G. album) of the permanent grassland ecosystem of the Giessen free-air carbon dioxide enrichment (GiFACE) system: The abundance of Sphingomonas, Pseudomonas and Methylobacterium spp. will increase due to the increase in plant substrate (sugar, methanol). Increased CO2 concentration will lead to an adaptation of oligo/heterotrophic and methylotrophic bacteria, especially to a functional adaptation. To prove these hypotheses, oligo/heterotrophic and methylotrophic bacteria of the phyllosphere of both plant species (A. elatius, G. album) were cultivated for the first time using the dilution-to-extinction method in 96-well microtiter plates. The concentration of cultured bacteria was determined using the most probable number (MPN) method. The shift in the composition of the most enriched bacteria assemblages were presented using denaturing gradient gel electrophoreses (DGGE) and non-metric multidimensional scaling (NMDS) approaches. For analysis of a bacterial adaptation, the most abundant enriched bacteria were first isolated and then phylogenetically identified (using partial 16S rRNA gene sequence analysis). Isolates were assigned to phylotypes, and their occurrence was correlated with environmental factors (eCO2, plants) was presented using canonical correspondence analysis (CCA). Pink-pigmented, facultative methylotrophic (PPFM) phylotypes, as abundant inhabitants of the phyllosphere, were analyzed at functional adaptation using changes of partial nucleotide sequences and especially amino acid sequences of mxaF-gene to find specifically adapted ecotypes. Another aim of this study was to establish a highly efficient DNA extraction method for the phyllosphere-associated bacteria of the two plant species (A. elatius, G. album) without plant co-extracts (chloroplast and mitochondrial DNA) to enable the analysis of the phyllosphere microbiota of the two plant species using cultivation-independent methods (e.g. DGGE, next-generation-sequencing (NGS)) without co-amplification. For this establishment, direct DNA extraction (such as cetyltrimethylammonium bromide (CTAB)-methods, various commercial kits) and alternative DNA extraction (bacterial cells harvesting before DNA extraction) methods on total fresh and frozen, frozen-homogenized, as well as freeze-dried leaves were tested. For alternative DNA extraction methods, bacterial cells were first homogenized or detached from the leaves and then collected by a centrifugation density gradient medium (Percoll, Nycodenz), centrifugation or Sterivex filtration with/without prefilter, and then DNA was extracted using kit or CTAB method. The efficiency of the DNA methods was checked on co-extracts by polymerase chain reaction (PCR) with the universal bacterial primer systems. The results showed that long-term elevated CO2 concentration affected the composition and abundance of the cultured oligo/heterotrophic and methylotrophic bacterial communities of the two plant species (A. elatius, G. album). The abundance of oligo/heterotrophic and methylotrophic bacteria of the two plant species was different and plant genotype specific. Sphingomonas and Pseudomonas were the most abundant genera only on G. album leaves and their abundance increased under elevated CO2 concentrations. Methylobacterium spp. were the most abundant methylotrophs on the two plants and their abundance was not affected by elevated CO2 concentrations. Among the PPFM phylotypes, single plant-specific isolates were found, indicating a potential functional plant-specific adaptation and potential genetic adaptation to elevated CO2 concentrations. No highly efficient DNA extraction method without co-extracts could be established from A. elatius and G. album leaves. DNA extraction using the NucleoSpin® for Soil Kit (Macherey-Nagel) was the most efficient method and showed the best detection of extracted bacterial DNA from the G. album leaves in the presence of co-extracts and proved to be reliable. The kit was one of the few kits that allowed bacterial community analysis of the two plants (A. elatus, G. album) after the integrated co-extract removal step. This study was the first to demonstrate a correlation between the long-term elevated CO2 effect and the oligo/heterotrophic and methylotrophic bacterial communities of A. elatius and G. album phyllosphere and provided the first indication for the potential functional adaptation of PPFMs to elevated CO2 concentrations.