Doreen Fischer

Molecular characterisation of the diazotrophic bacterial community in uninoculated and inoculated field-grown sugarcane (Saccharum sp.)

To identify active diazotrophs in sugarcane, 16S rRNA and nifH transcript analyses were applied. This should help to better understand the basis of the biological nitrogen fixation (BNF) activity of a high nitrogen fixing sugarcane variety. A field experiment using the sugarcane variety RB 867515 was conducted in Seropédica, RJ, Brazil, receiving the following treatments: unfertilised and fertilised controls without inoculation, unfertilised with inoculation. The five-strain mixture developed by EMBRAPA-CNPAB was used as inoculum. Root and leaf sheath samples were harvested in the third year of cultivation to analyse the 16S rRNA and nifH transcript diversity. In addition to nifH expression from Gluconacetobacter spp. and Burkholderia spp., a wide diversity of nifH sequences from previously uncharacterised Ideonella/Herbaspirillum related phylotypes in sugarcane shoots as well as Bradyrhizobium sp. and Rhizobium sp. in roots was found. These results were confirmed using 16S cDNA analysis. From the inoculated bacteria, only nifH transcripts from G. diazotrophicus and B. tropica were detected in leaf sheaths and roots. Known as well as yet uncultivated diazotrophs were found active in sugarcane roots and stems using molecular analyses. Two strains of the inoculum mix were identified at the late summer harvest.

Dynamic regulation of N‐acyl‐homoserine lactone production and degradation in Pseudomonas putida IsoF

The biocontrol strain Pseudomonas putida IsoF, which was isolated from a tomato rhizosphere, is a known N-acyl-homoserine lactone (AHL) producer with only one LuxI/LuxR-like quorum-sensing (QS) system. The production and degradation of AHLs were analysed in different growth phases of the bacterium. Using the analytical tools of ultra performance liquid chromatography and high resolution MS, it was possible to determine not only the various AHLs synthesized over time but also their degradation products. 3-oxo-decanoyl-homoserine lactone was found to be the dominant AHL, which reached its maximum in the early logarithmic growth phase. Although the pH of the medium was neutral, the AHLs were degraded thereafter rapidly to the corresponding homoserines and other metabolites. The proposed lactonase gene of P. putida IsoF could not be identified, because it is apparently quite different from hitherto described lactonases. The analytical data were used to calculate the rates and thresholds of AHL production by mathematical modelling, allowing quantitative predictions and a further understanding of the QS-based regulations in this bacterium. This study, combining microbiological, chemical and mathematical approaches, suggests that AHL degradation is an integral part of the whole autoinducer circuit of P. putida IsoF.

Restoration of Growth of Durum Wheat (Triticum durum var. waha) Under Saline Conditions Due to Inoculation with the Rhizosphere Bacterium Azospirillum brasilense NH and Extracts of the Marine Alga Ulva lactuca

Inoculation with the rhizosphere bacterium Azospirillum brasilense NH, originally isolated from salt-affected soil in northern Algeria, greatly enhanced growth of durum wheat (Triticum durum var. waha) under saline soil conditions. Important plant parameters like the rate of germination, stem height, spike length, dry weight of roots and shoots, chlorophyll a and b content, proline and total sugar contents, 1000-seed weight, seed number per spike, and weight of seeds per spike were measured. At salt stress conditions (160 and 200xa0mM NaCl) A. brasilense NH restored almost completely vegetative growth and seed production. The combination with extracts of the marine alga Ulva lactuca resulted in even more improved salt tolerance of durum wheat. Proline and total sugar accumulation, a sign of physiological plant stress under inhibitory salt conditions, was reduced in plants inoculated with A. brasilense NH with and without addition of algal extracts. Inoculation with the salt-sensitive A. brasilense strain Sp7 could not restore salt-affected plant growth at 200xa0mM NaCl. Furthermore, it could be demonstrated by fluorescence in situ hybridization and confocal laser scanning microscopy that A. brasilense NH is able to colonize roots of durum wheat endophytically under salt-stressed conditions. Thus, the salt-tolerant rhizobacterium A. brasilense NH could effectively provide alone or in combination with extracts of U. lactuca a promising solution to overcome salt inhibition which is a major threat hindering productive wheat cultivation in arid saline soils.

Biogeography and phylogenetic diversity of a cluster of exclusively marine myxobacteria.

Myxobacteria are common in terrestrial habitats and well known for their formation of fruiting bodies and production of secondary metabolites. We studied a cluster of myxobacteria consisting only of sequences of marine origin (marine myxobacteria cluster, MMC) in sediments of the North Sea. Using a specific PCR, MMC sequences were detected in North Sea sediments down to 2.2u2009m depth, but not in the limnetic section of the Weser estuary and other freshwater habitats. In the water column, this cluster was only detected on aggregates up to a few meters above the sediment surface, but never in the fraction of free-living bacteria. A quantitative real-time PCR approach revealed that the MMC constituted up to 13% of total bacterial 16S rRNA genes in surface sediments of the North Sea. In a global survey, including sediments from the Mediterranean Sea, the Atlantic, Pacific and Indian Ocean and various climatic regions, the MMC was detected in most samples and to a water depth of 4300u2009m. Two fosmids of a library from sediment of the southern North Sea containing 16S rRNA genes affiliated with the MMC were sequenced. Both fosmids have a single unlinked 16S rRNA gene and no complete rRNA operon as found in most bacteria. No synteny to other myxobacterial genomes was found. The highest numbers of orthologues for both fosmids were assigned to Sorangium cellulosum and Haliangium ochraceum. Our results show that the MMC is an important and widely distributed but largely unknown component of marine sediment-associated bacterial communities.

pH as a Driver for Ammonia-Oxidizing Archaea in Forest Soils

In this study, we investigated the impact of soil pH on the diversity and abundance of archaeal ammonia oxidizers in 27 different forest soils across Germany. DNA was extracted from topsoil samples, the amoA gene, encoding ammonia monooxygenase, was amplified; and the amplicons were sequenced using a 454-based pyrosequencing approach. As expected, the ratio of archaeal (AOA) to bacterial (AOB) ammonia oxidizers’ amoA genes increased sharply with decreasing soil pH. The diversity of AOA differed significantly between sites with ultra-acidic soil pH (<3.5) and sites with higher pH values. The major OTUs from soil samples with low pH could be detected at each site with a soil pH <3.5 but not at sites with pH >4.5, regardless of geographic position and vegetation. These OTUs could be related to the Nitrosotalea group 1.1 and the Nitrososphaera subcluster 7.2, respectively, and showed significant similarities to OTUs described from other acidic environments. Conversely, none of the major OTUs typical of sites with a soil pH >4.6 could be found in the ultra- and extreme acidic soils. Based on a comparison with the amoA gene sequence data from a previous study performed on agricultural soils, we could clearly show that the development of AOA communities in soils with ultra-acidic pH (<3.5) is mainly triggered by soil pH and is not influenced significantly by the type of land use, the soil type, or the geographic position of the site, which was observed for sites with acido-neutral soil pH.

Spatial variability of hydrolytic and oxidative potential enzyme activities in different subsoil compartments

The spatial heterogeneity of nutrient turnover in subsoils has been rarely studied in the past, although drilosphere and rhizosphere are found to be important microbial hotspots in this oligotrophic environment. In this study, we measured different potential enzyme activities in different soil compartments of subsoil and topsoil. It could be shown that the activities of hydrolases, which cleave readily available organic substrates, are significantly higher in samples from the drilosphere and rhizosphere both in topsoil and subsoil. In bulk soil, hydrolase activities decrease with depth. In contrast, oxidative enzymes, which are involved in the decay of recalcitrant organic material, are released from the microbial community especially in the bulk fraction of subsoil. This emphasizes the importance of subsoil for nutrient acquisition and gives evidence for a distinct spatial separation of microbes with diverging lifestyles.

Endophytic Bradyrhizobium spp. isolates from sugarcane obtained through different culture strategies.

Brazilian sugarcane has been shown to obtain part of its nitrogen via biological nitrogen fixation (BNF). Recent reports, based on the culture independent sequencing of bacterial nifH complementary DNA (cDNA) from sugarcane tissues, have suggested that members of the Bradyrhizobium genus could play a role in sugarcane-associated BNF. Here we report on the isolation of Bradyrhizobium spp. isolates and a few other species from roots of sugarcane cultivar RB867515 by two cultivation strategies: direct isolation on culture media and capture of Bradyrhizobium spp. using the promiscuous legume Vigna unguiculata as trap-plant. Both strategies permitted the isolation of genetically diverse Bradyrhizobium spp. isolates, as concluded from enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) fingerprinting and 16S ribosomal RNA, nifH and nodC sequence analyses. Several isolates presented nifH phylotypes highly similar to nifH cDNA phylotypes detected in field-grown sugarcane by a culture-independent approach. Four isolates obtained by direct plate cultivation were unable to nodulate V.u2009unguiculata and, based on PCR analysis, lacked a nodC gene homologue. Acetylene reduction assay showed in vitro nitrogenase activity for some Bradyrhizobium spp. isolates, suggesting that these bacteria do not require a nodule environment for BNF. Therefore, this study brings further evidence that Bradyrhizobium spp. may play a role in sugarcane-associated BNF under field conditions.

Short-term dynamics of bacterial communities in a tidally affected coastal ecosystem.

Tidal effects on the composition of free-living (FL) and particle-associated (PA) bacterial communities were studied in a tidal flat ecosystem in the southern North Sea. Denaturing gradient gel electrophoresis targeting the 16S rRNA gene and the 16S rRNA of Bacteria, Bacteroidetes, Alphaproteobacteria and the Roseobacter clade was applied. Despite strong tidal variations in the quantity and, depending on the season, also the quality of suspended matter as well as variations in bacterial activity, the bacterial community composition remained rather stable. FISH showed some variations of the community composition, but these were not related to typical tidal situations. Variations were higher during tidal cycles in May and July compared with November. Bacteroidetes, Alpha- and Gammaproteobacteria constituted the majority of the bacterial communities but relative proportions of the different groups varied considerably. On particles, Betaproteobacteria were also detected to substantial proportions. The Roseobacter clade constituted up to 90% of FL but only 30% of PA Alphaproteobacteria. Banding patterns of the Bacteroidetes-specific amplicons, and in particular those targeting the 16S rRNA, revealed tidally induced effects, as several bands appeared or disappeared at distinct events such as slack water or resuspension. Sequencing of prominent bands revealed predominantly phylotypes reported previously from this ecosystem.

Prokaryotes in Subsoil-Evidence for a Strong Spatial Separation of Different Phyla by Analysing Co-occurrence Networks.

Microbial communities in soil provide a wide range of ecosystem services. On the small scale, nutrient rich hotspots in soil developed from the activities of animals or plants are important drivers for the composition of microbial communities and their functional patterns. However, in subsoil, the spatial heterogeneity of microbes with differing lifestyles has been rarely considered so far. In this study, the phylogenetic composition of the bacterial and archaeal microbiome based on 16S rRNA gene pyrosequencing was investigated in the soil compartments bulk soil, drilosphere, and rhizosphere in top- and in the subsoil of an agricultural field. With co-occurrence network analysis, the spatial separation of typically oligotrophic and copiotrophic microbes was assessed. Four bacterial clusters were identified and attributed to bulk topsoil, bulk subsoil, drilosphere, and rhizosphere. The bacterial phyla Proteobacteria and Bacteroidetes, representing mostly copiotrophic bacteria, were affiliated mainly to the rhizosphere and drilosphere—both in topsoil and subsoil. Acidobacteria, Actinobacteria, Gemmatimonadetes, Planctomycetes, and Verrucomicrobia, bacterial phyla which harbor many oligotrophic bacteria, were the most abundant groups in bulk subsoil. The bacterial core microbiome in this soil was estimated to cover 7.6% of the bacterial sequencing reads including both oligotrophic and copiotrophic bacteria. In contrast the archaeal core microbiome includes 56% of the overall archaeal diversity. Thus, the spatial variability of nutrient quality and quantity strongly shapes the bacterial community composition and their interaction in subsoil, whereas archaea build a stable backbone of the soil prokaryotes due to their low variability in the different soil compartments.

Abundance of ammonia oxidizing microbes and denitrifiers in different soil horizons of an agricultural soil in relation to the cultivated crops

The role of subsoils and their microbial communities for the nutrient supply for plants is to a large extent unknown, especially in comparison to well investigated topsoil layers. Therefore, in this study, the influence of three different plant species with different rooting systems and different N uptake strategies on ammonium and nitrate levels and microbial communities involved in ammonia oxidation and denitrification was investigated in different soil horizons. Overall, our results show a higher genetic potential for both processes in topsoils than in subsoils independent of the present plant. Although we found accumulation of N in top and subsoils in plots with legumes, we could not observe an impact of the higher nitrate content on the genetic potential of denitrification and ammonia oxidation. However, differences in the ratios of ammonia oxidizing archaea to bacteria and also between denitrifying bacteria harboring genes for copper- (nirK) or cytochrome- (nirS) dependent nitrite reductase in top and subsoil samples reveal different ecophysiologies of microbes involved in N turnover in top and subsoil habitats.