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Some European ash trees show tolerance towards dieback caused by the invasive pathogen

Three strains (H4-D09T, S2-D11 and S9-F39) of a member of the genus Paracoccus attributed to a novel species were isolated from topsoil of temperate grasslands. The genome sequence of the type strain H4-D09T exhibited a complete set of genes required for denitrification as well as methylotrophy. The genome of H4-D09T included genes for two alternative pathways of formaldehyde oxidation. Besides the genes for the canonical glutathione (GSH)-dependent formaldehyde oxidation pathway, all genes for the tetrahydrofolate–formaldehyde oxidation pathway were identified. The strain has the potential to utilize methanol and/or methylamine as a single carbon source as evidenced by the presence of methanol dehydrogenase (mxaFI) and methylamine dehydrogenase (mau) genes. Apart from dissimilatory denitrification genes (narA, nirS, norBC and nosZ), genes for assimilatory nitrate (nasA) and nitrite reductases (nirBD) were also identified. The results of phylogenetic analysis based on 16S rRNA genes coupled with riboprinting revealed that all three strains represented the same species of genus Paracoccus . Core genome phylogeny of the type strain H4-D09T indicated that Paracoccus thiocyanatus and Paracoccus denitrificans are the closest phylogenetic neighbours. The average nucleotide index (ANI) and digital DNA–DNA hybridization (dDDH) with the closest phylogenetic neighbours revealed genetic differences at the species level, which were further substantiated by differences in several physiological characteristics. The major respiratory quinone is Q-10, and the predominant cellular fatty acids are C18 : 1ω7c, C19 : 0cyclo ω7c, and C16 : 0, which correspond to those detected in other members of the genus. The polar lipid profile consists of a diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), aminolipid (AL), glycolipid (GL) and an unidentified lipid (L). On the basis of our results, we concluded that the investigated isolates represent a novel species of the genus Paracoccus , for which the name Paracoccus methylovorus sp. nov. (type strain H4-D09T=LMG 31941T= DSM 111585T) is proposed.

Microorganisms acting as sinks for the greenhouse gas nitrous oxide (N

A group of isolates of the genus Luteimonas was characterised, which represented a specific component of the healthy core microbiome of Fraxinus excelsior in forest districts with a high infection rate of H. fraxineus, the causal agent of ash dieback. Based on phylogenomic and phenotypic analyses, a clear differentiation from related Luteimonas species was shown. Comparisons of the overall genome relatedness indices with the closest phylogenetic neighbours resulted in values below the recommended species cut-off levels. In addition, differences in several physiological and chemotaxonomic traits allowed a clear demarcation from the type strains of closely related species. Conclusively, the strain group was considered to represent a novel species in the genus Luteimonas, for which the name Luteimonas fraxinea sp. nov. is proposed, with strain D4P002

Some European ash trees show tolerance towards dieback caused by the invasive pathogen Hymenoscyphus fraxineus. The microbiome of these trees harbours a range of specific bacterial groups. One of these groups belonging to the species Aureimonas altamirensis was studied in detail by genome analysis and a plant inoculation trial. The strain group was shown to be phylogenetically distinct from clinical isolates by 16S rRNA analysis and phylogenomics. Genome analysis of a representative strain C2P003 resulted in a large number of unique gene sequences in comparison to other well-studied strains of the species. A functional analysis of the genome revealed features associated with the synthesis of exopolysaccharides, protein secretion and biofilm production as well as genes for stress adaptation, suggesting the ability of C2P003 to effectively colonize ash leaves. The inoculation of ash seedlings with C2P003 showed a significant positive effect on the plant health of the seedlings that were exposed to H. fraxineus infection. This effect was maintained over a period of three years and was accompanied by a significant shift in the bacterial microbiome composition one year after inoculation. Overall, the results indicate that C2P003 may suppress H. fraxineus in or on ash leaves via colonization resistance or indirectly by affecting the microbiome.

Fluorescent pseudomonads colonizing wheat ears have a high antagonistic potential against phytopathogenic fungi. To check this hypothesis, the bacterial antagonist Pseudomonas simiae 9 rif+/kan+ was spray-inoculated onto the ears of winter wheat in a locally demarcated experimental field plot. Fusarium and Alternaria fungi naturally occurring on the ears and the formation of their mycotoxins in the ripe grains were investigated. Inoculated bacteria were recovered from the plants in the inoculation cell, but not in the untreated neighboring plots or in the air above the plants. Growth of fusaria and alternaria on the ears was not influenced by the bacterial antagonist. Wheat kernels were co-inoculated in vitro with the antagonist and one mycotoxin-producing strain of Fusarium and Alternaria, respectively. Mycotoxin production was almost completely suppressed in these approaches. Concentrations of zearalenone, deoxynivalenol, alternariol, and tenuazonic acid were also significantly reduced in ripe grains in the field, but to a lesser extent than in vitro. The results of this and previous studies suggest that widespread biological control of the growth of fusaria and alternaria and their mycotoxin formation by naturally occurring pseudomonads with antagonistic activity is rather unlikely.

Peat soils can be strong sources of atmospheric nitrous oxide (N2O), but at the same time act as sinks for the greenhouse gas N2O. However, the role of N2O reduction to dinitrogen (N2) here is still not fully understood. In particular, this applies to pristine or weakly disturbed fen mires. These types of peatland ecosystems are characterised by anoxic soil conditions and special N dynamics restricted to ammonium (NH4+) turnover and very low nitrate (NO3−) availability. N2O and N2 fluxes from intact soil cores from three weakly disturbed fen mire types and two soil habitats (tussocks and hollows) were investigated using the helium (He) incubation approach. Ambient air in headspaces were first substituted with a He-O2 trace gas mixture to quantify N2O and N2 exchanges under prevailing soil oxygen (O2) conditions, and then with an anoxic He trace gas mixture (99.9% He) for establishing the maximum possible denitrification rate. Changing from the He-O2 mixture to a pure He trace gas mixture led to strong increase of N2 fluxes (up to 2916 μg N m−2 h−1) and negative N2O fluxes of up to −72 μg N m−2 h−1. Whilst small differences in N gas fluxes were found between all types of fen mires, an analysis of the denitrifier abundance based on nirK, nirS and nosZ genes indicated respectively more pronounced variation. The structure of denitrifier communities exhibited a strong plot specificity driven by water-filled pore space, soil organic matter and soil pH. This short-term He incubation experiment revealed that weakly disturbed fen mires act as considerable N2O sinks under anoxic conditions and improved our knowledge of the original N dynamics in this peatland ecosystem.

The genera

The genera Proteus and Cosenzaea are closely related members of the family Morganellaceae . The genus Cosenzaea consists of the species Cosenzaea myxofaciens originally separated from the genus Proteus by rpoB gene analysis. Due to the high similarity of the 16S rRNA genes between species of both genera, the taxonomic status is here re-evaluated by a genome-based approach. Based on a core genome phylogeny and genome relatedness indices, it is shown that the taxonomy and nomenclature given for the basonym Proteus myxofaciens is more appropriate. Therefore, we propose to use this name in preference. Furthermore, the species status of Proteus terrae and Proteus cibarius was reassessed. Both species are related at subspecies level by digital DNA–DNA hybridization (dDDH) analysis. Additionally, average amino acid identity (AAI) and average nucleotide identity (ANI) do not support a separate species status, and therefore it is proposed to classify P. cibarius as a subspecies of P. terrae . Consequently, both species are being renamed Proteus terrae subsp. cibarius subsp. nov. and Proteus terrae subsp. terrae subsp. nov., respectively. The genome relatedness indices revealed a close relationship of the Proteus genomospecies 5 with P. terrae subsp. terrae. Thus, it has been assigned to the same subspecies.

Most vegetated land on earth has been used for agriculture for hundreds of years, shaping the land’s features and functions. Agricultural (rural) landscapes are the basis for feeding the global population and meeting their many other demands. In the globalised world of the twenty-first century, agriculture is facing some crucial challenges: feeding more people whilst maintaining natural resources, biodiversity and landscape heritage, and mitigating climate change. This requires new knowledge, innovations and decision tools for taking responsible action. Research on agricultural landscapes provides analyses to help understand landscape processes and elaborates sustainable scientific, technical and cultural solutions. We report on recent progress in some topics of measuring, modelling and monitoring processes, and on organismic diversity in agricultural landscapes, focusing on developments in Western Europe. Information is also given about long-term experimental agricultural research in Russia and neighbouring countries. Results indicate that there has been considerable progress in understanding landscape processes based on experimental and modelling research on different scales. A number of landscape-related research methods and tools have been developed in recent years. These include high-tech measurement systems, models and decision aids, and tools for monitoring and optimising agricultural systems and for agri-environmental monitoring. Many of them also have potential for successfully tackling existing problems with the sustainability of agricultural systems in other landscapes and regions. Transdisciplinary international research projects are required to make innovations operable.

In the last few years, the alarming spread of Hymenoscyphus fraxineus, the causal agent of ash dieback, has resulted in a substantial threat to native ash stands in central and northern Europe. Since leaves and leaf petioles are the primary infection sites, phyllosphere microorganisms are presumed to interact with the pathogen and are discussed as a source of biocontrol agents. We studied compound leaves from susceptible and visible infection-free trees in four ash stands with a high likelihood of infection to assess a possible variation in the bacterial microbiota, depending on the health status of the trees. The bacterial community was analyzed by culture-independent 16S rRNA gene amplicon sequencing and through the isolation and taxonomic classification of 2,589 isolates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The bacterial community structure did not show significant differences. However, a set of amplicon sequence variants (ASVs) and MALDI groups belonging to Luteimonas, Aureimonas, Pseudomonas, Bacillus, and Paenibacillus were distinctly increased in tolerant trees, which may be associated with the ability of the tree to resist the pathogen. The most obvious differences were observed for Luteimonas, a genus that is also exclusively present in the healthy core microbiome. In a first in vitro screen of antagonists, approximately 11% of total isolates suppressed the growth of H. fraxineus, but a statistical test with two different H. fraxineus strains confirmed only the antagonistic activity of 8% of these isolates. The antagonistic isolates were assigned to Bacillus velezensis, Pantoea vagans, and Pseudomonas caspiana. Overall, our study provides a set of isolates or phylogenetic groups that might be involved in the process that prevents the penetration and spread of H. fraxineus. In the next step, in planta experiments are required with a longer period of exposure to H. fraxineus to evaluate effective isolates or consortia of isolates acting through direct antagonism or competition or indirectly by inducing resistance.

In the last few years, the alarming spread of Hymenoscyphus fraxineus, the causal agent of ash dieback, has resulted in a substantial threat to native ash stands in central and northern Europe. Since leaves and leaf petioles are the primary infection sites, phyllosphere microorganisms are presumed to interact with the pathogen and are discussed as a source of biocontrol agents. We studied compound leaves from susceptible and visible infection-free trees in four ash stands with a high likelihood of infection to assess a possible variation in the bacterial microbiota, depending on the health status of the trees. The bacterial community was analyzed by culture-independent 16S rRNA gene amplicon sequencing and through the isolation and taxonomic classification of 2,589 isolates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The bacterial community structure did not show significant differences. However, a set of amplicon sequence variants (ASVs) and MALDI groups belonging to Luteimonas, Aureimonas, Pseudomonas, Bacillus, and Paenibacillus were distinctly increased in tolerant trees, which may be associated with the ability of the tree to resist the pathogen. The most obvious differences were observed for Luteimonas, a genus that is also exclusively present in the healthy core microbiome. In a first in vitro screen of antagonists, approximately 11% of total isolates suppressed the growth of H. fraxineus, but a statistical test with two different H. fraxineus strains confirmed only the antagonistic activity of 8% of these isolates. The antagonistic isolates were assigned to Bacillus velezensis, Pantoea vagans, and Pseudomonas caspiana. Overall, our study provides a set of isolates or phylogenetic groups that might be involved in the process that prevents the penetration and spread of H. fraxineus. In the next step, in planta experiments are required with a longer period of exposure to H. fraxineus to evaluate effective isolates or consortia of isolates acting through direct antagonism or competition or indirectly by inducing resistance.

Worldwide excessive use of chemical pesticides with harmful side effects to environment and humans has increased the call for a pesticide-free agriculture. Biological control of plant pathogens by antagonistic microorganisms may be an alternative. These should also include plant-associated pseudomonads. They are adapted to live on and inside plants due to their diverse metabolic and ecological abilities. Many Pseudomonas strains exhibit a diverse spectrum of antagonistic traits enabling them to suppress various phytopathogens by direct antibiosis and/or by triggering defence responses in the host plants. Although a certain proportion of strains within the indigenous populations of pseudomonads on and inside plants are able to do so, the spread of these bacteria in the field is rather restricted and individuals with antagonistic activity are unevenly and patchily distributed. Natural biological control of fungal and bacterial plant diseases by pseudomonads possibly supported by adapted management systems therefore, seems unlikely. However, selected Pseudomonas strains can be artificially introduced into the soil–plant ecosystem and have proven to be promising microbial antagonists. But, more substantial in-depth studies to improve the efficacy and survival of biocontrol agents are needed to use them in pesticide-free agriculture on a field scale. Potential biocontrol strains have to undergo a targeted selection process that also takes into account their mode of action. Their application in consortia of multiple strains or with other microbial taxa that are complementary in their action may have advantages in biocontrol efficiency compared to single strains. Biotechnological solutions have to ensure both efficient antagonistic performance and sufficient persistence of the introduced bacteria in the field. With today's knowledge, seed inoculation seems to be a practicable approach to exploit the biocontrol potential of pseudomonads in crop farming.

Natural control of phytopathogenic microorganisms is assumed as a priority function of the commensal plant microbiota. In this study, the suitability of fluorescent pseudomonads in the phyllosphere of crop plants as natural control agents was evaluated. Under field conditions, ears of winter wheat were found to be colonized with high consistency and at a high density by pseudomonads at the late milk dough stage. Isolates of these bacteria were evaluated for their potential to protect the plants from phytopathogenic Alternaria and Fusarium fungi. More Pseudomonas isolates were antagonistically active against alternaria than against fusaria in the dual culture test. The alternaria responded species-specifically and more sensitively to bacterial antagonism than the strain-specific reacting fusaria. A total of 110 randomly selected Pseudomonas isolates were screened for genes involved in the biosynthesis of the antibiotics 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid, pyoluteorin, and pyrrolnitrin. The key gene for production of the phloroglucinol was found in none of these isolates. At least one of the genes, encoding the biosynthesis of the other antibiotics was detected in 81% of the isolates tested. However, the antagonistic effect found in the dual culture assay was not necessarily associated with the presence of these antibiotic genes. Wheat grains as natural substrate were inoculated with selected antagonistic Pseudomonas isolates and Alternaria and Fusarium strains, respectively. The fungal growth was only slightly delayed, but the mycotoxin production was significantly reduced in most of these approaches. In conclusion, the distribution of phytopathogenic fungi of the genera Alternaria and Fusarium in the field is unlikely to be inhibited by naturally occurring pseudomonads, also because the bacterial antagonists were not evenly distributed in the field. However, pseudomonads can reduce the production of Alternaria and Fusarium mycotoxins in wheat grains and thus have the potential to improve the crop quality.

Fluorescent pseudomonads isolated from wheat leaves were characterized regarding their antagonistic potential and taxonomy in relation to protect crop plants from infestation by Fusarium and Alternaria fungi causing diseases in wheat. Using a dual culture assay, inhibition of fungal growth was found for 40 isolates of 175 fluorescent pseudomonads. Twenty-two of the antagonists were able to suppress strains of Fusarium as well as Alternaria. By means of real-time qPCR, the phlD gene encoding the antibiotic 2,4-diacetylphloroglucinol was detected in 20 isolates. On the basis of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry spectral patterns, the isolates with antagonistic activity were assigned to the phylogenetic subgroup Pseudomonas fluorescens and the closely related Pseudomonas gessardii subgroup. The results of the study suggest that pseudomonads in the phyllosphere of crop plants may possibly contribute to natural plant protection.

In the context of studying the influence of N-fertilization on N2 and N2O flux rates in relation to the soil bacterial community composition in fen peat grassland, a group of bacterial strains was isolated that performed dissimilatory nitrate reduction to ammonium and concomitantly produced N2O. The amount of nitrous oxide produced was influenced by the C/N ratio of the medium. The potential to generate nitrous oxide was increased by higher availability of nitrate-N. Phylogenetic analysis based on the 16S rRNA and the rpoB gene sequences demonstrated that the investigated isolates belong to the genus Proteus, showing high similarity with the respective type strains of Proteus vulgaris and Proteus penneri. DNA-DNA hybridization studies revealed differences at the species level. These differences were substantiated by MALDI-TOF MS analysis and several distinct physiological characteristics. On the basis of these results, it was concluded that the soil isolates represent a novel species for which the name Proteus terrae sp. nov. (type strain N5/687(T) =DSM 29910(T) =LMG 28659(T)) is proposed.

In the context of studying the bacterial community involved in nitrogen transformation processes in arable soils exposed to different extents of erosion and sedimentation in a long-term experiment (CarboZALF), a strain was isolated that reduced nitrate to nitrous oxide without formation of molecular nitrogen. The presence of the functional gene nirK, encoding the respiratory copper-containing nitrite reductase, and the absence of the nitrous oxide reductase gene nosZ indicated a truncated denitrification pathway and that this bacterium may contribute significantly to the formation of the important greenhouse gas N2O. Phylogenetic analysis based on the 16S rRNA gene sequence and the housekeeping genes recA and atpD demonstrated that the investigated soil isolate belongs to the genus Rhizobium. The closest phylogenetic neighbours were the type strains of Rhizobium. subbaraonis and Rhizobium. halophytocola. The close relationship with R. subbaraonis was reflected by similarity analysis of the recA and atpD genes and their amino acid positions. DNA-DNA hybridization studies revealed genetic differences at the species level, which were substantiated by analysis of the whole-cell fatty acid profile and several distinct physiological characteristics. Based on these results, it was concluded that the soil isolate represents a novel species of the genus Rhizobium, for which the name Rhizobium azooxidifex sp. nov. (type strain Po 20/26T=DSM 100211T=LMG 28788T) is proposed.

The taxonomic position of a group of actinobacterial strains isolated from the phyllosphere of potato plants was investigated by using a polyphasic approach. Although the similarity values for their 16S rRNA gene sequences suggested an intermediate position between Microbacterium and Agrococcus, the phylogenetic tree demonstrated a clear clustering of the representative strain, K 114/01(T), within the genus Agrococcus. The presence of 2,4-diaminobutyric acid as the diagnostic diamino acid in the cell-wall peptidoglycan of strain K 114/01(T) substantiated the affiliation to the genus Agrococcus. An analysis performed using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry revealed highly similar spectral patterns for the isolated strains, which, together with their conformity regarding a multitude of phenotypic features, supported their affiliation to the same species. Differences in several physiological features, peptidoglycan and menaquinone composition and whole-cell fatty acid profiles enabled discrimination of the phyllosphere isolates with respect to recognized Agrococcus species. As the 16S rRNA gene sequence similarity values were below 97 %, the strains isolated from the phyllosphere of potato plants represent a novel species of the genus Agrococcus, for which the name Agrococcus versicolor sp. nov. is proposed. The type strain is K 114/01(T) (=DSM 19812(T) =LMG 24386(T)).

In the course of studying the composition of the microbial community in the phyllosphere of potato plants, a Gram-positive bacterium, strain K 70/01(T), was isolated. On the basis of 16S rRNA gene sequence analysis, it was shown that the investigated isolate represents a novel species in the genus Leucobacter; the closest phylogenetic neighbour was Leucobacter chromiireducens subsp. chromiireducens, with a sequence similarity of 96.8 % to the type strain. Chemotaxonomic characteristics supported affiliation to the genus Leucobacter. Strain K 70/01(T) displayed the diagnostic diamino acid 2,4-diaminobutyric acid in the cell-wall peptidoglycan. The major fatty acids were anteiso-C(15 : 0,) anteiso-C(17 : 0) and iso-C(16 : 0), which are typical of the genus. The quinone system consisted of the menaquinones MK-10, MK-11 and MK-9. Differences in several physiological features and the absence of the cell-wall amino acids gamma-aminobutyric acid and threonine enabled the isolate to be differentiated from all recognized Leucobacter species. Based on these phylogenetic and phenotypic results, it can be concluded that the isolate from the phyllosphere of potato plants represents a novel species, for which the name Leucobacter tardus sp. nov. is proposed. The type strain is K 70/01(T) (=DSM 19811(T) =LMG 24388(T)).

Three isolates obtained from grass samples were investigated by means of a polyphasic taxonomic study and were shown to represent a novel species within the genus Chryseobacterium. Comparison of 16S rRNA gene sequences and phenotypic features indicated that the three isolates belonged to a single species. On the basis of 16S rRNA gene sequence analysis, the closest phylogenetic neighbours were Chryseobacterium shigense and Chryseobacterium vrystaatense, which formed a stable cluster with the isolates; this phylogeny was supported by a high bootstrap value and was obtained using different treeing methods. A DNA–DNA hybridization study with the closest neighbour, C. shigense DSM 17126T (98.3 % 16S rRNA gene sequence similarity), clearly demonstrated a separate species status for the grass isolate strain P 456/04T. Comparisons involving physiological properties and whole-cell fatty acid profiles confirmed this result at the phenotypic level. On the basis of these results, strain P 456/04T represents a novel species of the genus Chryseobacterium, for which the name Chryseobacterium luteum sp. nov. is proposed. The type strain is P 456/04T (=DSM 18605T =LMG 23785T).

Comparative analysis of 16S rDNA sequences revealed a close phylogenetic relationship (99.6 % similarity) between Subtercola pratensis Behrendt et al. 2002 and Agreia bicolorata Evtushenko et al. 2001. The two species were found to share genus-specific chemotaxonomic characteristics such as the occurrence of D-ornithine and L-2,4-diaminobutyric acid in the peptidoglycan and the profile of cellular fatty acids and 1,1-dimethoxy-alkanes. DNA-DNA relatedness of only 47.8 % and differences in phenotypic features such as the menaquinone profile and oxidase and Voges-Proskauer reactions confirmed the distinct species status of S. pratensis and A. bicolorata. On the basis of the data from phylogenetic and phenotypic analyses, the reclassification of S. pratensis as Agreia pratensis comb. nov. is proposed. As a result of this reclassification, the two genera are coherent, in that the cell wall composition and 1,1-dimethoxy-alkane spectrum are significant genus-specific characteristics.

The community composition of epiphytic heterotrophic bacteria on leaves of beech and oak, which were either damaged by lepidopterous larvae or remained undamaged, was investigated. In addition, the ability of these bacteria to utilize inorganic nitrogen was studied. The bacteria were isolated on nutrient agar and systematically identified with biochemical and physiological tests. Rarefaction plots and the Shannon-Wiener function revealed that species diversity was significantly higher on leaves of damaged beech compared to undamaged leaves, but no differences were found on leaves of oak. The portion of bacterial isolates showing a strong response to ammonia and nitrate was significantly larger on leaves of oak than on those of beech. Furthermore, significantly more isolates with a high capability to assimilate both nitrogen compounds were found on leaves attacked by the folivorous larvae compared to those not attacked on oak. It is suggested that the changes in the microbial community in response to folivorous insects might affect the extent of nutrient cycling exceeding eventually the scale of a leaf.

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).

Mikroorganismen‐Gesellschaften auf dem Grunland konnen sich in Abhangigkeit von Umweltfaktoren hinsichtlich ihrer Populationsstruktur und ‐dynamik verandern und so die Futterqualitat der Aufwuchse und folgender Konservierungsprodukte beeinflussen. Die Populationsentwicklung relevanter eukaryotischer und prokaryotischer Organismengruppen, die zur autochthonen Mikroflora gehoren oder als potenziell tierpathogen bekannt sind, wird unter Berucksichtigung von Einfiussfaktoren behandelt und deren mogliche Auswirkungen auf Qualitatsparameter des Futters diskutiert. Abschliesend wird auf Grund des bisherigen Kenntnisstandes weiterer Forschungsbedarf abgeleitet, um eine Bewertung von Bewirtschaftungsmasnahmen des Grunlandes mit Blick auf die mikrobiologische Qualitat des Futters zu ermoglichen.

Phyllospharen‐ und Rhizospharenbakterien (Pseudomonas spp., Pantoea spp., Agrobacterium spp., Rhizobium spp. bzw. Stenotrophomonas spp.) stimulierten unabhangig von der Herkunft (Getreide, Mais, Graser, Raps, Rotklee, Kiefern) das Wachstum von W.‐Weizen, Mais und Erbsen in gemasigten Klimaten. Sie uberlebten nach Spros‐ und Wurzelinokulation bevorzugt im Rhizospharenraum. Auch Phyllospharenbakterien wanderten nach Sprosinokulation in die Rhizosphare ab. In gemasigten Klimaten sind Wachstumsstimulierungen nicht an eine Nitrogenaseaktivitat der Bakterien gebunden. Phytoeffektive Bakterien bildeten meist Phytohormone, Auxine und z.T. Cytokinine, die in den bevorzugt besiedelten Seitenwurzelzonen und Wurzelspitzen das Wurzelwachstum und die Nahrstoffaufnahme aus dem Boden forderten. Das wirkte sich auch positiv auf den Chlorophyllgehalt in Maisblattern aus. Mogliche Unterschiede zur Bedeutung des Besiedlungsverhaltens und mikrobieller Stoffwechselleistungen in tropischen Klimaten werden diskutiert.

In einer kurzen Ubersicht werden die Besiedlung der Oberflache gruner Blatter (Phyllosphare) durch Bakterien, Hefen und filamentose Pilze, die sie beeinflussenden okologischen Faktoren, die Bedeutung dieser Mikoorganismen fur den naturlichen Stoffhaushalt und fur die Gesunderhaltung der Kulturpflanzenbestande sowie mikrobiologisch bedingte okonomische Aspekte bei der Verwertung der Pflanzen angesprochen. Daraus ableitend werden Forschungsdefizite hinsichtlich der Erfassung der mikrobiellen Diversitat in der Phyllosphare, der Beziehungen zwischen Phyllospharen‐Mikroorganis‐men und Stoffkreislaufen in Ackerland, Grunland und Wald sowie der Auswirkungen von Landnutzungsanderungen auf die mikrobiellen Organismen‐Gemeinschaften aufgezeigt.

Es werden an Beispielen aktuelle Forschungsarbeiten des ZALF, insbesondere des Institutes fur Mikrobielle Okologie und Bodenbiologie, zu den Moglichkeiten des genetischen Monitorings diskutiert. Dabei erfolgt die Betrachtung in zwei grundsatzlich verschiedenen Untersuchungsebenen, zum einen die standortbezogene, vergleichende Bewertung von Einflusfaktoren auf die genetische Anpassungsfahigkeit von ausgewahlten Populationen und zum anderen die Analyse der Besiedlungsstruktur auf regionaler Ebene. In zwei mehrjahrig angelegten Experimenten konnte die genetische Anpassung der Rhizobium‐Population auf eine Gulleablagerung und der VA‐Mykorrhiza auf eine mineralische Stickstoffdungung an zwei Standorten nachgewiesen werden. Diese eindeutigen und sensitiven Reaktionen der mikrobiellen Populationen erlauben die Abschatzung von mikroevolutionaren Entwicklungsrichtungen (Ward u.a. 1998). Daraus leitet sich die Frage ab, welche Faktoren die genetische Struktur von Populationen in Landschaften beeinflussen, wie sich ...

Comparing different methods of grassland management, the microbial community of grasses was analysed throughout the growing season. The quantitative development of heterotrophic bacteria, yeasts, filamentous fungi, streptomycetes and Listeria has been investigated. Furthermore, the community structure of heterotrophic bacteria was analysed by identifying isolated strains by means of classical biochemical and morphological examination supported by an application of multitest systems based on enzymatic reactions (Biolog, API). The population of heterotrophic bacteria and filamentous fungi was influenced by the age of plant material as well as by the density in the field. Thus, bacteria and fungi reached significantly larger numbers on plants of the late cut utilization (first cut at the beginning of July) in comparison with the regrowth of conventional cutting in mid May. These higher densities of micro-organisms exceeded threshold values that has been fixed for feedstuff s and therefore suggest an affect on the forage quality of this material. The population of yeasts neither showed a continual development over the season nor seemed to be influenced by any kind of management. Streptomycetes were only determined in larger numbers at the beginning of sampling in April, followed by a sporadical occurrence in the following course of investigation. Species of the genera Listeria have not been found at any time of sampling. The community structure of heterotrophic bacteria was numerically predominated by the genera Pseudomonas, Stenotrophomonas, Pantoea, Clavibacter and Curtobacterium . Generally, the population was characterized by low diversity. An influence of management on the composition of bacteria did not become obvious.

A total of 256 strains of lactic acid bacteria isolated from forage grasses were screened for antagonistic activities under conditions where the effects of organic acids and hydrogen peroxide were minimised. Thirty seven strains were shown to inhibit the growth of other lactic acid bacteria in the agar spot test with viable cells. Cell free supernatants from 16 of these strains, most of them belonging to the species Lactobacillus plantarum and Enterococcus faecium , exhibited inhibitory activity against indicator strains. Crude protein extracts containing different bacteriocin-like substances were made from cell free supernatants of Lactobacillus plantarum P1331, Enterococcus faecium P5977 and Leuconostoc mesenteroides P6007, and sensitivity to these extracts of 53 strains of lactic acid bacteria and 25 strains of other phylloplane bacteria were tested. Forty four strains of lactic acid bacteria, and all but one strain of the Gram-positive heterotrophic aerobic bacteria tested were inhibited by at least one of the antagonists. All Gram-negative strains tested were resistant. Inhibitory activity against sensitive indicators was also detected when the strains P1331, P5977 and P6007 grew on “phylloplane agar”, a medium which simulates the low level nutrient conditions found on the phylloplane, in a temperature range between 4 and 37°C. It therefore seems to be likely that strains of lactic acid bacteria able to produce bacteriocin-like substances can influence the development of the population of lactic acid bacteria on plants.

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.

In the context of studying the effects of transgenic fructan-producing potatoes on the community structure of phyllosphere bacteria, a group of strains closely related to the species Leifsonia ginsengi was isolated. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the new isolates and L. ginsengi DSM 19088T formed a lineage at the genus level and this finding was supported by chemotaxonomic characterization. The peptidoglycan type of the representative isolate, K134/01T, and L. ginsengi DSM 19088T was B2γ, with d- and l-diaminobutyric acid as the diagnostic diamino acid and glycine, alanine and threo-3-hydroxyglutamic acid. The almost-complete substitution of glutamic acid by threo-3-hydroxyglutamic acid supported the differentiation of the new strains from recognized species of the genus Leifsonia. Furthermore, the detection of substantial amounts of the fatty acid cyclohexyl-C17 : 0 in the new isolates and L. ginsengi DSM 19088T was a prominent chemotaxonomic feature for a clear demarcation of these strains from all genera of the family Microbacteriaceae that display the B2γ cell-wall type. Comparative phylogenetic and phenotypic analyses of the isolates and L. ginsengi DSM 19088T revealed the separate species status of the isolates. On the basis of these results, it is proposed that L. ginsengi should be classified as the type species of a novel genus, Herbiconiux gen. nov., with the name Herbiconiux ginsengi gen. nov., comb. nov. (type strain wged11T = CGMCC 4.3491T = JCM 13908T = DSM 19088T = NBRC 104580T). The phyllosphere isolates are assigned to a novel species, Herbiconiux solani sp. nov. (type strain K134/01T = DSM 19813T = LMG 24387T = NBRC 106740T).

The activity of the leucine arylamidase (EC 3.4.11.2) was measured in washings of green needles from the canopy and dead needles from the litter of Scots pine throughout one year. It was highest in the litter and markedly higher in 2-year-old needles than in young ones, which were colonized by only a few bacteria. The leucine arylamidase activity largely arose from microbial epiphytes. Screenings for the potential of the enzyme activity among strains of a collection of phyllosphere microorganisms isolated from forest trees revealed that the leucine arylamidase activity was more abundant among bacteria (79%) and yeasts (57%) than among filamentous fungi, whereas the opposite was true in degrading complex proteins by proteinases.

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.

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).

The taxonomic position of a group of fluorescent pseudomonad strains isolated from the phyllosphere of grasses was investigated through a polyphasic approach. Riboprinting analysis revealed highly similar patterns for the investigated strains which supported, together with the agreement of many phenotypic characteristics, their affiliation to the same species. A comparison of 16S rRNA gene sequences of strain P 513/18T, a representative strain from the grass isolates, revealed that it was affiliated to the cluster of the ‘Pseudomonas fluorescens group’, with Pseudomonas costantinii as the closest phylogenetic neighbour. However, DNA–DNA hybridization showed a clear demarcation at the species level between strain P 513/18T and P. costantinii. Furthermore, a comparison of riboprint patterns with Pseudomonas species clustering next to the novel grass isolates on the basis of 16S rRNA gene sequences supported their separate species status at the phylogenetic level. Based on phenotypic features, the novel isolates could also be differentiated from the other fluorescent Pseudomonas species that share positive arginine dihydrolase and oxidase reactions. As a consequence of these phenotypic and phylogenetic analyses, the isolates from the grass pyllosphere represent a novel species for which the name Pseudomonas lurida sp. nov. is proposed. The type strain is P 513/18T (=DSM 15835T=LMG 21995T).

Milchsaurebakterien (MSB) sind die entscheidenden Mikroorganismen der Silierung. Sie kommen normalerweise in der epiphytischen Mikroflora von Futterpflanzen vor und gelangen zusammen mit anderen Keimgruppen in den Silo, wo sie unter siliertechnisch gunstigen Ausgangsbedingungen durch rasche Vermehrung und aktiven Stoffwechsel den konservierenden Prozes der Futtergarung erwirken.

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.

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 (Cfol, Haelll, Alul, Hinfl, Mspl, Sau3A and ScrFl) 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.