Accumulation of sulfonamide resistance genes in arable soils due to repeated application of manure containing sulfadiazine.

Two soils were amended three times with pig manure. The abundance of sulfonamide resistance genes was determined by quantitative PCR 2 months after each application. In both soils treated with sulfadiazine-containing manure, the numbers of copies of sul1 and sul2 significantly increased compared to numbers after treatments with antibiotic-free manure or a control and accumulated with repeated applications.

Changes in diversity and functional gene abundances of microbial communities involved in nitrogen fixation, nitrification, and denitrification in a tidal wetland versus paddy soils cultivated for different time periods.

In many areas of China, tidal wetlands have been converted into agricultural land for rice cultivation. However, the consequences of land use changes for soil microbial communities are poorly understood. Therefore, we investigated bacterial and archaeal communities involved in inorganic nitrogen turnover (nitrogen fixation, nitrification, and denitrification) based on abundances and relative species richness of the corresponding functional genes along a soil chronosequence ranging between 50 and 2,000 years of paddy soil management compared to findings for a tidal wetland. Changes in abundance and diversity of the functional groups could be observed, reflecting the different chemical and physical properties of the soils, which changed in terms of soil development. The tidal wetland was characterized by a low microbial biomass and relatively high abundances of ammonia-oxidizing microbes. Conversion of the tidal wetlands into paddy soils was followed by a significant increase in microbial biomass. Fifty years of paddy management resulted in a higher abundance of nitrogen-fixing microbes than was found in the tidal wetland, whereas dominant genes of nitrification and denitrification in the paddy soils showed no differences. With ongoing rice cultivation, copy numbers of archaeal ammonia oxidizers did not change, while that of their bacterial counterparts declined. The nirK gene, coding for nitrite reductase, increased with rice cultivation time and dominated its functionally redundant counterpart, nirS, at all sites under investigation. Relative species richness showed significant differences between all soils with the exception of the archaeal ammonia oxidizers in the paddy soils cultivated for 100 and 300 years. In general, changes in diversity patterns were more pronounced than those in functional gene abundances.

Effect of sulfadiazine-contaminated pig manure on the abundances of genes and transcripts involved in nitrogen transformation in the root-rhizosphere complexes of maize and clover.

The antibiotic sulfadiazine (SDZ) can enter the environment by application of manure from antibiotic-treated animals to arable soil. Because antibiotics are explicitly designed to target microorganisms, they likely affect microbes in the soil ecosystem, compromising important soil functions and disturbing processes in nutrient cycles. In a greenhouse experiment, we investigated the impact of sulfadiazine-contaminated pig manure on functional microbial communities involved in key processes of the nitrogen cycle in the root-rhizosphere complexes (RRCs) of maize (Zea mays) and clover (Trifolium alexandrinum). At both the gene and transcript level, we performed real-time PCR using nifH, amoA (in both ammonia-oxidizing bacteria and archaea), nirK, nirS, and nosZ as molecular markers for nitrogen fixation, nitrification, and denitrification. Sampling was performed 10, 20, and 30 days after the application. SDZ affected the abundance pattern of all investigated genes in the RRCs of both plant species (with stronger effects in the RRC of clover) 20 and 30 days after the addition. Surprisingly, effects on the transcript level were less pronounced, which might indicate that parts of the investigated functional groups were tolerant or resistant against SDZ or, as in the case of nifH and clover, have been protected by the nodules.

Effect of sulfadiazine on abundance and diversity of denitrifying bacteria by determining nirK and nirS genes in two arable soils.

Sulfadiazine (SDZ) is an antibiotic frequently used in agricultural husbandry. Via manuring of excrements of medicated animals, the drug reaches the soil and might impair important biochemical transformation processes performed by microbes, e.g., the nitrogen turnover. We studied the effect of pig manure and SDZ-spiked pig manure on denitrifying bacteria by quantifying nirK and nirS nitrite reductase genes in two arable soils. Addition of manure entailed mainly an increase of nirK-harboring denitrifiers in both soils, whereas in the SDZ-amended treatments, primarily the nirS denitrifiers increased in abundance after the bioavailable SDZ had declined. However, the community composition of nirS nitrite reducers investigated by denaturing gradient gel electrophoresis did not change despite the observed alterations in abundance.

Abundance of microbes involved in nitrogen transformation in the rhizosphere of Leucanthemopsis alpina (L.) Heywood grown in soils from different sites of the Damma glacier forefield.

Glacier forefields are an ideal playground to investigate the role of development stages of soils on the formation of plant-microbe interactions as within the last decades, many alpine glaciers retreated, whereby releasing and exposing parent material for soil development. Especially the status of macronutrients like nitrogen differs between soils of different development stages in these environments and may influence plant growth significantly. Thus, in this study, we reconstructed major parts of the nitrogen cycle in the rhizosphere soil/root system of Leucanthemopsis alpina (L.) HEYWOOD: as well as the corresponding bulk soil by quantifying functional genes of nitrogen fixation (nifH), nitrogen mineralisation (chiA, aprA), nitrification (amoA AOB, amoA AOA) and denitrification (nirS, nirK and nosZ) in a 10-year and a 120-year ice-free soil of the Damma glacier forefield. We linked the results to the ammonium and nitrate concentrations of the soils as well as to the nitrogen and carbon status of the plants. The experiment was performed in a greenhouse simulating the climatic conditions of the glacier forefield. Samples were taken after 7 and 13 weeks of plant growth. Highest nifH gene abundance in connection with lowest nitrogen content of L. alpina was observed in the 10-year soil after 7 weeks of plant growth, demonstrating the important role of associative nitrogen fixation for plant development in this soil. In contrast, in the 120-year soil copy numbers of genes involved in denitrification, mainly nosZ were increased after 13 weeks of plant growth, indicating an overall increased microbial activity status as well as higher concentrations of nitrate in this soil.

Effects of repeated application of sulfadiazine-contaminated pig manure on the abundance and diversity of ammonia and nitrite oxidizers in the root-rhizosphere complex of pasture plants under field conditions.

In a field experiment, the impact of repeated application of the antibiotic sulfadiazine (SDZ)-contaminated pig manure was assessed on functional microbial communities involved in ammonia and nitrite oxidation in the root-rhizosphere complexes (RRCs) of diverse plants composing a pasture. We surveyed the abundance of ammonia-oxidizing archaea (AOA) and bacteria (AOB) as well as Nitrobacter- and Nitrospira-like nitrite-oxidizing bacteria (NOB) by quantitative PCR (qPCR), and the diversity of amoA AOA and Nitrobacter-like nxrA amplicons using a cloning-sequencing approach. Whereas the first SDZ-contaminated manure application caused only slight effects on the investigated microbial communities and did not change the diversity and abundance pattern significantly, the second application of SDZ-contaminated manure induced an up to 15-fold increased ratio of AOA:AOB and a reduction of nrxA genes. The diversity of AOA amoA increased after the second application of SDZ-contaminated manure compared to the control treatment whereas a clear reduction of nrxA OTUs was visible in the same samples. The results indicate that the application of SDZ may principally affect nitrite oxidation by NOB and alternative pathways like nitrite reduction might be favored under these conditions.

Abundances and potential activities of nitrogen cycling microbial communities along a chronosequence of a glacier forefield.

Glacier forefields are ideal ecosystems to study the development of nutrient cycles as well as single turnover processes during soil development. In this study, we examined the ecology of the microbial nitrogen (N) cycle in bulk soil samples from a chronosequence of the Damma glacier, Switzerland. Major processes of the N cycle were reconstructed on the genetic as well as the potential enzyme activity level at sites of the chronosequence that have been ice-free for 10, 50, 70, 120 and 2000 years. In our study, we focused on N fixation, mineralization (chitinolysis and proteolysis), nitrification and denitrification. Our results suggest that mineralization, mainly the decomposition of deposited organic material, was the main driver for N turnover in initial soils, that is, ice-free for 10 years. Transient soils being ice-free for 50 and 70 years were characterized by a high abundance of N fixing microorganisms. In developed soils, ice-free for 120 and 2000 years, significant rates of nitrification and denitrification were measured. Surprisingly, copy numbers of the respective functional genes encoding the corresponding enzymes were already high in the initial phase of soil development. This clearly indicates that the genetic potential is not the driver for certain functional traits in the initial phase of soil formation but rather a well-balanced expression of the respective genes coding for selected functions.

Nitrogen turnover in soil and global change.

Nitrogen management in soils has been considered as key to the sustainable use of terrestrial ecosystems and a protection of major ecosystem services. However, the microorganisms driving processes like nitrification, denitrification, N-fixation and mineralization are highly influenced by changing climatic conditions, intensification of agriculture and the application of new chemicals to a so far unknown extent. In this review, the current knowledge concerning the influence of selected scenarios of global change on the abundance, diversity and activity of microorganisms involved in nitrogen turnover, notably in agricultural and grassland soils, is summarized and linked to the corresponding processes. In this context, data are presented on nitrogen-cycling processes and the corresponding microbial key players during ecosystem development and changes in functional diversity patterns during shifts in land use. Furthermore, the impact of increased temperature, carbon dioxide and changes in precipitation regimes on microbial nitrogen turnover is discussed. Finally, some examples of the effects of pesticides and antibiotics after application to soil for selected processes of nitrogen transformation are also shown.

Improved protocol for the simultaneous extraction and column-based separation of DNA and RNA from different soils.

We developed an improved protocol, allowing the simultaneous extraction of DNA and RNA from soil using phenol-chloroform with subsequent column-based separation of DNA and RNA (PCS). We compared this new approach with the well established protocol published by Griffiths et al. (2000), where DNA and RNA are separated by selective enzymatic digestions and two commercial kits used for DNA or RNA extraction, respectively, using four different agricultural soils. We compared yield and purity of the nucleic acids as well as abundance and diversity profiles of the soil bacterial communities targeting the nosZ gene via quantitative real-time PCR and terminal restriction fragment length polymorphism on DNA and RNA level. The newly developed protocol provided purer nucleic acid extracts compared to the used kit-based protocols. All protocols were suitable for DNA- and RNA-based gene quantification, however high variations between replicates were obtained for RNA samples using the original Griffiths protocol. Diversity patterns of nosZ were highly influenced by the extraction protocol used both on the DNA and RNA level. Finally, our data showed that the new protocol allows a simultaneous and reproducible extraction and separation of DNA and RNA, which were suitable for reliable analyses of gene and transcript copy numbers and diversity pattern.

Influence of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on ammonia-oxidizing bacteria and archaea in rhizosphere and bulk soil.

In agricultural plant production nitrification inhibitors like 3,4-dimethylpyrazole phosphate (DMPP) are used to retard the microbial nitrification process of fertilized ammonium to enhance the nitrogen supply for cultivated crops and to reduce nitrogen losses from the production system. Besides the well-known ammonia-oxidizing bacteria (AOB) it is known for a few years that also ammonia-oxidizing archaea (AOA) are able to perform the first step in nitrification, hence being also a target for a nitrification inhibitor. However, so far no information are available concerning the effectiveness of DMPP and its extent towards AOB and AOA, neither in bulk soil nor in the root-rhizosphere complex. We investigated in a field experiment performed according to agricultural practice the effect of DMPP on the abundance of AOB and AOA two, four and eight weeks after fertilization. We observed impaired abundances of AOB but not of AOA in both soil compartments that were still visible eight weeks after application, possibly indicating a reduced effectiveness of the nitrification inhibitor in our study.

Differences in amplification efficiency of standard curves in quantitative real-time PCR assays and consequences for gene quantification in environmental samples.

High and comparable efficiency values are the key for reliable quantification of target genes from environmental samples using real-time PCR. Therefore it was the aim of this study to investigate if PCR amplification efficiencies of plasmid DNA used for the calculation of standard curves (i) remain constant along a logarithmic scale of dilutions and (ii) if these values are comparable to those of DNA extracted from environmental samples. It could be shown that comparable efficiency values within the standards cannot be achieved using log scale serial dilutions and a comparison of gene copy numbers from DNA extracted from environmental samples and standard DNA extracted from plasmids is only possible in a very small interval.