Methyl-mercury affects microbial activity and biomass,bacterial community structure but rarely the fungal community structure

Monomethyl-mercury is one of the most toxic compounds. Methylation of Hg usually appears under anoxic conditions. In Swiss forest soils, methyl-Hg concentrations of up to 3 μg kg⁻¹ soil dw have been observed, but the impact of methyl-Hg on soil microorganisms have rarely been examined so far. In this study, we investigated the effect of increasing concentrations of methyl-Hg (0, 5, 20, 90 μg kg⁻¹ soil dw) on the microbial communities in various forest soils differing in their physico-chemical properties. Experiments were conducted in microcosms under controlled conditions and the basal respiration (BR), the microbial biomass carbon (MBC) and the bacterial and fungal community structures using T-RFLP-profiling were investigated. BR was significantly affected by methyl-Hg. In general, the BR increased with increasing methyl-Hg concentrations, whereas the MBC was significantly reduced. Bacterial communities were more sensitive to methyl-Hg than fungal communities. In five out of seven soils, the bacterial community structures differed significantly between the treatments whereas the fungal communities did not. The impact of methyl-Hg on the soil bacterial communities was site specific. In one soil, a methyl-Hg concentration of already 5 μg kg⁻¹ soil dw significantly affected the relative abundance of 13% bacterial operational taxonomic units (OTU), whereas in other soils concentrations of even 90 μg kg⁻¹ soil dw rarely affected the abundance of OTUs. In this study, for the first time, the impact of methyl-Hg on soil bacterial and fungal communities in forest soils was assessed. We showed that its impact strongly depends on the physico-chemical conditions of the soil and that bacterial communities were more sensitive to methyl-Hg than fungi.     

Physiological, biochemical and molecular responses of the soil microbial community after afforestation of pastures with Pinus radiata

Afforestation and deforestation are key land-use changes across the world, and are considered to be dominant factors controlling ecosystem functioning and biodiversity. However, the responses of soil microbial communities to these land-use changes are not well understood. Because changes in soil microbial abundance and community structure have consequences for nutrient cycling, C-sequestration and long-term sustainability, we investigated impacts of land-use change, age of stand and soil physico-chemical properties on fungal and bacterial communities and their metabolic activities. This study was carried out at four sites in two geographical locations that were afforested on long-established pastures with Pinus radiata D. Don (pine). Two of the sites were on volcanic soils and two on non-volcanic soils and stand age ranged from 5 to 20 y. Microbial communities were analysed by biochemical (phospho-lipid fatty acids; PLFA) and molecular (multiplex-terminal restriction fragment length polymorphism; M-TRFLP) approaches. Both site and stand age influenced microbial properties, with changes being least detectable in the 5-y-old stand. Land use was a key factor influencing soil metabolic activities as measured by physiological profiling using MicroResp. Pasture soils had higher microbial biomass (P < 0.001), and metabolic activities (P < 0.001), and basal respiration rates were up to 2.8-times higher than in the pine soils. Microbial abundance analysis by PLFA showed that the fungal to bacterial ratio was higher in the pine soils (P < 0.01). Community analysis suggested that soil bacterial communities were more responsive to site (principal component 1; P < 0.001) than to land use (principal component 5; P < 0.001). In contrast, the fungal community was more affected by land-use change (principal component 1; P < 0.001) than by site, although site still had some influence on fungal community structure (principal component 2; P < 0.001). Redundancy analysis also suggested that bacterial and fungal communities responded differently to various soil abiotic properties, land-use change and location of sites. Overall, our results indicate that the change in land use from pasture to P. radiata stands had a direct impact on soil fungal communities but an indirect effect, through its effects on soil abiotic properties, on bacterial communities. Most of the changes in bacterial communities could be explained by altered soil physico-chemical properties associated with afforestation of pastures.     

Bacterial salt tolerance is unrelated to soil salinity across an arid agroecosystem salinity gradient

In arid and semi-arid ecosystems, salinization is a major threat to the productivity of agricultural land. While the influence of other physical and chemical environmental factors on decomposer microorganisms have been intensively studied in soil, the influence of salinity has been less exhaustively assessed. We investigated the influence of soil salinity on soil bacterial communities in soils covering a range of salt levels. We assessed tolerance of the bacterial communities from Libyan agricultural soils forming a salinity gradient to salt (NaCl), by extracting bacterial communities and instantaneously monitoring the concentration-response to added NaCl with the Leucine incorporation technique for bacterial growth. To maximise our ability to detect differences in bacterial salt tolerance between the soils, we also repeated the assessment of bacterial growth tolerance after one month incubation with 1 or 2% added organic matter additions to stimulate microbial growth levels. We could establish clear concentration-response relationships between bacterial growth and soil salinity, demonstrating an accurate assessment of bacterial tolerance. The in situ soil salinity in the studied soils ranged between 0.64 and 2.73 mM Na (electrical conductivities of 0.74-4.12 mS cm⁻¹; cation exchange capacities of 20-37 mmol_c kg⁻¹) and the bacterial tolerance indicated by the concentration inhibiting 50% of the bacterial growth (EC₅₀) varied between 30 and 100 mM Na or between electrical conductivities of 3.0 and 10.7 mS cm⁻¹. There was no relationship between in situ soil salinity and the salt tolerance of the soil bacterial communities. Our results suggest that soil salinity was not a decisive factor for bacterial growth, and thus for structuring the decomposer community, in the studied soils.     

Carbon uptake by a microbial community during 30-day treatment with 13C-glucose of a sandy loam soil fertilized for 20 years with NPK or compost as determined by a GC–C–IRMS analysis of phospholipid fatty acids

To investigate the uptake by the microbial community of easily decomposable exogenous organic C and the proportion of this organic C remaining in soils under long-term fertilization schemes, ¹³C-glucose was supplied to arable soils (aquic inceptisol) following a 20-year (1989–2009) application of compost (CM) or inorganic NPK (NPK), along with a control (no fertilizer). Phospholipid fatty acids (PLFAs) were used as biomarkers for actinobacteria, bacteria and fungi. Gas chromatography–combustion–stable isotope ratio mass spectrometry (GC–C–IRMS) was used to determine the incorporation of ¹³C into individual PLFAs. The concentrations of soil microbial PLFAs significantly (P < 0.05) increased in all three soils after the addition of ¹³C-glucose. Over a 30-day incubation period, the highest PLFA concentrations were on day 7 (control) or day 15 (NPK and CM) for bacteria, and on day 30 for both fungi and actinobacteria. The added ¹³C-glucose was incorporated into bacterial PLFAs first, whilst an increase of ¹³C in fungal and actinobacterial PLFAs was measured on day 7 and 15, respectively. The mean amounts of ¹³C in bacterial, actinobacterial and fungal PLFAs in CM-treated soil during the 30-day incubation period were 0.589, 0.030 and 0.056 μg g⁻¹ soil, respectively, which were significantly (P < 0.05) higher than levels measured in the NPK and control soils. Among the bacterial groups, the amount of ¹³C in Gram-positive (G⁺) bacteria over the entire incubation ranged from 0.326 to 0.440 μg g⁻¹ soil in the CM scheme, which was significantly (P < 0.05) higher than levels detected in the NPK and control regimes. In contrast, ¹³C concentrations in monounsaturated PLFAs (aerobic microorganisms) in the CM-treated soil were 0.030–0.045 μg g⁻¹ soil, which was significantly (P < 0.05) lower than in the NPK schemes. The proportion of glucose-derived ¹³C remaining in soils was ranked as follows: CM (53%) > NPK (41%) > control (28%) after 30 days of incubation. Easily decomposable exogenous organic C was thus more effectively maintained under the CM regime, primarily because, after 20 years, CM had altered the microbial community by reducing the ratio of aerobic to anaerobic microorganisms whilst increasing levels of G⁺ bacteria in soil compared to the control and NPK soils. This study aids our understanding of the transformation and maintenance of easily decomposable organic C in soil over long-term fertilization regimes.     

Ethion degradation and its correlation with microbial and biochemical parameters of tea soils

Ethion, a highly persistent insecticide in soil, is extensively used in tea cultivation in the tropics. The studies on the environmental impact of ethion in tea soil ecosystems are scanty. Silty loam and sandy loam soils from tea fields of Dooars (Typic Uderthents) and Hill (Typic Dystrudepts), respectively, were investigated for the degradation and effect of ethion application on soil microbial and biochemical variables under controlled laboratory conditions. Ethion degraded faster in the Hill soil than in the Dooars soil. Higher temperature (30°C) aided in faster degradation due to the increased microbial activity in the soils. Ethion application at field rate (FR) had lower half-lives (70 days at 20°C and 42.3 days at 30°C for Dooars soil; 65.4 days at 20°C and 39 days at 30°C for Hill soil) than at ten times FR (10FR; 75.2 days at 20°C and 44.2 days at 30°C for Dooars soil; 70 days at 20°C and 41.8 days at 30°C for Hill soil). Soil microbial biomass carbon, ergosterol content, fluorescein diacetate hydrolyzing and β-glucosidase activities declined in all the treatment combinations up to day 60 for both FR and 10FR doses at 20°C, irrespective of the soil types. At 30°C, the decreasing trend was observed up to day 30 for both the soils. The toxicological effect of ethion on microbiological and biochemical parameters persisted till their corresponding half-lives. The microbial metabolic quotient and microbial respiration quotient were altered, but was short-lived, indicating ethion induced disturbances. The recovery of the depressive action at 10FR ethion spiking on the studied variables was of slightly longer duration than noticed at FR application, although the depressive effect was overcoming after the respective half-lives of ethion. The microbial and biochemical soil parameters were negatively correlated with application of ethion up to day 60 of incubation.     

Grassland management influences spatial patterns of soil microbial communities

Soil micro-organisms play a vital role in grassland ecosystem functioning but little is known about the effects of grassland management on spatial patterns of soil microbial communities. We compared plant species composition with terminal restriction fragment length polymorphism (T-RFLP) fingerprints of soil bacterial and fungal communities in unimproved, restored and improved wet grasslands. We assessed community composition of soil micro-organisms at distances ranging from 0.01 m to 100 m and determined taxa–area relationships from field- to landscape level. We show that land management type influenced bacterial but not fungal community composition. However, extensive grassland management to restore aboveground diversity affected spatial patterns of soil fungi. We found distinct distance–decay and small-scale aggregation of fungal populations in extensively managed grasslands restored from former arable use. There were no clear spatial patterns in bacterial communities at the field-scale. However, at the landscape level there was a moderate increase in bacterial taxa and a strong increase in fungal taxa with the number of sites sampled. Our results suggest that grassland management affects soil microbial communities at multiple scales; the observed small-scale variation may facilitate plant species coexistence and should be taken into account in field studies of soil microbial communities.     

Microbial community composition and rhizodeposit-carbon assimilation in differently managed temperate grassland soils

Rhizodeposit-carbon provides a major energy source for microbial growth in the rhizosphere of grassland soils. However, little is known about the microbial communities that mediate the rhizosphere carbon dynamics, especially how their activity is influenced by changes in soil management. We combined a ¹³CO₂ pulse-labeling experiment with phospholipid fatty acid (PLFA) analysis in differently managed Belgian grasslands to identify the active rhizodeposit-C assimilating microbial communities in these grasslands and to evaluate their response to management practices. Experimental treatments consisted of three mineral N fertilization levels (0, 225 and 450 kg N ha⁻¹ y⁻¹) and two mowing frequencies (3 and 5 times y⁻¹). Phospholipid fatty acids were extracted from surface (0-5 cm) bulk (BU) and root-adhering (RA) soil samples prior to and 24 h after pulse-labeling and were analyzed by gas chromatography-combustion-isotope ratio mass spectrometry (GC-c-IRMS). Soil habitats significantly differed in microbial community structure (as revealed by multivariate analysis of mol% biomarker PLFAs) as well as in gram-positive bacterial rhizodeposit-C uptake (as revealed by greater ¹³C-PLFA enrichment following pulse-labeling in RA compared to BU soil in the 450N/5M treatment). Mowing frequency did not significantly alter the relative abundance (mol%) or activity (¹³C enrichment) of microbial communities. In the non-fertilized treatment, the greatest ¹³C enrichment was seen in all fungal biomarker PLFAs (C16:1ω5, C18:1ω9, C18:2ω6,9 and C18:3ω3,6,9), which demonstrates a prominent contribution of fungi in the processing of new photosynthate-C in non-fertilized grassland soils. In all treatments, the lowest ¹³C enrichment was found in gram-positive bacterial and actinomycetes biomarker PLFAs. Fungal biomarker PLFAs had significantly lower ¹³C enrichment in the fertilized compared to non-fertilized treatments in BU soil (C16:1ω5, C18:1ω9) as well as RA soil (all fungal biomarkers). While these observations clearly indicated a negative effect of N fertilization on fungal assimilation of plant-derived C, the effect of N fertilization on fungal abundance could only be detected for the arbuscular mycorrhizal fungal (AMF) PLFA (C16:1ω5). On the other hand, increases in the relative abundance of gram-positive bacterial PLFAs with N fertilization were found without concomitant increases in ¹³C enrichment following pulse-labeling. We conclude that in situ¹³C pulse-labeling of PLFAs is an effective tool to detect functional changes of those microbial communities that are dominantly involved in the immediate processing of new rhizosphere-C.     

Structural and functional diversity of soil microbes is affected by elevated [CO<Subscript>2</Subscript>] and N addition in a poplar plantation

Background, Aims, and Scope  The genetic structure and the functionality of soil microbes are both important when studying the role of soil in the C cycle in elevated CO₂ scenarios. The aim of this work was to investigate the genetic composition of the fungal community by means of PCR-DGGE and the functional diversity of soil micro-organisms in general with MicroResp-based community level physiological profiling (CLPP) in a poplar plantation (POPFACE) grown under elevated [CO₂] with and without nitrogen fertilization. Materials and Methods  The POPFACE experimental plantation and FACE facility are located in central Italy, Tuscania (VT). Clones of Populus alba, Populus nigra and Populus x euramericana were grown, from 1999 to 2004, in six 314 m² plots treated either with atmospheric (control) or enriched (550 μmol mol⁻¹) CO₂ with FACE (Free Air CO₂ Enrichment) technology in each growing season. Each plot is divided into six triangular sectors, with two sectors per poplar genotype: three species × two nitrogen levels. After removal of the litter layer one soil core per genotype (10 cm wide, 20 cm depth) was taken inside each of the three sectors in each plot, for a total of 36 soil cores (3 replicates × 2 [CO₂] × 2 fertilization × 3 species) in October 2004 and in July 2005. DNA was extracted with a bead beating procedure. 18S rDNA gene fragments were amplified with PCR using fungal primers (FR1 GC and FF390). Analysis of CLPP was performed using the MicroResp method. Carbon substrates were selected depending on their ecological relevance to soil and their solubility in water. In particular rhizospheric C sources (carboxylic acids and carbohydrates) were chosen considering the importance of root inputs for microbial metabolism. Results  The fertilization treatment differentiated the fungal community composition regardless of elevated [CO₂] or the poplar species; moreover the number of fungal species was lower in fertilized soil. The effect of elevated [CO₂] on the fungal community composition was evident only as interaction with the fertilization treatment as, in N-sufficient soils, the elevated [CO₂] selected a different microbial community. For CLPP, the differ ent poplar species were the main factors of variation. The FACE treatment, on average, resulted in lower C utilization rates in un-fertilized soils and higher in fertilized soils. Discussion  Fungal biomass and fungal composition depend on different factors: from previous studies we know that the greater quantity and the higher C/N ratio of organic inputs under elevated [CO₂] influenced positively the fungal biomass both in fertilized and in un-fertilized soil, whereas nitrogen availability resulted to be the main determinant of fungal community composition in this work. Whole active microbial community was directly influenced by the soil nutrient availability and the poplar species. Under elevated CO₂ the competition for N with plants strongly affected the microbial communities, which were not able to benefit from added rhizospheric substrates. Under Nsufficient conditions, the increase of microbial activity due to [CO₂] enrichment was related to a more active microbial community, favoured by the current availability of C and N. Conclusions  Different factors influenced the microbial community at different levels: poplar species and root exudates affected the functional properties of the microbial community, while the fungal specific composition (as seen with DGGE) remained unaffected. On the other hand, factors such as N and C availability had a strong impact on the community functionality and composition. Fungal community structure reflected the availability of N in soils and the effect of elevated [CO₂] on community structure and function was evident only in N-sufficient soils. The simultaneous availability of C and N was therefore the main driving force for microbial structure and function in this plantation. Recommendations and Perspectives  Using the soil instead of soil extracts for CLPP determination provides a direct measurement of substrate catabolism by microbial communities and reflects activity rather than growth because more immediate responses to substrates are measured. Further applications of this approach could include selective inhibition of different microbial functional groups to investigate specific CLPPs. To combine the structural analysis and the catabolic responses of specific microbial communities (i.e. fungi or bacteria) could provide new outlooks on the role of microbes on SOM decomposition. ESS-Submission Editor: Dr. Kirk Semple (k.semple@lancaster.ac.uk)     

Development of pollution-induced community tolerance is linked to structural and functional resilience of a soil bacterial community following a five-year field exposure to copper

Copper (Cu) is accumulating in agricultural soils worldwide creating concern for adverse impacts on soil microbial communities and associated ecosystem services. In order to evaluate the structural and functional resilience of soil microbial communities to increasing Cu levels, we compared a Cu-adapted and a corresponding non-adapted soil microbial community for their abilities to resist experimental Cu pollution. Laboratory soil microcosms were set-up with either High-Cu soil from Cu-amended field plots (63 g Cu m⁻²) or with Low-Cu control soil from the same five-year field experiment. Laboratory treatments consisted of Cu amendments in the presence or absence of pig manure. Microbial activities (soil respiration, substrate-induced respiration, [³H]leucine incorporation), bacterial community structure (terminal restriction fragment length polymorphism, T-RFLP), community-level physiological profiles, and pollution-induced bacterial community tolerance (PICT detected using the [³H]leucine incorporation technique) were monitored for 12 weeks. The High-Cu and Low-Cu soil microbial communities initially exhibited almost identical structure and function and could only be distinguished from each other by their differential Cu tolerance. Experimental Cu pollution inhibited microbial activities, affected bacterial community structure, and induced further bacterial community tolerance to Cu. However, Low-Cu and High-Cu soil microbial communities showed essentially identical responses. Manure amendment did not protect against Cu toxicity and slightly increased Cu bioavailability as measured by a Cu-specific whole-cell bacterial biosensor. Our results indicate convergence of bacterial community structure and function in the High-Cu and Low-Cu soils during the five-year field experiment. We conclude that soil bacterial communities can exhibit structural and functional resilience to a five-year Cu exposure by virtue of their ability to develop Cu tolerance without affecting overall community structure. The observed increased Cu tolerance may involve phenotypic adaptation or selection at the micro-diversity level, for example an increased proportion of Cu-resistant strains within each bacterial species, which go undetected by T-RFLP community fingerprinting. Finally, our results indicate that Cu-dissolved organic matter complexes contribute to microbial toxicity in manure-amended soils implying that free Cu may comprise a poor predictor of metal toxicity.     

Influence of grass species and soil type on rhizosphere microbial community structure in grassland soils

A number of studies have reported species specific selection of microbial communities in the rhizosphere by plants. It is hypothesised that plants influence microbial community structure in the rhizosphere through rhizodeposition. We examined to what extent the structure of bacterial and fungal communities in the rhizosphere of grasses is determined by the plant species and different soil types. Three grass species were planted in soil from one site, to identify plant-specific influences on rhizosphere microbial communities. To quantify the soil-specific effects on rhizosphere microbial community structure, we planted one grass species (Lolium perenne L.) into soils from three contrasting sites. Rhizosphere, non-rhizosphere (bulk) and control (non-planted) soil samples were collected at regular intervals, to examine the temporal changes in soil microbial communities. Rhizosphere soil samples were collected from both root bases and root tips, to investigate root associated spatial influences. Both fungal and bacterial communities were analysed by terminal restriction fragment length polymorphism (TRFLP). Both bacterial and fungal communities were influenced by the plant growth but there was no evidence for plant species selection of the soil microbial communities in the rhizosphere of the different grass species. For both fungal and bacterial communities, the major determinant of community structure in rhizospheres was soil type. This observation was confirmed by cloning and sequencing analysis of bacterial communities. In control soils, bacterial composition was dominated by Firmicutes and Actinobacteria but in the rhizosphere samples, the majority of bacteria belonged to Proteobacteria and Acidobacteria. Bacterial community compositions of rhizosphere soils from different plants were similar, indicating only a weak influence of plant species on rhizosphere microbial community structure.     

Structural and functional diversity of bacterial community in soil treated with the herbicide napropamide estimated by the DGGE,CLPP and r/K-strategy approaches

Napropamide is one of the most commonly used herbicide in agricultural practice and can exhibit toxic effect to soil microorganisms. Therefore, the main objective of this study was to examine the genetic and functional diversity of microbial communities in soil treated with napropamide at field rate (FR, 2.25 mg kg⁻¹ of soil) and 10 times the FR (10 × FR, 22.5 mg kg⁻¹ of soil) by the denaturing gradient gel electrophoresis (DGGE) and the community level physiological profile (CLPP) methods. In addition, the r/K-strategy approach was used to evaluate the effect of this herbicide on the community structure of the culturable soil bacteria. DGGE patterns revealed that napropamide affected the structure of microbial community; however, the richness (S) and genetic diversity (H) values indicated that the FR dosage of napropamide experienced non-significant changes. In turn, the 10 × FR dosage of herbicide caused significant changes in the S and H values of dominant soil bacteria. DGGE profiles suggest an evolution of bacteria capable of degrading napropamide among indigenous microflora. Analysis of the CLPPs indicated that the catabolic activity of microbial community expressed as AWCD (average well-color development) was temporary positively affected after napropamide application and resulted in an increase of the substrate richness (S_R) as well as functional biodiversity (H) values. Analysis of the bacterial growth strategy revealed that napropamide affected the r- or K-type bacterial classes (ecotypes). In treated-soil samples K-strategists dominated the population, as indicated by the decreased ecophysiological (EP) index. Napropamide significantly affected the physiological state of culturable bacteria and caused a reduction in the rate of colony formation as well as a prolonged time of growth rate. Obtained results indicate that application of napropamide may poses a potential risk for soil functioning.     

A DGGE analysis shows that crop rotation systems influence the bacterial and fungal communities in soils

A better understanding of the relationships among different cropping systems, their effects on soil microbial ecology, and their effects on crop health and productivity is necessary for the development of more efficient, sustainable crop production systems. We used denaturing gradient gel electrophoresis (DGGE) to determine the impacts of crop rotations and crop types on bacterial and fungal communities in the soil. The communities of bacterial 16S rRNA genes and fungal 18S rRNA genes were analyzed in experimental field plots that were kept under 4 different crop rotation systems from 1999 to 2008 (continuous cabbage (Brassica oleracea var. capitata L.), cabbage–lettuce (Lactuca sativa L.) rotation, cabbage–radish (Raphanus sativus L. var. longipinnatus L.H. Bailey) rotation, and a 3-year crop rotation). A principal component analysis (PCA) and a canonical correspondence analysis (CCA) revealed that both the bacterial and fungal communities in bulk soils were influenced by the crop rotation systems. However, the primary factors influencing each community differed: bacterial communities were most affected by soil properties (especially carbon content), while fungal communities were influenced most strongly by rotation times. To elucidate factors that may cause differences in crop rhizosphere microbial communities, the microbial communities in the harvested cabbage rhizospheres were also analyzed. The results suggest that the fungal communities in bulk soil are related to the rhizosphere fungal communities. Our present study indicates that the microbial communities in bulk and rhizosphere soils could be managed by crop rotation systems.     

Nitrogen losses from two grassland soils with different fungal biomass

Nitrogen losses from agricultural grasslands cause eutrophication of ground- and surface water and contribute to global warming and atmospheric pollution. It is widely assumed that soils with a higher fungal biomass have lower N losses, but this relationship has never been experimentally confirmed. With the increased interest in soil-based ecosystem services and sustainable management of soils, such a relationship would be relevant for agricultural management. Here we present a first attempt to test this relationship experimentally. We used intact soil columns from two plots from a field experiment that had consistent differences in fungal biomass (68 ± 8 vs. 111 ± 9 μg C g⁻¹) as a result of different fertilizer history (80 vs. 40 kg N ha⁻¹ y⁻¹ as farm yard manure), while other soil properties were very similar. We performed two greenhouse experiments: in the main experiment the columns received either mineral fertilizer N or no N (control). We measured N leaching, N₂O emission and denitrification from the columns during 4 weeks, after which we analyzed fungal and bacterial biomass and soil N pools. In the additional ¹⁵N experiment we traced added N in leachates, soil, plants and microbial biomass. We found that in the main experiment, N₂O emission and denitrification were lower in the high fungal biomass soil, irrespective of the addition of fertilizer N. Higher ¹⁵N recovery in the high fungal biomass soil also indicated lower N losses through dentrification. In the main experiment, N leaching after fertilizer addition showed a 3-fold increase compared to the control in low fungal biomass soil (11.9 ± 1.0 and 3.9 ± 1.0 kg N ha⁻¹, respectively), but did not increase in high fungal biomass soil (6.4 ± 0.9 after N addition vs. 4.5 ± 0.8 kg N ha⁻¹ in the control). Thus, in the high fungal biomass soil more N was immobilized. However, the ¹⁵N experiment did not confirm these results; N leaching was higher in high fungal biomass soil, even though this soil showed higher immobilization of ¹⁵N into microbial biomass. However, only 3% of total ¹⁵N was found in the microbial biomass 2 weeks after the mineral fertilization. Most of the recovered ¹⁵N was found in plants (approximately 25%) and soil organic matter (approximately 15%), and these amounts did not differ between the high and the low fungal biomass soil. Our main experiment confirmed the assumption of lower N losses in a soil with higher fungal biomass. The additional ¹⁵N experiment showed that higher fungal biomass is probably not the direct cause of higher N retention, but rather the result of low nitrogen availability. Both experiments confirmed that higher fungal biomass can be considered as an indicator of higher nitrogen retention in soils.     

The influence of soil properties on the structure of bacterial and fungal communities across land-use types

Land-use change can have significant impacts on soil conditions and microbial communities are likely to respond to these changes. However, such responses are poorly characterized as few studies have examined how specific changes in edaphic characteristics do, or do not, influence the composition of soil bacterial and fungal communities across land-use types. Soil samples were collected from four replicated (n = 3) land-use types (hardwood and pine forests, cultivated and livestock pasture lands) in the southeastern US to assess the effects of land-use change on microbial community structure and distribution. We used quantitative PCR to estimate bacterial–fungal ratios and clone libraries targeting small-subunit rRNA genes to independently characterize the bacterial and fungal communities. Although some soil properties (soil texture and nutrient status) did significantly differ across land-use types, other edaphic factors (e.g., pH) did not vary consistently with land-use. Bacterial–fungal ratios were not significantly different across the land-uses and distinct land-use types did not necessarily harbor distinct soil fungal or bacterial communities. Rather, the composition of bacterial and fungal communities was most strongly correlated with specific soil properties. Soil pH was the best predictor of bacterial community composition across this landscape while fungal community composition was most closely associated with changes in soil nutrient status. Together these results suggest that specific changes in edaphic properties, not necessarily land-use type itself, may best predict shifts in microbial community composition across a given landscape. In addition, our results demonstrate the utility of using sequence-based approaches to concurrently analyze bacterial and fungal communities as such analyses provide detailed phylogenetic information on individual communities and permit the robust assessment of the biogeographical patterns exhibited by soil microbial communities.     

Development of soil microbial communities during tallgrass prairie restoration

Soil microbial communities were examined in a chronosequence of four different land-use treatments at the Konza Prairie Biological Station, Kansas. The time series comprised a conventionally tilled cropland (CTC) developed on former prairie soils, two restored grasslands that were initiated on former agricultural soils in 1998 (RG₉₈) and 1978 (RG₇₈), and an annually burned native tallgrass prairie (BNP), all on similar soil types. In addition, an unburned native tallgrass prairie (UNP) and another grassland restored in 2000 (RG₀₀) on a different soil type were studied to examine the effect of long-term fire exclusion vs. annual burning in native prairie and the influence of soil type on soil microbial communities in restored grasslands. Both 16S rRNA gene clone libraries and phospholipid fatty acid analyses indicated that the structure and composition of bacterial communities in the CTC soil were significantly different from those in prairie soils. Within the time series, soil physicochemical characteristics changed monotonically. However, changes in the microbial communities were not monotonic, and a transitional bacterial community formed during restoration that differed from communities in either the highly disturbed cropland or the undisturbed original prairie. The microbial communities of RG₉₈ and RG₀₀ grasslands were also significantly different even though they were restored at approximately the same time and were managed similarly; a result attributable to the differences in soil type and associated soil chemistry such as pH and Ca. Burning and seasonal effects on soil microbial communities were small. Similarly, changing plot size from 300 m² to 150 m² in area caused small differences in the estimates of microbial community structure. In conclusion, microbial community structure and biochemical properties of soil from the tallgrass prairie were strongly impacted by cultivation, and the microbial community was not fully restored even after 30 years.     

Changes in the genetic structure of Bacteria and microbial activity in an agricultural soil amended with tannery sludge

The application of tannery sludge to soils is a form of recycling; however, few studies have examined the impacts of this practice on soil microbial properties. We studied effects of two applications (2006 and 2007) of tannery sludge (with a low chromium content) on the structure of the bacterial community and on the microbial activity of soils. We fertilized an agricultural area in Rolândia, Paraná state, Brazil with different doses of sludge based on total N content, which ranged from 0 to 1200 kg N ha⁻¹. Sludge remained on the soil surface for three months before being plowed. Soils were sampled seven times during the experiment. Bacterial community structure, assessed by denaturing gradient gel electrophoresis (DGGE), was modified by the application of tannery sludge. Soon after the first application, there was clear separation between the bacterial communities in different treatments, such that each dose of sludge was associated with a specific community. These differences remained until 300 days after application and also after the second sludge application, but 666 days after the beginning of the experiment no differences were found in the bacterial communities of the lowest doses and the control. The principal response curve (PRC) analysis showed that the first sludge application strongly stimulated biological activity even 300 days after application. The second application also stimulated activity, but at a lower magnitude and for a shorter time, given that 260 days after the second application there was no difference in biological activity among treatments. PRC also showed that the properties most influenced by the application of tannery sludge were enzymatic activities related to N cycling (asparaginase and urease). The redundancy analysis (RDA) showed that tannery sludge’s influence on microbial activity is mainly related to increases in inorganic N and soil pH. Results showed that changes in the structure of the bacterial community in the studied soils were directly related to changes of their biological activity.     

Soil microbial communities response to herbicide 2,4-dichlorophenoxyacetic acid butyl ester

2,4-Dichlorophenoxyacetic acid butyl ester (2,4-D butyl ester) is extensively applied for weed control in cultivation fields in China, but its effect on soil microbial community remains obscure. This study investigated the microbial response to 2,4-D butyl ester application at different concentrations (CK, 10, 100 and 1000 μg g^?1) in the soils with two fertility levels, using soil dilution plate method and phospholipid fatty acid (PLFA) analysis. Culturable microorganisms were affected by the herbicide in both soils, particularly at the higher concentration. After treating soil with 100 μg g^?1 herbicide, culturable bacteria and actinomycetes were significantly higher, compared to other treatments. Treatment of soil with 1000 μg g^?1 2,4-D butyl ester caused a decline in culturable microbial counts, with the exception of fungal numbers, which increased over the incubation time. PLFA profiles showed that fatty acids for Gram-negative (GN) bacteria, Gram-positive (GP) bacteria, total bacteria and total fungi, as well as total PLFAs, varied with herbicide concentration for both soil samples. As herbicide concentration increased, the GN/GP ratio decreased dramatically in the two soils. The higher stress level was in the treatments with high concentrations of herbicide (1000 μg g^?1) for both soils. Principal component analysis of PLFAs showed that the addition of 2,4-D butyl ester significantly shifted the microbial community structure in the two soils. These results showed that the herbicide 2,4-D butyl ester might have substantial effects on microbial population and microbial community structure in agricultural soils. In particular, the effects of 2,4-D butyl ester were greater in soil with low organic matter and fertility level than in soil with high organic matter and fertility level.     

Do growth yield efficiencies differ between soil microbial communities differing in fungal:bacterial ratios? Reality check and methodological issues

Soil communities dominated by fungi such as those of no-tillage (NT) agroecosystems are often associated with greater soil organic matter (SOM) storage. This has been attributed in part to fungi having a higher growth yield efficiency (GYE) compared to bacteria. That is, for each unit of substrate C utilized, fungi invest a greater proportion into biomass and metabolite production than do bacteria. The assumption of higher fungal efficiency may be unfounded because results from studies in which fungal and bacterial efficiencies have been characterized are equivocal and because few studies have measured microbial GYE directly. In this study, we measured microbial GYE in agricultural soils by following ¹³C-labeled glucose loss, total CO₂-C, and ¹³CO₂-C evolution at 2 h intervals for 20 h in two experiments (differing in N amendment levels) in which the fungal:bacterial biomass ratios (F:B) were manipulated. No differences in efficiency were observed for communities with high versus low F:B in soils with or without added inorganic N. When calculated using ¹³CO₂-C (in contrast to total CO₂-C) evolution, growth yield efficiencies of soils having high and low F:B were 0.69±0.01 and 0.70±0.01, respectively. When soils were amended with N, soils with high and low F:B had growth yield efficiencies of 0.78±0.01 and 0.76±0.01, respectively. Our experiments do not support the widely held assumption that soil fungi have greater growth efficiency than soil bacteria. Thus, claims of greater fungal efficiency may be unsubstantiated and should be evoked cautiously when explaining the mechanisms underlying greater C storage and slower C turnover in fungal-dominated soils.     

Long-term impacts of zinc and copper enriched sewage sludge additions on bacterial, archaeal and fungal communities in arable and grassland soils

Long-term impacts of metal contamination derived from sewage sludge on soil microbial communities have been widely evaluated, but confounding effects have made it difficult to draw firm conclusions and thus to advise on safe metal limits. Here we used Multiplex-terminal restriction length fragment polymorphism (M-TRFLP) to assess the long-term impact of sludge-borne Zn and Cu contamination on the structure of bacterial, fungal and archaeal communities across seven different soils at metal levels relevant to current guideline limits. Despite strong effects of site on microbial community structure, analysis of similarity (ANOSIM) demonstrated a small but significant effect of Zn on bacteria (P < 0.001), archaea (P < 0.001), and fungi (P < 0.001). Significant effects of Cu on bacteria (P < 0.001), archaea (P < 0.001) and fungi (P < 0.001) were also observed. Several bacterial and fungal T-RFs were identified as responding to Zn or Cu. For example the bacterial T-RF 72 was negatively correlated with Zn and Cu, and T-RF 259 was positively correlated with Zn. Attempts to identify these bacterial markers of Zn and Cu contamination suggest a negative impact of Cu on Acidobacteria in arable soils. These results demonstrate for the first time, that despite a strong influence of site on microbial community structure, effects of Zn and Cu derived from sewage sludge can be detected as shifts in bacterial, fungal and archaeal communities indicating a common response more than 11 years after sludge addition.     

Parent material and vegetation influence bacterial community structure and nitrogen functional genes along deep tropical soil profiles at the Luquillo Critical Zone Observatory

Microbial communities mediate every step of the soil nitrogen cycle, yet the structure and associated nitrogen cycle functions of soil microbial communities remain poorly studied in tropical forests. Moreover, tropical forest soils are often many meters deep, but most studies of microbial nitrogen cycling have focused exclusively on surface soils. The objective of our study was to evaluate changes in bacterial community structure and nitrogen functional genes with depth in soils developed on two contrasting geological parent materials and two forest types that occur at different elevations at the Luquillo Critical Zone Observatory in northeast Puerto Rico. We excavated three soil pits to 140 cm at four different sites representing the four soil × forest combinations (n = 12), and collected samples at ten-centimeter increments from the surface to 140 cm. We used bacterial 16S rRNA gene-DGGE (denaturant gradient gel electrophoresis) to fingerprint microbial community structures, and quantitative PCR to measure the abundance of five functional genes involved in various soil nitrogen transformations: nifH (nitrogen fixation), chiA (organic nitrogen decomposition), amoA (ammonia oxidation), nirS (nitrite reduction) and nosZ (nitrous oxide reduction). Multivariate analyses of DGGE fingerprinting patterns revealed differences in bacterial community structure across the four soil × forest types that were strongly correlated with soil pH (r = 0.69, P < 0.01) and nutrient stoichiometry (r² ≥ 0.36, P < 0.05). Across all soil and forest types, nitrogen functional genes declined significantly with soil depth (P < 0.001). Denitrification genes (nirS and nosZ) accounted for the largest proportion of measured nitrogen functional genes. Measured nitrogen functional genes were positively correlated with soil carbon, nitrogen and phosphorus concentrations (P < 0.001) and all genes except amoA were significantly more abundant in the Inceptisol soil type compared with the Oxisol soil type (P < 0.03). Greater abundances and a stronger vertical zonation of nitrogen functional genes in Inceptisols suggest more dynamic nitrogen transformation processes in this soil type. As the first study to examine bacterial nitrogen functional gene abundances below the surface 20 cm in tropical forest soils, our work provides insight into how pedogenically-driven vertical gradients control the nitrogen-cycling capacity of soil microbial communities. While previous studies have shown evidence for redox-driven hotspots in tropical nitrogen cycling on a watershed scale, our study corroborates this finding on a molecular scale.