Phosphate Addition and Plant Species Alters Microbial Community Structure in Acidic Upland Grassland Soil

Agricultural improvement (addition of fertilizers, liming) of seminatural acidic grasslands across Ireland and the UK has resulted in significant shifts in floristic composition, soil chemistry, and microbial community structure. Although several factors have been proposed as responsible for driving shifts in microbial communities, the exact causes of such changes are not well defined. Phosphate was added to grassland microcosms to investigate the effect on fungal and bacterial communities. Plant species typical of unimproved grasslands (Agrostis capillaris, Festuca ovina) and agriculturally improved grasslands (Lolium perenne) were grown, and phosphate was added 25 days after seed germination, with harvesting after a further 50 days. Phosphate addition significantly increased root biomass (p < 0.001) and shoot biomass (p < 0.05), soil pH (by 0.1 U), and microbial activity (by 5.33 mg triphenylformazan [TPF] g⁻¹ soil; p < 0.001). A slight decrease (by 0.257 mg biomass-C g⁻¹ soil; p < 0.05) in microbial biomass after phosphate addition was found. The presence of plant species significantly decreased soil pH (p < 0.05; by up to 0.2 U) and increased microbial activity (by up to 6.02 mg TPF g⁻¹ soil) but had no significant effect on microbial biomass. Microbial communities were profiled using automated ribosomal intergenic spacer analysis. Multidimensional scaling plots and canonical correspondence analysis revealed that phosphate addition and its interactions with upland grassland plant species resulted in considerable changes in the fungal and bacterial communities of upland soil. The fungal community structure was significantly affected by both phosphate (R = 0.948) and plant species (R = 0.857), and the bacterial community structure was also significantly affected by phosphate (R = 0.758) and plant species (R = 0.753). Differences in microbial community structure following P addition were also revealed by similarity percentage analysis. These data suggest that phosphate application may be an important contributor to microbial community structural change during agricultural management of upland grasslands.     

Vegetation Affects the Relative Abundances of Dominant Soil Bacterial Taxa and Soil Respiration Rates in an Upland Grassland Soil

Plant-derived organic matter inputs are thought to be a key driver of soil bacterial community composition and associated soil processes. We sought to investigate the role of acid grassland vegetation on soil bacterial community structure by assessing bacterial diversity in combination with other soil variables in temporally and spatially distinct samples taken from a field-based plant removal experiment. Removal of aboveground vegetation resulted in reproducible differences in soil properties, soil respiration and bacterial diversity. Vegetated soils had significantly increased carbon and nitrogen concentrations and exhibited higher rates of respiration. Molecular analyses revealed that the soils were broadly dominated by Alphaproteobacterial and Acidobacterial lineages, with increased abundances of Alphaproteobacteria in vegetated soils and more Acidobacteria in bare soils. This field-based study contributes to a growing body of evidence documenting the effect of soil nutrient status on the relative abundances of dominant soil bacterial taxa, with Proteobacterial taxa dominating over Acidobacteria in soils exhibiting higher rates of C turnover. Furthermore, we highlight the role of aboveground vegetation in mediating this effect by demonstrating that plant removal can alter the relative abundances of dominant soil taxa with concomitant changes in soil CO₂-C efflux.     

Soil Bacterial and Fungal Community Structure Across a Range of Unimproved and Semi-Improved Upland Grasslands

Changes in soil microbial community structure due to improvement are often attributed to concurrent shifts in floristic community composition. The bacterial and fungal communities of unimproved and semi-improved (as determined by floristic classification) grassland soils were studied at five upland sites on similar geological substrata using both broad-scale (microbial activity and fungal biomass) and molecular [terminal restriction fragment length polymorphism (TRFLP), automated ribosomal intergenic spacer analysis (ARISA)] approaches. It was hypothesized that microbial community structure would be similar in soils from the same grassland type, and that grassland vegetation classifications could thus be used as predictors of microbial community structure. Microbial community measurements varied widely according to both site and grassland type, and trends in the effect of grassland improvement differed between sites. These results were consistent with those from similar studies, and indicated that floristic community composition was not a stable predictor of microbial community structure across sites. This may indicate a lack of correlation between grassland plant composition and soil microbial community structure, or that differences in soil chemistry between sites had larger impacts on soil microbial populations than plant-related effects.     

Spatial Analysis of Archaeal Community Structure in Grassland Soil

The complex structure of soil and the heterogeneity of resources available to microorganisms have implications for sampling regimens when the structure and diversity of microbial communities are analyzed. To assess the heterogeneity in community structure, archaeal communities, which typically contain sequences belonging to the nonthermophilic Crenarchaeota, were examined at two contrasting spatial scales by using PCR-denaturing gradient gel electrophoresis (DGGE) analysis followed by unweighted pair group method with arithmetic mean analysis of 16S rRNA- and ribosomal DNA-derived profiles. A macroscale analysis was carried out with soil cores taken at 2-m intervals along triplicate 8-m transects from both managed (improved) and natural (unimproved) grassland rhizosphere soils. A microscale analysis was carried out with a single soil core by assessing the effects of both sample size (10, 1, and 0.1 g) and distance between samples. The much reduced complexity of archaeal profiles compared to the complexity typical of the bacterial community facilitated visual comparison of profiles based on band presence and revealed different levels of heterogeneity between sets of samples. At the macroscale level, heterogeneity over the transect could not be related to grassland type. Substantial heterogeneity was observed across both improved and unimproved transects, except for one improved transect that exhibited substantial homogeneity, so that profiles for a single core were largely representative of the entire transect. At the smaller scale, the heterogeneity of the archaeal community structure varied with sample size within a single 8- by 8-cm core. The archaeal DGGE profiles for replicate 10-g soil samples were similar, while those for 1-g samples and 0.1-g samples showed greater heterogeneity. In addition, there was no relationship between the archaeal profiles and the distance between 1- or 0.1-g samples, although relationships between community structure and distance of separation may occur at a smaller scale. Our findings demonstrate the care required when workers attempt to obtain a representative picture of microbial community structure in the soil environment.     

Molecular Analysis of a Bacterial Chitinolytic Community in an Upland Pasture

The effects of agricultural-improvement treatments on the chitinolytic activity and diversity of a microbial community were investigated within an upland pasture. The treatments of interest were lime and treated sewage sludge, both commonly applied to pasture land to improve fertility. Burial of chitin-containing litter bags at the field site resulted in enrichment of bacteria according to 16S rRNA fingerprinting. Chitinolytic-activity measurements showed that the highest activity occurred in those bags recovered from sludge-amended plots, which correlated well with increased counts of actinobacteria in samples from these chitin bags. Our findings suggest that sewage sludge increases the fertility of the soil in terms of chitinase activity. Ten clone libraries were constructed from family 18 subgroup A chitinases, PCR amplified from litter bags buried in soil in July 2000 or in September 2000, in a separate study. Analysis of these libraries by restriction fragment length polymorphism and sequencing showed that they were dominated by actinobacterium-like chitinase sequences. This suggests that actinobacteria have an important chitinolytic function in this soil ecosystem. Our findings showed that sludge application increased chitinolytic activity but decreased the diversity of chitinases present.     

Streptomycin Application Has No Detectable Effect on Bacterial Community Structure in Apple Orchard Soil

Streptomycin is commonly used to control fire blight disease on apple trees. Although the practice has incited controversy, little is known about its nontarget effects in the environment. We investigated the impact of aerial application of streptomycin on nontarget bacterial communities in soil beneath streptomycin-treated and untreated trees in a commercial apple orchard. Soil samples were collected in two consecutive years at 4 or 10 days before spraying streptomycin and 8 or 9 days after the final spray. Three sources of microbial DNA were profiled using tag-pyrosequencing of 16S rRNA genes: uncultured bacteria from the soil (culture independent) and bacteria cultured on unamended or streptomycin-amended (15 μg/ml) media. Multivariate tests for differences in community structure, Shannon diversity, and Pielou''s evenness test results showed no evidence of community response to streptomycin. The results indicate that use of streptomycin for disease management has minimal, if any, immediate effect on apple orchard soil bacterial communities. This study contributes to the profile of an agroecosystem in which antibiotic use for disease prevention appears to have minimal consequences for nontarget bacteria.     

Effect of Sugarcane Burning or Green Harvest Methods on the Brazilian Cerrado Soil Bacterial Community Structure

Background

The Brazilian Cerrado is one of the most important biodiversity reservoirs in the world. The sugarcane cultivation is expanding in this biome and necessitates the study of how it may impact the soil properties of the Cerrado. There is a lack of information especially about the impacts of different sugarcane management on the native bacterial communities of Cerrado soil. Therefore, our objective was to evaluate and compare the soil bacterial community structure of the Cerrado vegetation with two sugarcane systems.

Methods

We evaluated samples under native vegetation and the impact of the two most commonly used management strategies for sugarcane cultivation (burnt cane and green cane) on this diversity using pyrosequencing and quantitative PCR of the rrs gene (16S rRNA).

Results and Conclusions

Nineteen different phyla were identified, with Acidobacteria (≈35%), Proteobacteria (≈24%) and Actinobacteria (≈21%) being the most abundant. Many of the sequences were represented by few operational taxonomic units (OTUs, 3% of dissimilarity), which were found in all treatments. In contrast, there were very strong patterns of local selection, with many OTUs occurring only in one sample. Our results reveal a complex bacterial diversity, with a large fraction of microorganisms not yet described, reinforcing the importance of this biome. As possible sign of threat, the qPCR detected a reduction of the bacterial population in agricultural soils compared with native Cerrado soil communities. We conclude that sugarcane cultivation promoted significant structural changes in the soil bacterial community, with Firmicutes phylum and Acidobacteria classes being the groups most affected.     

Changes in Soil Bacterial Community Structure with Increasing Disturbance Frequency

Little is known of the responsiveness of soil bacterial community structure to disturbance. In this study, we subjected a soil microcosm to physical disturbance, sterilizing 90 % of the soil volume each time, at a range of frequencies. We analysed the bacterial community structure using 454 pyrosequencing of the 16S rRNA gene. Bacterial diversity was found to decline with the increasing disturbance frequencies. Total bacterial abundance was, however, higher at intermediate and high disturbance frequencies, compared to low and no-disturbance treatments. Changing disturbance frequency also led to changes in community composition, with changes in overall species composition and some groups becoming abundant at the expense of others. Some phylogenetic groups were found to be relatively more disturbance-sensitive or tolerant than others. With increasing disturbance frequency, phylogenetic species variability (an index of community composition) itself became more variable from one sample to another, suggesting a greater role of chance in community composition. Compared to the tightly clustered community of the original undisturbed soil, in all the aged disturbed soils the lists of most abundant operational taxonomic units (OTUs) in each replicate were very different, suggesting a possible role of stochasticity in resource colonization and exploitation in the aged and disturbed soils. For example, colonization may be affected by whichever localized concentrations of bacterial populations happen to survive the last disturbance and be reincorporated in abundance into each pot. Overall, it appears that the soil bacterial community is very sensitive to physical disturbance, losing diversity, and that certain groups have identifiable ‘high disturbance’ vs. ‘low disturbance’ niches.     

Cumulative Effects of Short-Term Polymetal Contamination on Soil Bacterial Community Structure

In this study we evaluated the short-term effects of copper, cadmium, and mercury, added singly or in combination at different doses, on soil bacterial community structure using the bacterial automated ribosomal intergenic spacer analysis (B-ARISA) fingerprinting technique. Principal-component analysis of B-ARISA profiles allowed us to deduce the following order of impact: (Cu + Cd + Hg) >> Hg ≥ Cd > Cu. These results demonstrated that there was a cumulative effect of metal toxicity. Furthermore, the trend of modifications was consistent with the “hump-backed” relationships between biological diversity and disturbance described by Giller et al. (K. E. Giller, E. Witler, and S. P. McGrath, Soil Biol. Biochem. 30:1389-1414, 1998).     

Characterization of Bacterial Community Structure in Rhizosphere Soil of Grain Legumes

Molecular techniques were used to characterize bacterial community structure, diversity (16S rDNA), and activity (16S rRNA) in rhizospheres of three grain legumes: faba beans (Vicia faba L., cv. Scirocco), peas (Pisum sativum L., cv. Duel) and white lupin (Lupinus albus L., cv. Amiga). All plants were grown in the same soil under controlled conditions in a greenhouse and sampled after fruiting. Amplified 16S rDNA and rRNA products (using universal bacterial primers) were resolved by denaturing gradient gel electrophoresis (DGGE). Distinct profiles were observed for the three legumes with most of the bands derived from RNA being a subset of those derived from DNA. Comparing the total bacterial profiles with actinomycete-specific ones (using actinomycete-specific primers) highlighted the dominance of this group in the three rhizospheres. 16S PCR and RT-PCR products were cloned to construct libraries and 100 clones from each library were sequenced. Actinomycetes and proteobacteria dominated the clone libraries with differences in the groups of proteobacteria. Absence of β-subdivision members in pea and γ-subdivision members of proteobacteria in faba bean rhizosphere was observed. Plant-dependent rhizosphere effects were evident from significant differences in the bacterial community structure of the legume rhizospheres under study. The study gives a detailed picture of both residing and „active” bacterial community in the three rhizospheres. The high abundance of actinomycetes in the rhizospheres of mature legumes indicates their possible role in soil enrichment after the legumes are plowed into the soil as biofertilizers.     

The Relationship between Microbial Community Structure and Functional Stability,Tested Experimentally in an Upland Pasture Soil

Soil collected from an upland pasture was manipulated experimentally in ways shown previously to alter microbial community structure. One set of soil was subjected to chloroform fumigation for 0, 0.5, 2, or 24 h and the other was sterilised by gamma-irradiation and inoculated with a 10^–2, 10^–4, 10^–6, or 10^–8 dilution of a soil suspension prepared from unsterilized soil. Following incubation for 8 months, to allow for the stabilization of microbial biomass and activity, the resulting microbial community structure (determined by PCR-DGGE of bacterial specific amplification products of total soil DNA) was assessed. In addition, the functional stability (defined here as the resistance and resilience of short-term decomposition of plant residues to a transient heat or a persistent copper perturbation) was determined. Changes in the active bacterial population following perturbation (determined by RT-PCR-DGGE of total soil RNA) were also monitored. The manipulations resulted in distinct shifts in microbial community structure as shown by PCR-DGGE profiles, but no significant decreases in the number of bands. These shifts in microbial community structure were associated with a reduction in functional stability. The clear correlation between altered microbial community structure and functional stability observed in this upland pasture soil was not evident when the same protocols were applied to soils in other studies. RT-PCR-DGGE profiles only detected a shift in the active bacterial population following heat, but not copper, perturbation. We conclude that the functional stability of decomposition is related to specific components of the microbial community.     

Bacterial Community Structure of Biofilms on Artificial Surfaces in an Estuary

This study examined bacterial community structure of biofilms on stainless steel and polycarbonate in seawater from the Delaware Bay. Free-living bacteria in the surrounding seawater were compared to the attached bacteria during the first few weeks of biofilm growth. Surfaces exposed to seawater were analyzed by using 16S rDNA libraries, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE). Community structure of the free-living bacterial community was different from that of the attached bacteria according to FISH and DGGE. In particular, alpha-proteobacteria dominated the attached communities. Libraries of 16S rRNA genes revealed that representatives of the Rhodobacterales clade were the most abundant members of biofilm communities. Changes in community structure during biofilm growth were also examined by DGGE analysis. We hypothesized that bacterial communities on dissimilar surfaces would initially differ and become more similar over time. In contrast, the compositions of stainless steel and polycarbonate biofilms were initially the same, but differed after about 1 week of biofilm growth. These data suggest that the relationship between surface properties and biofilm community structure changes as biofilms grow on surfaces such as stainless steel and polycarbonate in estuarine water.     

Effect of Red Clay on Diesel Bioremediation and Soil Bacterial Community

Red clay is a type of soil, the red color of which results from the presence of iron oxide. It is considered an eco-friendly material, with many industrial, cosmetic, and architectural uses. A patented method was applied to red clay in order to change its chemical composition and mineral bioavailability. The resulting product was designated processed red clay. This study evaluates the novel use of red clay and processed red clay as biostimulation agents in diesel-contaminated soils. Diesel biodegradation was enhanced in the presence of red clay and processed red clay by 4.9- and 6.7-fold, respectively, and the number of culturable bacterial cells was correlated with the amount of diesel biodegradation. The growth of Acinetobacter oleivorans DR1, Pseudomonas putida KT2440, and Cupriavidus necator was promoted by both types of red clays. Culture-independent community analysis determined via barcoded pyrosequencing indicated that Nocardioidaceae, Xanthomonadaceae, Pseudomonadaceae, and Caulobacteraceae were enriched by diesel contamination. Bacterial strain isolation from naphthalene- and liquid paraffin-amended media was affiliated with enriched taxa based on 16S rRNA gene sequence identity. We suggest that the biostimulating mechanism of red clay and processed red clay is able to support bacterial growth without apparent selection for specific bacterial species.     

Community Structure of Actively Growing Bacterial Populations in Plant Pathogen Suppressive Soil

The bacterial community in soil was screened by using various molecular approaches for bacterial populations that were activated upon addition of different supplements. Plasmodiophora brassicae spores, chitin, sodium acetate, and cabbage plants were added to activate specific bacterial populations as an aid in screening for novel antagonists to plant pathogens. DNA from growing bacteria was specifically extracted from the soil by bromodeoxyuridine immunocapture. The captured DNA was fingerprinted by terminal restriction fragment length polymorphism (T-RFLP). The composition of the dominant bacterial community was also analyzed directly by T-RFLP and by denaturing gradient gel electrophoresis (DGGE). After chitin addition to the soil, some bacterial populations increased dramatically and became dominant both in the total and in the actively growing community. Some of the emerging bands on DGGE gels from chitin-amended soil were sequenced and found to be similar to known chitin-degrading genera such as Oerskovia, Kitasatospora, and Streptomyces species. Some of these sequences could be matched to specific terminal restriction fragments on the T-RFLP output. After addition of Plasmodiophora spores, an increase in specific Pseudomonads could be observed with Pseudomonas-specific primers for DGGE. These results demonstrate the utility of microbiomics, or a combination of molecular approaches, for investigating the composition of complex microbial communities in soil.     

The Effects of Soil Bacterial Community Structure on Decomposition in a Tropical Rain Forest

Soil microorganisms are key drivers of terrestrial biogeochemical cycles, yet it is still unclear how variations in soil microbial community composition influence many ecosystem processes. We investigated how shifts in bacterial community composition and diversity resulting from differences in carbon (C) availability affect organic matter decomposition by conducting an in situ litter manipulation experiment in a tropical rain forest in Costa Rica. We used bar-coded pyrosequencing to characterize soil bacterial community composition in litter manipulation plots and performed a series of laboratory incubations to test the potential functional significance of community shifts on organic matter decomposition. Despite clear effects of the litter manipulation on soil bacterial community composition, the treatments had mixed effects on microbial community function. Distinct communities varied in their ability to decompose a wide range of C compounds, and functional differences were related to both the relative abundance of the two most abundant bacterial sub-phyla (Acidobacteria and Alphaproteobacteria) and to variations in bacterial alpha-diversity. However, distinct communities did not differ in their ability to decompose native dissolved organic matter (DOM) substrates that varied in quality and quantity. Our results show that although resource-driven shifts in soil bacterial community composition have the potential to influence decomposition of specific C substrates, those differences may not translate to differences in DOM decomposition rates in situ. Taken together, our results suggest that soil bacterial communities may be either functionally dissimilar or equivalent during decomposition depending on the nature of the organic matter being decomposed.     

Bacterial Community Structure in a Semi-Arid Haloalkaline Soil Using Culture Independent Method

Bacterial community structures in two physicochemically different soils from the coastal region of Gujarat, India were investigated using PCR, 16S rRNA gene clone libraries and sequencing methods. The aim of the study was to determine the diversity of bacterial communities inhabiting haloalkaline soil from a semi-arid coastal region. The phylogenetic diversity of bacteria in a haloalkaline soil (EC 20 dS/m; pH 9.5) was compared with a normal soil (EC 0.93 dS/m; pH 7.2). Clones representing phyla Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Actinobacteria, Acidobacteria and Planctomycetes were found in both soils. Cyanobacteria, Verrucomicrobia, OP10 and Bacteria incertae sedis were detected in normal soil whereas Nitrospira was found only in haloalkaline soil. The dominant phylum in the haloalkaline soil was Bacteroidetes followed by Proteobacteria whereas normal soil was dominated by Proteobacteria and Actinobacteria. About 82% of the sequences from the haloalkaline library were related to those previously retrieved from various saline, alkaline and oil-natural gas field ecosystems whereas 50% of the sequences from normal soil resembled sequences of bacteria retrieved from agriculture-related habitats viz. agriculture fields, rhizosphere and grasslands. One third of the total sequences from both soil samples showed low BLAST identities (<95%) suggesting that these soils may harbor unique, novel taxa. Further, the correlation analysis revealed negative correlations of Shannon diversity indices and species evenness with salinity (EC) and pH but positive correlations with total carbon and total nitrogen contents of the soil samples. The haloalkaline soil exhibited less bacterial diversity and communities were significantly different from those of normal soil. In this study, the haloalkaline soil from a semi-arid region supports oligotrophic microbes.

Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

Impact of Protists on the Activity and Structure of the Bacterial Community in a Rice Field Soil

Flooded rice fields have become a model system for the study of soil microbial ecology. In Italian rice fields, in particular, aspects from biogeochemistry to molecular ecology have been studied, but the impact of protistan grazing on the structure and function of the prokaryotic community has not been examined yet. We compared an untreated control soil with a γ-radiation-sterilized soil that had been reinoculated with a natural bacterial assemblage. In order to verify that the observed effects were due to protistan grazing and did not result from sterilization, we set up a third set of microcosms containing sterilized soil that had been reinoculated with natural assemblage bacteria plus protists. The spatial and temporal changes in the protistan and prokaryotic communities were examined by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis, respectively, both based on the small-subunit gene. Sequences retrieved from DGGE bands were preferentially affiliated with Cercozoa and other bacteriovorous flagellates. Without protists, the level of total DNA increased with incubation time, indicating that the level of the microbial biomass was elevated. Betaproteobacteria were preferentially preyed upon, while low-G+C-content gram-positive bacteria became more dominant under grazing pressure. The bacterial diversity detectable by T-RFLP analysis was greater in the presence of protists. The level of extractable NH₄⁺ was lower and the level of extractable SO₄²⁻ was higher without protists, indicating that nitrogen mineralization and SO₄²⁻ reduction were stimulated by protists. Most of these effects were more obvious in the partially oxic surface layer (0 to 3 mm), but they could also be detected in the anoxic subsurface layer (10 to 13 mm). Our observations fit well into the overall framework developed for protistan grazing, but with some modifications pertinent to the wetland situation: O₂ was a major control, and O₂ availability may have limited directly and indirectly the development of protists. Although detectable in the lower anoxic layer, grazing effects were much more obvious in the partially oxic surface layer.     

连作对麦冬根际土壤细菌群落的影响

以中草药麦冬为研究对象,采用变性梯度凝胶电泳(DGGE)方法,考察连作对麦冬根际土壤细菌群落的影响。结果表明,重茬麦冬根际细菌群落的Shannon-Wiener指数从苗期至快速生长期、再到块根膨大期分别为3.36-3.40-3.69,丰富度指数为55.0-61.5-63.5,均匀度指数为0.84-0.82-0.89;而同期正茬根际细菌群落的上述指数值分别为3.66-3.33-3.72、67.5-53.5-63.5和0.87-0.84-0.90。通过对土壤细菌群落的DGGE图谱进行主成分分析,结果显示,上述各个时期重茬与正茬样本均发生显著分离,表明连作改变了麦冬根际细菌群落的多样性变化趋势和结构组成。进一步比较麦冬块根膨大期根际主要功能菌群的数量变化,发现连作后氨化细菌和好气性纤维素分解菌数量增加,而硝化细菌和固氮菌的数量减少。对比发现重茬麦冬的产量仅为正茬的70.6%,表明麦冬连作减产与其根际土壤细菌群落的变化相关。     

Seasonal Changes in an Alpine Soil Bacterial Community in the Colorado Rocky Mountains

The period when the snowpack melts in late spring is a dynamic time for alpine ecosystems. The large winter microbial community begins to turn over rapidly, releasing nutrients to plants. Past studies have shown that the soil microbial community in alpine dry meadows of the Colorado Rocky Mountains changes in biomass, function, broad-level structure, and fungal diversity between winter and early summer. However, little specific information exists on the diversity of the alpine bacterial community or how it changes during this ecologically important period. We constructed clone libraries of 16S ribosomal DNA from alpine soil collected in winter, spring, and summer. We also cultivated bacteria from the alpine soil and measured the seasonal abundance of selected cultured isolates in hybridization experiments. The uncultured bacterial communities changed between seasons in diversity and abundance within taxa. The Acidobacterium division was most abundant in the spring. The winter community had the highest proportion of Actinobacteria and members of the Cytophaga/Flexibacter/Bacteroides (CFB) division. The summer community had the highest proportion of the Verrucomicrobium division and of β-Proteobacteria. As a whole, α-Proteobacteria were equally abundant in all seasons, although seasonal changes may have occurred within this group. A number of sequences from currently uncultivated divisions were found, including two novel candidate divisions. The cultured isolates belonged to the α-, β-, and γ-Proteobacteria, the Actinobacteria, and the CFB groups. The only uncultured sequences that were closely related to the isolates were from winter and spring libraries. Hybridization experiments showed that actinobacterial and β-proteobacterial isolates were most abundant during winter, while the α- and γ-proteobacterial isolates tested did not vary significantly. While the cultures and clone libraries produced generally distinct groups of organisms, the two approaches gave consistent accounts of seasonal changes in microbial diversity.     

Effect of Agricultural Management Regime on Burkholderia Community Structure in Soil

The main objective of this study was to determine the Burkholderia community structure associated with areas under different agricultural management and to evaluate to which extent this community structure is affected by changes in agricultural management. Two fields with distinct soil history (arable land and permanent grassland) were exposed to three agricultural management regimes (crop rotation, maize monoculture, and grassland). By using a culture-independent approach, based on a Burkholderia-specific polymerase chain reaction–denaturing gradient gel electrophoresis system, it was possible to observe the conversion of Burkholderia communities typical for permanent grassland to those of arable land after four consecutive years. However, the time needed to achieve the reverse transition, i.e., converting the Burkholderia community associated with arable land to that of grassland, was beyond the duration of the field experiment. In addition, by applying principal response curves, the direction and extent of the conversion from grassland to arable land (maize monoculture and to crop rotation) were determined. Hence, the results suggested that agricultural practices, such as fertilization and tillage, were more effective in changing the Burkholderia community structure than agricultural management regime. To determine the effect of agricultural management on the Burkholderia population with biocontrol abilities, the culturable fraction of the Burkholderia community was assessed. The areas under permanent grassland and grassland converted to maize monoculture had the highest percentages of Burkholderia strains with antagonistic activity against Rhizoctonia solani AG-3, mainly Burkholderia pyrrocinia and Burkholderia sp. LMG 22929. The isolation frequency of antagonistic isolates from arable land was extremely low. Our results indicate that (changes in) agricultural management, mainly crop rotation, affect the frequency of isolation of antagonistic Burkholderia strains and that grassland represents a reservoir of Burkholderia species with great potential for agricultural applications.