Andreas Ulrich

Diversity of endophytic bacterial communities in poplar grown under field conditions

Bacterial endophytes may be important for plant health and other ecologically relevant functions of poplar trees. The composition of endophytic bacteria colonizing the aerial parts of poplar was studied using a multiphasic approach. The terminal restriction fragment length polymorphism analysis of 16S rRNA genes demonstrated the impact of different hybrid poplar clones on the endophytic community structure. Detailed analysis of endophytic bacteria using cultivation methods in combination with cloning of 16S rRNA genes amplified from plant tissue revealed a high phylogenetic diversity of endophytic bacteria with a total of 53 taxa at the genus level that included Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. The community structure displayed clear differences in terms of the presence and relative proportions of bacterial taxa between the four poplar clones studied. The results showed that the genetic background of the hybrid poplar clones corresponded well with the endophytic community structure. Out of the 513 isolates and 209 clones identified, Actinobacteria, in particular the family Microbacteriaceae, made up the largest fraction of the isolates, whereas the clone library was dominated by Alpha- and Betaproteobacteria. The most abundant genera among the isolates were Pseudomonas and Curtobacterium, while Sphingomonas prevailed among the clones.

Identification of plant-associated enterococci.

Aims: Enterococcus isolates from forage grass were subjected to taxonomical investigations and tested for antibiotic resistance.

Diversity of grass-associated Microbacteriaceae isolated from the phyllosphere and litter layer after mulching the sward; polyphasic characterization of Subtercola pratensis sp. nov., Curtobacterium herbarum sp. nov. and Plantibacter flavus gen. nov., sp. nov.

A representative selection of coryneform bacteria, isolated from the phyllosphere of grasses and the litter layer after mulching the sward, was characterized by a polyphasic approach to clarify their taxonomic position in the family Microbacteriaceae, with particular reference to potentially plant-pathogenic bacteria. On the basis of 16S rDNA analysis, the isolates can be classified into six genotypes representing the genera Curtobacterium, Clavibacter, Subtercola and a subgroup, which was not affiliated to a known genus. One genotype, belonging to the genus Curtobacterium, had an identical 16S rDNA sequence to reference strains of the Curtobacterium flaccumfaciens pathovars. Another genotype, closely related to the potentially pathogenic Curtobacterium flaccumfaciens, could be distinguished from known species of the genus on the basis of phylogenetic and phenotypic characterization and is consequently proposed as a novel species, Curtobacterium herbarum sp. nov. (type strain P 420/07T DSM 14013T = LMG 19917T). Two genotypes assigned to Clavibacter showed a close relationship to Clavibacter michiganensis subsp. tessellarius, a pathogenic bacterium causing foliar lesions on wheat. A further genotype, which clustered clearly in the genus Subtercola by comparison of 16S rDNA sequences, showed a hitherto undescribed B-type of peptidoglycan containing the diagnostic diamino acids ornithine and 2,4-diaminobutyric acid, in the cell wall; this genotype is proposed as Subtercola pratensis sp. nov. (type strain P 229/10T = DSM 14246T = LMG 21000T). For one genotype, which formed a phylogenetically separate branch in the family of Microbacteriaceae showing chemotaxonomic similarities to the genus Rathayibacter, a novel genus, Plantibacter gen. nov., is proposed; the type species is Plantibacter flavus sp. nov. (type strain P 297/02T = DSM 14012T = LMG 19919T).

A taxonomic study of bacteria isolated from grasses: a proposed new species Pseudomonas graminis sp. nov.

The taxonomic position of a yellow-pigmented group of bacteria, isolated from the phyllosphere of grasses was investigated. Results obtained from restriction analysis of amplified 16S rDNA with seven endonucleases (CfoI, HaeIII, AluI, HinfI, MspI, Sau3A and ScrFI) showed identical restriction patterns for each enzyme of all isolates studied, which suggests that all strains belong to the same species. The grass isolates displayed the characteristics of the genus Pseudomonas. They were Gram-negative, aerobic and rod-shaped with polar flagella. Isolates were catalase-positive and oxidase-negative, and unable to oxidize or ferment glucose with the production of acid. The isolates did not reduce nitrate to nitrite but were able to utilize a wide range of compounds individually as a sole carbon source, with preference being given to the utilization of monosaccharides. The disaccharides tested were not utilized as substrates. The DNA base compositions of the tested strains ranged from 60 to 61 mol% G+C. The major isoprenoid quinone of each was ubiquinone Q-9 and hydroxy fatty acids were represented by 3-hydroxydodecanoic acid and 2-hydroxydodecanoic acid. Comparison of 16S rDNA sequences showed that the bacteria were members of the genus Pseudomonas, with similarity values between 91.5 and 97.7%. DNA-DNA hybridization studies with closely related neighbours revealed a low level of homology (< 27%), indicating that the isolates represent an individual species. On the basis of phenotypic and phylogenetic analyses a new species, Pseudomonas graminis sp. nov. (type strain DSM 11363T), is proposed.

Description of Microbacterium foliorum sp. nov. and Microbacterium phyllosphaerae sp. nov., isolated from the phyllosphere of grasses and the surface litter after mulching the sward, and reclassification of Aureobacterium resistens (Funke et al. 1998) as Microbacterium resistens comb. nov..

The taxonomic position of a group of coryneform bacteria isolated from the phyllosphere of grasses and the surface litter after sward mulching was investigated. On the basis of restriction analyses of 16S rDNA, the isolates were divided into two genotypes. According to the 16S rDNA sequence analysis, representatives of both genotypes were related at a level of 99.2% similarity and clustered within the genus Microbacterium. Chemotaxonomic features (major menaquinones MK-12, MK-11 and MK-10; predominating iso- and anteiso-branched cellular fatty acids; G+C content 64-67 mol%; peptidoglycan-type B2beta with glycolyl residues) corresponded to this genus as well. DNA-DNA hybridization studies showed a reassociation value of less than 70% between representative strains of both subgroups, suggesting that two different species are represented. Although the extensive morphological and physiological analyses did not reveal any differentiating feature for the genotypes, differences in the presence of the cell-wall sugar mannose enabled the subgroups to be distinguished from one another. DNA-DNA hybridization with type strains of closely related Microbacterium spp. indicated that the isolates represent two individual species, which can also be differentiated from previously described species of Microbacterium on the basis of biochemical features. As a result of phenotypic and phylogenetic analyses, the species Microbacterium foliorum sp. nov., type strain P 333/02T (= DSM 12966T = LMG 19580T), and Microbacterium phyllosphaerae sp. nov., type strain P 369/06T (= DSM 13468T = LMG 19581T), are proposed. Furthermore, the reclassification of Aureobacterium resistens (Funke et al. 1998) as Microbacterium resistens (Funke et al. 1998) comb. nov. is proposed.

Characterization of heterotrophic nitrifying bacteria with respiratory ammonification and denitrification activity – Description of Paenibacillus uliginis sp. nov., an inhabitant of fen peat soil and Paenibacillus purispatii sp. nov., isolated from a spacecraft assembly clean room☆

In the course of studying the influence of N-fertilization on N(2) and N(2)O flux rates in relation to soil bacterial community composition of a long-term fertilization experiment in fen peat grassland, a strain group was isolated that was related to a strain isolated from a spacecraft assembly clean room during diversity studies of microorganisms, which withstood cleaning and bioburden reduction strategies. Both the fen soil isolates and the clean room strain revealed versatile physiological capacities in N-transformation processes by performing heterotrophic nitrification, respiratory ammonification and denitrification activity. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that the investigated isolates belonged to the genus Paenibacillus. Sequence similarities lower than 97% in comparison to established species indicated a separate species position. Except for the peptidoglycan type (A4alpha L-Lys-D-Asp), chemotaxonomic features of the isolates matched the genus description, but differences in several physiological characteristics separated them from related species and supported their novel species status. Despite a high 16S rRNA gene sequence similarity between the clean room isolate ES_MS17(T) and the representative fen soil isolate N3/975(T), DNA-DNA hybridization studies revealed genetic differences at the species level. These differences were substantiated by MALDI-TOF MS analysis, ribotyping and several distinct physiological characteristics. On the basis of these results, it was concluded that the fen soil isolates and the clean room isolate ES_MS17(T) represented two novel species for which the names Paenibacillus uliginis sp. nov. (type strain N3/975(T)=DSM 21861(T)=LMG 24790(T)) and Paenibacillus purispatii sp. nov. (type strain ES_MS17(T)=DSM 22991(T)=CIP 110057(T)) are proposed.

Vegetation cover of forest, shrub and pasture strongly influences soil bacterial community structure as revealed by 16S rRNA gene T-RFLP analysis

Bacterial community structure is influenced by vegetation, climate and soil chemical properties. To evaluate these influences, terminal restriction fragment length polymorphism (T-RFLP) and cloning of the 16S rRNA gene were used to analyze the soil bacterial communities in different ecosystems in southwestern China. We compared (1) broad-leaved forest, shrub and pastures in a high-plateau region, (2) three broad-leaved forests representing a climate gradient from high-plateau temperate to subtropical and tropical regions and (3) the humus and mineral soil layers of forests, shrub lands and pastures with open and restricted grazing activities, having varied soil carbon and nutrient contents. Principal component analysis of the T-RFLP patterns revealed that soil bacterial communities of the three vegetation types were distinct. The broad-leaved forests in different climates clustered together, and relatively minor differences were observed between the soil layers or the grazing regimes. Acidobacteria dominated the broad-leaved forests (comprising 62% of the total clone sequences), but exhibited lower relative abundances in the soils of shrub (31%) and pasture (23%). Betaproteobacteria was another dominant taxa of shrub land (31%), whereas Alpha- (19%) and Gammaproteobacteria (13%) and Bacteriodetes (16%) were major components of pasture. Vegetation exerted more pronounced influences than climate and soil chemical properties.

Effects of transgenic fructan-producing potatoes on the community structure of rhizosphere and phyllosphere bacteria

The rhizosphere and phyllosphere microbial communities of transgenic potatoes producing fructan were studied in comparison with isogenic controls and conventional varieties in a field release experiment over a period of 3 years. Population densities and 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) analysis of the rhizosphere bacterial community only displayed the influence of annual and seasonal effects and the influence of field heterogeneity. In contrast, the T-RFLP analysis of the phyllosphere bacteria revealed in two of the 3 years significant differences in the community structure between the transgenic lines producing inulin and the other variants. This effect was studied in more detail through the analysis of bacterial isolates and a 16S rRNA gene clone library obtained from a transgenic line and the control. Both methods revealed a lower genetic diversity in the transgenic line and changes in the abundance of several bacterial groups. The isolates of the transgenic line were dominated by Bacilli, whereas most of the control isolates represented Actinobacteria. The clones were dominated by Proteobacteria, with main differences between both variants in Deltaproteobacteria, Bacilli and Bacteroidetes. However, all in all, the impact of the transgenic lines did not exceed the natural variability of the phyllosphere community structure on potato plants.

Chryseobacterium gregarium sp. nov., isolated from decaying plant material

In this study, strain P 461/12T, isolated from decaying plant material after mulching a pasture, was shown to represent a novel species of the genus Chryseobacterium by means of a polyphasic approach. The closest phylogenetic neighbours to the novel strain, as determined by 16S rRNA gene sequence analysis, were Chryseobacterium daeguense K105T and Chryseobacterium soldanellicola PSD1-4T with gene sequence similarities of 97.4 % and 97.2 %, respectively. Strain P 461/12T could be differentiated by means of its RiboPrint pattern from the type strains of all recognized Chryseobacterium species belonging to the same cluster as determined by 16S rRNA gene sequence comparisons. The nearest phylogenetic neighbours, and in particular the closest relatives C. daeguense and C. soldanellicola, could be distinguished from the novel isolate by means of several physiological features and also by the remarkably lower proportion of anteiso-C15 : 0 in the whole-cell fatty acid profile. Based on these findings, the new isolate represents a novel species, for which the name Chryseobacterium gregarium sp. nov. is proposed. The type strain is P 461/12T (=DSM 19109T=LMG 24052T).

Soil bacterial community structure responses to precipitation reduction and forest management in forest ecosystems across Germany.

Soil microbial communities play an important role in forest ecosystem functioning, but how climate change will affect the community composition and consequently bacterial functions is poorly understood. We assessed the effects of reduced precipitation with the aim of simulating realistic future drought conditions for one growing season on the bacterial community and its relation to soil properties and forest management. We manipulated precipitation in beech and conifer forest plots managed at different levels of intensity in three different regions across Germany. The precipitation reduction decreased soil water content across the growing season by between 2 to 8% depending on plot and region. T-RFLP analysis and pyrosequencing of the 16S rRNA gene were used to study the total soil bacterial community and its active members after six months of precipitation reduction. The effect of reduced precipitation on the total bacterial community structure was negligible while significant effects could be observed for the active bacteria. However, the effect was secondary to the stronger influence of specific soil characteristics across the three regions and management selection of overstorey tree species and their respective understorey vegetation. The impact of reduced precipitation differed between the studied plots; however, we could not determine the particular parameters being able to modify the response of the active bacterial community among plots. We conclude that the moderate drought induced by the precipitation manipulation treatment started to affect the active but not the total bacterial community, which points to an adequate resistance of the soil microbial system over one growing season.