Evelyn Hackl

Comparison of Diversities and Compositions of Bacterial Populations Inhabiting Natural Forest Soils

ABSTRACT The diversity and composition of soil bacterial communities were compared among six Austrian natural forests, including oak-hornbeam, spruce-fir-beech, and Austrian pine forests, using terminal restriction fragment length polymorphism (T-RFLP, or TRF) analysis and sequence analysis of 16S rRNA genes. The forests studied differ greatly in soil chemical characteristics, microbial biomass, and nutrient turnover rates. The aim of this study was to relate these differences to the composition of the bacterial communities inhabiting the individual forest soils. Both TRF profiling and clone sequence analysis revealed that the bacterial communities in soils under Austrian pine forests, representing azonal forest types, were distinct from those in soils under zonal oak-hornbeam and spruce-fir-beech forests, which were more similar in community composition. Clones derived from an Austrian pine forest soil were mostly affiliated with high-G+C gram-positive bacteria (49%), followed by members of the α-Proteobacteria (20%) and the Holophaga/Acidobacterium group (12%). Clones in libraries from oak-hornbeam and spruce-fir-beech forest soils were mainly related to the Holophaga/Acidobacterium group (28 and 35%), followed by members of the Verrucomicrobia (24%) and the α-Proteobacteria (27%), respectively. The soil bacterial communities in forests with distinct vegetational and soil chemical properties appeared to be well differentiated based on 16S rRNA gene phylogeny. In particular, the outstanding position of the Austrian pine forests, which are determined by specific soil conditions, was reflected in the bacterial community composition.

Nitrifiers and denitrifiers respond rapidly to changed moisture and increasing temperature in a pristine forest soil

Complete cycling of mineral nitrogen (N) in soil requires the interplay of microorganisms performing nitrification and denitrification, whose activity is increasingly affected by extreme rainfall or heat brought about by climate change. In a pristine forest soil, a gradual increase in soil temperature from 5 to 25 degrees C in a range of water contents stimulated N turnover rates, and N gas emissions were determined by the soil water-filled pore space (WFPS). NO and N(2)O emissions dominated at 30% WFPS and 55% WFPS, respectively, and the step-wise temperature increase resulted in a threefold increase in the NO(3)(-) concentrations and a decrease in the NH(4)(+) concentration. At 70% WFPS, NH(4)(+) accumulated while NO(3)(-) pools declined, indicating gaseous N loss. AmoA- and nirK-gene-based analysis revealed increasing abundance of bacterial ammonia oxidizers (AOB) with increasing soil temperature and a decrease in the abundance of archaeal ammonia oxidizers (AOA) in wet soil at 25 degrees C, suggesting the sensitivity of the latter to anaerobic conditions. Denitrifier (nirK) community structure was most affected by the water content and nirK gene abundance rapidly increased in response to wet conditions until the substrate (NO(3)(-)) became limiting. Shifts in the community structure were most pronounced for nirK and most rapid for AOA, indicating dynamic populations, whereas distinct adaptation of the AOB communities required 5 weeks, suggesting higher stability.

Dynamics of ammonia-oxidizing communities in barley-planted bulk soil and rhizosphere following nitrate and ammonium fertilizer amendment.

Oxidation of ammonia by nitrifying microorganisms is a major pathway that fertilizer nitrogen (N) may take upon application to agricultural soils, but the relative roles of bacterial (AOB) vs. archaeal (AOA) ammonia oxidizers are controversial. We explored the effects of various forms of mineral N fertilizer on the AOB and AOA community dynamics in two different soils planted with barley. Ammonia oxidizers were monitored via real-time PCR and terminal restriction fragment length polymorphism analysis of bacterial and archaeal amoA genes following the addition of either [NH₄]₂SO₄, NH₄NO₃ or KNO₃. AOB and AOA communities were also studied specifically in the rhizospheres of two different barley varieties upon [NH₄]₂SO₄ vs. KNO₃ addition. AOB changed in community composition and increased in abundance upon ammonium amendment in bulk soil and rhizosphere, with changes in bacterial amoA copy numbers lagging behind relative to changes in soil ammonium. In both soils, only T-RFs corresponding to phylotypes related to Nitrosospira clade 3a underwent significant community changes. Increases in AOB abundance were generally stronger in the bulk soil than in the rhizosphere, implying significant ammonia uptake by plant roots. AOA underwent shifts in the community composition over time and fluctuated in abundance in all treatments irrespective of ammonia availability. AOB were thus considered as the main agents responsible for fertilizer ammonium oxidation, while the functions of AOA in soil N cycling remain unresolved.

Rapid and dissimilar response of ammonia oxidizing archaea and bacteria to nitrogen and water amendment in two temperate forest soils

Biochemical processes relevant to soil nitrogen (N) cycling are performed by soil microorganisms affiliated with diverse phylogenetic groups. For example, the oxidation of ammonia, representing the first step of nitrification, can be performed by ammonia oxidizing bacteria (AOB) and, as recently reported, also by ammonia oxidizing archaea (AOA). However, the contribution to ammonia oxidation of the phylogenetically separated AOA versus AOB and their respective responsiveness to environmental factors are still poorly understood. The present study aims at comparing the capacity of AOA and AOB to momentarily respond to N input and increased soil moisture in two contrasting forest soils. Soils from the pristine Rothwald forest and the managed Schottenwald forest were amended with either NH(4)(+)-N or NO(3)(-)-N and were incubated at 40% and 70% water-filled pore space (WFPS) for four days. Nitrification rates were measured and AOA and AOB abundance and community composition were determined via quantitative PCR (qPCR) and terminal restriction length fragment polymorphism (T-RFLP) analysis of bacterial and archaeal amoA genes. Our study reports rapid and distinct changes in AOA and AOB abundances in the two forest soils in response to N input and increased soil moisture but no significant effects on net nitrification rates. Functional microbial communities differed significantly in the two soils and responded specifically to the treatments during the short-term incubation. In the Rothwald soil the abundance and community composition of AOA were affected by the water content, whereas AOB communities responded to N amendment. In the Schottenwald soil, by contrast, AOA responded to N addition. These results suggest that AOA and AOB may be selectively influenced by soil and management factors.

Greenhouse gas fluxes respond to different N fertilizer types due to altered plant-soil-microbe interactions

The application of inorganic nitrogen (N) fertilizers strongly influences the contribution of agriculture to the greenhouse effect, especially by potentially increasing emissions of nitrous oxide (N2O), carbon dioxide (CO2) and methane (CH4) from soils. The present microcosm-study investigates the effect of different forms of inorganic N fertilizers on greenhouse gas (GHG) emissions from two different agricultural soils. The relationship between greenhouse gas emissions and soil microbial communities, N transformation rates and plant (Hordeum vulgare L. cv. Morex) growth were investigated. Repeated N fertilization led to increased N2O emissions. In a parallel survey of functional microbial population dynamics we observed a stimulation of bacterial and archaeal ammonia oxidisers accompanied with these N2O emissions. The ratio of archaeal to bacterial ammonium monooxygenase subunit A (amoA) gene copies (data obtained from Inselsbacher et al., 2010) correlated positively with N2O fluxes, which suggests a direct or indirect involvement of archaea in N2O fluxes. Repeated N fertilization also stimulated methane oxidation, which may also be related to a stimulation of ammonia oxidizers. The fertilizer effects differed between soil types: In the more organic Niederschleinz soil N-turnover rates increased more strongly after fertilization, while in the sandy Purkersdorf soil plant growth and soil respiration were accelerated depending on fertilizer N type. Compared to addition of NH4+ and NO3−, addition of NH4NO3 fertilizer resulted in the largest increase in global warming potential as a summary indicator of all GHG related effects. This effect resulted from the strongest increase of both N2O and CO2 emission while plant growth was not equally stimulated, compared to e.g. KNO3 fertilization. In order to decrease N losses from agricultural ecosystems and in order to minimize soil derived global warming potential, this study points to the need for interdisciplinary investigations of the highly complex interactions within plant-soil-microbe-atmosphere systems. By understanding the microbial processes underlying fertilizer effects on GHG emissions the N use efficiency of crops could be refined.

A cost-effective high-throughput microcosm system for studying nitrogen dynamics at the plant- microbe-soil interface

In the present study a new microcosm system was evaluated for its suitability to investigate nitrogen dynamics between soils, plants and microbes. Five different agricultural soils were homogenized and transferred in the test tubes, and kept under controlled conditions in a climate chamber for 4weeks. Soils differed clearly in nitrogen pools and microbial population structures but less in their activities. Bacterial and fungal community compositions and soil properties, except gross N transformation rates, remained stable and reproducible during the test period in all soils. 15N tracer studies showed that N uptake patterns of barley as well as plant growth were linear in the initial growth period. Overall, the presented microcosm system proved to be a powerful tool to elucidate N pathways in soil-plant-microbe systems. In future studies the microcosm system may greatly help generating new insights in the complex processes and controls of nitrogen biogeochemical cycle in agricultural systems.

Microbial Communities Show Parallels at Sites with Distinct Litter and Soil Characteristics

ABSTRACT Plant and microbial community composition in connection with soil chemistry determines soil nutrient cycling. The study aimed at demonstrating links between plant and microbial communities and soil chemistry occurring among and within four sites: two pine forests with contrasting soil pH and two grasslands of dissimilar soil chemistry and vegetation. Soil was characterized by C and N content, particle size, and profiles of low-molecular-weight compounds determined by high-performance liquid chromatography (HPLC) of soil extracts. Bacterial and actinobacterial community composition was assessed by terminal restriction fragment length polymorphism (T-RFLP) and cloning followed by sequencing. Abundances of bacteria, fungi, and actinobacteria were determined by quantitative PCR. In addition, a pool of secondary metabolites was estimated by erm resistance genes coding for rRNA methyltransferases. The sites were characterized by a stable proportion of C/N within each site, while on a larger scale, the grasslands had a significantly lower C/N ratio than the forests. A Spearmans test showed that soil pH was correlated with bacterial community composition not only among sites but also within each site. Bacterial, actinobacterial, and fungal abundances were related to carbon sources while T-RFLP-assessed microbial community composition was correlated with the chemical environment represented by HPLC profiles. Actinobacteria community composition was the only studied microbial characteristic correlated to all measured factors. It was concluded that the microbial communities of our sites were influenced primarily not only by soil abiotic characteristics but also by dominant litter quality, particularly, by percentage of recalcitrant compounds.

Epidemic multidrug-resistant (MDR-AmpC) Salmonella enterica serovar Newport strains contain three phage regions and a MDR resistance plasmid

Multidrug-resistant (MDR-AmpC) Salmonella enterica serovar Newport has caused serious disease in animals and humans in North America, whereas in the UK S. enterica serovar Newport is not associated with severe disease and usually sensitive to antibiotics; MDR S. Newport (not AmpC) strains have only been isolated from poultry. We found that UK poultry strains belonged to MLST type ST166 and were distinct from cattle isolates for being able to utilize D-tagotose and when compared by pulsed-field gel electrophoresis (PFGE), comparative genomic hybridization (CGH) and diversity arrays technology (DArT). Cattle strains belonged to the ST45 complex differing from ST166 at all seven loci. PFGE showed that 19 out of 27 cattle isolates were more than 85% similar to each other and some UK and US strains were indistinguishable. Both CGH and DArT identified genes (including phage-related ones) that were uniquely present in the US isolates and two such genes identified by DArT showed sequence similarities with the pertussis-like (artAB) toxin. This work demonstrates that MDR-AmpC S. Newport from the USA are genetically closely related to pan-susceptible strains from the UK, but contained three extra phage regions and a MDR plasmid.

Nutrient Turnover, Greenhouse Gas Exchange and Biodiversity in Natural Forests of Central Europe

We measured microbial turnover of carbon (C) and nitrogen (N) in 12 natural forest reserves in Austria, and estimated potential emission rates of nitrous oxide (N2O) and carbon dioxide (CO2), and uptake rates of methane (CH4). The community composition of soil microorganisms was investigated using PLFA (phospholipid fatty acid) analysis and molecular tools, and we examined the biodiversity of selected taxa of micro-, meso- and macrofauna. These characterizations of natural forests provide reference data for evaluating soil biology in managed, especially disturbed or damaged forests. Ecophysio-logical quotients were tested for their ability to make predictions about the carbon dynamics of forest soils. The 12 forests represented the six typical types in Central Europe: oak, beech, spruce-fir-beech, floodplain, and pine forests. Nitrogen turnover rates were high in moist soils with high pH. Nitrogen losses as nitrate or N2O were small unless N deposition exceeded 30 kg ha−1 yr−1. The fastest turnover of C and N occurred in the floodplain forests, based on microbial quotients, xylanase activity, the relative thickness of litter layer and 15N abundance in the organic soil. Carbon turnover was slowest in the beech forests on acidic bedrock, and slow turnover may lead to the largest net C accumulation. Tree species had distinct effects on microbial communities, but high soil biodiversity in these natural forests may not be greater than in managed forests.

Phage-type specific markers identified by Diversity Arrays Technology (DArT) analysis of Salmonella enterica ssp. enterica serovars Enteritidis and Typhimurium

Diversity Arrays Technology (DArT) was applied to differentiate between S. enterica serovar Enteritidis and Typhimurium strains, respectively. Ten and eleven, mainly phage and plasmid-related markers were identified for serovars Enteritidis and Typhimurium. In combination, these markers can be used for subtyping among and within phage types.