Global warming shifts the composition of the abundant bacterial phyllosphere microbiota as indicated by a cultivation-dependent and -independent study of the grassland phyllosphere of a long-term warming field experiment.

The leaf-colonizing bacterial microbiota was studied in a long-term warming experiment on a permanent grassland, which had been continuously exposed to increased surface temperature (+2°C) for more than six years. Two abundant plant species, Arrhenatherum elatius and Galium album, were studied. Surface warming reduced stomata opening and changed leaf metabolite profiles. Leaf surface colonization and the concentration of leaf-associated bacterial cells were not affected. However, bacterial 16S ribosomal RNA (rRNA) gene amplicon Illumina sequencing showed significant temperature effects on the plant species-specific phyllosphere microbiota. Warming partially affected the concentrations of cultured bacteria and had a significant effect on the composition of most abundant cultured plant species-specific bacteria. The abundance of Sphingomonas was significantly reduced. Sphingomonas isolates from warmed plots represented different phylotypes, had different physiological traits and were better adapted to higher temperatures. Among Methylobacterium isolates, a novel phylotype with a specific mxaFtype was cultured from plants of warmed plots while the most abundant phylotype cultured from control plots was strongly reduced. This study clearly showed a correlation of long-term surface warming with changes in the plant physiology and the development of a physiologically and genetically adapted phyllosphere microbiota.

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

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.

Proposal of Mucilaginibacter galii sp. nov. isolated from leaves of Galium album.

A pale-pink-pigmented, Gram-stain-negative, rod-shaped, non-spore-forming bacterial strain, PP-F2F-G47T, was isolated from the phyllosphere of the herbaceous plant Galium album. Phylogenetic analysis based on the nearly full-length 16S rRNA gene sequence revealed highest sequence similarity to the type strains of Mucilaginibacter daejeonensis (96.2 %), Mucilaginibacter dorajii (95.7 %) and Mucilaginibacter phyllosphaerae (95.5 %). 16S rRNA gene sequence similarities to all other type strains were below 95.5 %. The predominant cellular fatty acids of the strain were C16 : 1ω7c/iso-C15 : 0 2-OH (measured as summed feature 3) and iso-C15 : 0. The major compound in the polyamine pattern was sym-homospermidine and major quinone was menaquinone MK-7. The polar lipid profile was composed of phosphatidylethanolamine and several unidentified aminolipipids, phospholipids, aminophospholipids and lipids without a functional group. A sphingophospholipid could not be detected but a ninhydrin-positive alkaline-stable lipid was visible. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. Based on phylogenetic, chemotaxonomic and phenotypic analyses a novel species is proposed, Mucilaginibacter galii sp. nov., with PP-F2F-G47T (=CCM 8711T=CIP 111182T=LMG 29767T) as the type strain.

Proposal of Mucilaginibacter phyllosphaerae sp. nov. isolated from the phyllosphere of Galium album.

A pink-pigmented, Gram-stain-negative, rod-shaped, non-spore-forming bacterial strain, PP-F2F-G21T, was isolated from the phyllosphere of Galium album. Phylogenetic analysis of the nearly full-length 16S rRNA gene sequence of strain PP-F2F-G21T showed the closest relationship to type strains of the species Mucilaginibacter lutimaris (97.7 %), Mucilaginibacter soli (97.3 %) and Mucilaginibacter rigui (97.1 %). Sequence similarities to all other type strains were below 97 %. The predominant cellular fatty acids of strain PP-F2F-G21T are C16 : 1 ω7c/iso-C15 : 0 2-OH (measured as summed feature 3 fatty acids) and iso-C15 : 0 followed by iso-C17 : 0 3-OH, C16 : 1 ω5c and C16 : 0. The major compound in the polyamine pattern was sym-homospermidine and the diamino acid of the peptidoglycan was meso-diaminopimelic acid. The quinone system was exclusively composed of menaquinone MK-7. The polar lipid profile contained the major lipid phosphatidylethanolamine and in addition 18 unidentified lipids. Based on phylogenetic, chemotaxonomic and phenotypic analyses, we propose a novel species of the genus Mucilaginibacter named Mucilaginibacter phyllosphaeraesp. nov. The type strain is PP-F2F-G21T (=CCM 8625T=CIP 110921T=LMG 29118T).

Aureimonas galii sp. nov. and Aureimonas pseudogalii sp. nov. isolated from the phyllosphere of Galium album.

Four yellow-pigmented, Gram-stain-negative, rod-shaped bacteria, strains PP-WC-4G-234T, PP-CE-2G-454T, PP-WC-1G-202 and PP-CC-3G-650, were isolated from the phyllosphere of Galium album. The strains shared 99.7-100 % 16S rRNA gene sequence similarity but could be differentiated by genomic fingerprinting using rep- and random amplification of polymorphic DNA PCRs. Phylogenetic analysis based on the 16S rRNA gene placed the strains within the family Aurantimonadaceae with highest 16S rRNA gene sequence similarity of 97.2-97.3 % to the type strain of Aureimonas phyllosphaerae. Sequence similarities to all other Aurantimonadaceae were below 97 %. The main cellular fatty acids of the strains were C18 : 1ω7c as the predominant fatty acid followed by C16 : 0 and summed feature 3 (C16 : 1ω7c/C16 : 1ω8c). The polyamine patterns of strains PP-WC-4G-234T and PP-CE-2G-454T contained sym-homospermidine as a major compound, and the major respiratory quinone was ubiquinone Q-10. Predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, phosphatidylcholine, sulfoquinovosyldiacylglycerol, three unidentified phospholipids and one unidentified lipid only detectable after total lipid staining. The DNA G+C content was 66.4, 68.9, 67.4 and 70.5 mol% for strains PP-WC-4G-234T, PP-CE-2G-454T, PP-WC-1G-202 and PP-CC-3G-650, respectively. Based on phylogenetic, chemotaxonomic and phenotypic analyses we propose two novel species of the genus Aureimonas, Aureimonas galii sp. nov. with PP-WC-4G-234T (=LMG 28655T=CIP 110892T) as the type strain and Aureimonas pseudogalii sp. nov. with PP-CE-2G-454T (=LMG 29411T=CCM 8665T) as the type strain and two further strains representing the same species, PP-WC-1G-202 and PP-CC-3G-650.