Polyphasic characterization of the bacterial community in an urban soil profile with in situ and culture-dependent methods

Bacterial communities of urban soils have not been thoroughly investigated up to now. Therefore, soil samples from the urban park Tiergarten in the centre of Berlin were taken from a profile in 15, 30 and 90 cm depth. The total number of bacteria (4′,6-diamidino-2-phenylindole (DAPI) counts) as well as biomass declined one order of magnitude from topsoil to subsoil. Soil texture changed comparably and water content and amount of organic matter dropped 3–10-fold. The number of culturable bacteria (colony forming units = CFU) also decreased with increasing soil depth. Amplified ribosomal DNA restriction analysis (ARDRA) revealed similar bacterial communities in the two upper soil layers in contrast to the deepest layer. The number of bacterial cells which were detected with probe EUB338 in relation to total cell counts differed between 43 and 35% in the three soil layers. With the probe active count method (PAC) this number could be increased up to 72% of total cell counts in topsoil whereas activation of cells declined with increasing depth. In relation to total cell counts (DAPI) α-Proteobacteria and β-Proteobacteria are equally distributed in all three depths, whereas γ-Proteobacteria declined within the soil profile. With the BIOLOG system we observed the general trend that the capability of utilizing diverse substrates decreased with soil depth whereas a few substrates, such as Tween 40 and Tween 80 could be utilised by the bacteria of all soil depths.     

土壤质量评价方法述评

 为全面总结土壤质量评价方法的研究进展,从指数法、模型法及GIS的参与等方面综述当前一些定量评价土壤质量的方法。指数法计算方法较简单、实用;模型法具有较强的逻辑性和系统性;应用GIS评价土壤质量客观且潜力大,对土壤进行时空动态监测以实现土壤持续经营管理。在土壤质量评价工作中将定量与定性评价结合起来,选择适宜的土壤质量评价指标,不断完善现有的评价方法,发挥信息技术的优势,为合理开发利用土壤提供依据。     

Impact of irrigation water quality on soil nitrifying and total bacterial communities

Disturbance induced by two contrasting irrigation regimes (groundwater versus urban wastewater) was evaluated on a sandy agricultural soil through chemical and microbial analyses. Contrary to wastewater, groundwater displayed very high nitrate contents but small amounts of ammonium and organic matter. Despite these strong compositional shifts, soil organic carbon and nitrogen, nitrate and ammonium contents were not significantly different in both types of irrigated plot. Moreover, neither microbial biomass nor its activity, determined as fluorescein diacetate hydrolysis activity, was influenced by irrigation regimes. Bacterial community structure, assessed by denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments, was also weakly impacted as molecular fingerprints shared an overall similarity of 85%. Ammonia-oxidizing bacterial community (AOB) was monitored by DGGE of the functional molecular marker amoA gene (alpha subunit of the ammonia monooxygenase). Surprisingly, no amoA signals were obtained from plots irrigated with groundwater, whereas signal intensities were high in all plots under wastewater. Among the last, compositional shifts of the AOB community were weak. Overall, impact of irrigation water quality on soil chemistry could not be evidenced, whereas effects were low on the total bacterial compartment but marked on the AOB community.     

土壤微生物群落多样性研究方法及进展

微生物多样性是指群落中的微生物种群类型和数量、种的丰度和均度以及种的分布情况。研究土壤微生物群落多样性的方法包括传统的以生化技术为基础的方法(直接平板计数、单碳源利用模式等)和以现代分子生物技术为基础的方法(从土壤中提取DNA,进行G C%含量的分析,或杂交分析,或进行PCR,产物再进行DGGE/TGGE等分析)。现代生物技术与传统微生物研究方法的结合使用,为更全面地理解土壤微生物群落的多样性和生态功能提供了良好的前景。     

Ecological standardization of soil and soil quality control

Theoretical bases are offered for the ecological soil standardization presuming the scientific substantiating of the allowable ecological state (quality) of the soil and the allowable anthropogenic impact on it. The modern experience of these bases’ application in regulatory-methodological, nature-control, and managerial practices is presented. The criteria are found for determining the levels of the allowable ecological quality of soil and the anthropogenic impact on it. The sources of the anthropogenic impact on the soil are characterized. A unified system of indices of soil quality and anthropogenic impacts and a mechanism for determining the range of the allowable values of these parameters have been developed taking into account the natural conditions and types of land use. Scientific-methodological approaches are proposed that support a certain balance between the allowable ecological status of the soil and the effects on it in connection with the mechanisms of the soil quality control in particular land plots.     

IBQS: A synthetic index of soil quality based on soil macro-invertebrate communities

We developed a synthetic index of biological soil quality (IBQS) based on soil macro-invertebrate community patterns to assess soil quality. In 22 sites representing the diversity of agroecosystems encountered in France, invertebrate communities co-varied significantly with a set of 14 parameters describing the physical and chemical properties of soil (co-inertia, p < 0.001; RV = 0.70). Using hierarchical classification, sites could be separated into four homogeneous groups and, using the ‘indicator value’ method, 46 indicator taxa characteristic of one or another of these groups were identified. We then used a formula that takes into account the abundance of indicator species and their respective indicator values to score soils from 1 to 20. IBQS was able to detect the effects of management practices on soil quality. Soil quality varied from 6 to 20 in forests, 7 to 9 in pastures and 2 to 9 in crops respectively. This suggests that well-managed crops and pastures may have better soil quality than some forests. Our results confirm that soil macro-invertebrates provide an integrative measure of soil quality and that the proposed index can be used either in short- or long-term monitoring, provided that it is calibrated and validated with respect to the regional context of the study.     

Biostimulation and enrichment of 2,6-dichlorobenzamide-mineralising soil bacterial communities from dichlobenil-exposed soil

Eleven enrichment cultures of 2,6-dichlorobenzamide (BAM)-mineralising bacteria from a dichlobenil-exposed soil were enriched using three different media. Ten of the enriched mixed cultures had stimulated BAM mineralisation only in the presence of supplementary carbon and were able to utilise BAM as the sole nitrogen source, while only one was able to utilise BAM as the sole source of both carbon and nitrogen. Cultivation and DNA-based analysis of the mixed cultures suggested that different bacterial populations were stimulated by the three strategies. Our findings indicate that natural BAM-mineralising populations capable of using BAM as a source of nitrogen can be readily stimulated, enriched and maintained following sub-culturing from dichlobenil-treated soils by adding an alternative carbon source.     

The under-recognized dominance of Verrucomicrobia in soil bacterial communities

Verrucomicrobia are ubiquitous in soil, but members of this bacterial phylum are thought to be present at low frequency in soil, with few studies focusing specifically on verrucomicrobial abundance, diversity, and distribution. Here we used barcoded pyrosequencing to analyze verrucomicrobial communities in surface soils collected across a range of biomes in Antarctica, Europe, and the Americas (112 samples), as well as soils collected from pits dug in a montane coniferous forest (69 samples). Data collected from surface horizons indicate that Verrucomicrobia average 23% of bacterial sequences, making them far more abundant than had been estimated. We show that this underestimation is likely due to primer bias, as many of the commonly used PCR primers appear to exclude verrucomicrobial 16S rRNA genes during amplification. Verrucomicrobia were detected in 180 out of 181 soils examined, with members of the class Spartobacteria dominating verrucomicrobial communities in nearly all biomes and soil depths. The relative abundance of Verrucomicrobia was highest in grasslands and in subsurface soil horizons, where they were often the dominant bacterial phylum. Although their ecology remains poorly understood, Verrucomicrobia appear to be dominant in many soil bacterial communities across the globe, making additional research on their ecology clearly necessary.     

Impact of heavy metals on the degradative capabilities of soil bacterial communities

The degradative capabilities of six heavy-metal-affected and six unaffected bacterial communities from Canadian and German soils were determined by enumerating colony-forming units on 20 specific media. Each of these contained an aromatic substrate as the sole source of C and energy. Comparisons of plate counts revealed that heavy metal stress caused a decrease in the eveness of the distribution of the 20 degradative capabilities This suggests that in heavy-metal-affected bacterial communities, relatively rare degradative capabilities, irrespective of their nature, are even rarer than in unaffected communities, while the reverse is true for more common capabilities. The results are discussed with respect to the ease with which aromatic substrates can serve as C and energy sources.     

Effects of bio-organic fertilizer plus soil amendment on the control of tobacco bacterial wilt and composition of soil bacterial communities

Tobacco bacterial wilt (TBW) is caused by Ralstonia solanacearum (R. solanacearum), a severe pathogenic agent with a wide host range. In this study, lime?+?ammonium bicarbonate (L?+?AB), organic fertilizer (OF), bio-organic fertilizer (BOF), and integrated treatment (L?+?AB?+?BOF) were assessed for the ability to control TBW and to influence the composition of native soil bacterial communities. The results showed that disease incidence of L?+?AB?+?BOF for two growth seasons in pot experiment was the lowest, with only 15.56 and 11.11 % at seasons 1 and 2, respectively. The integrated treatment could also significantly suppress TBW in the field, with a disease incidence of only 14.27 % compared with 35.41, 50.03, and 31.32 % in L?+?AB, OF, and BOF treatments, respectively. With application of the integrated treatment in pot and field experiments, the abundances of R. solanacearum were both significantly lower than those with other treatments. Denaturing gradient gel electrophoresis (DGGE) patterns showed that application of BOF significantly affected composition of bacterial communities of rhizosphere. The analysis of 454 sequencing data showed that application of integrated treatment recruited more beneficial bacteria than other treatments, such as Bacillus, Paenibacillus, Arthrobacter, and Streptomyces, while the abundance of Ralstonia with the integrated treatment was decreased. Overall, these results suggested that application of integrated agricultural management could effectively suppress bacterial wilt by affecting the composition of bacterial community and reducing the population of R. solanacearum.     

DGGE fingerprinting of culturable soil bacterial communities complements culture-independent analyses

Culture-dependent DGGE (CD DGGE) fingerprinting of the 16S rRNA gene was used to characterize mixed bacterial communities recovered on agar plates. Using R2A Agar as a growth medium, CD DGGE analysis resulted in clear banding patterns of sufficient complexity (16-32 major bands) and reproducibility to investigate differences in bacterial communities in a silt loam soil. Replicate CD DGGE profiles from plates inoculated with less-dilute samples (10⁻³) had a higher band count and were more similar (72-77%) than profiles from more-dilute samples (51-61%). Different culture media and incubation conditions resulted in distinct community fingerprints and increased the cumulative number of unique bands detected. When CD DGGE fingerprints were compared to profiles constructed from 16S rRNA genes obtained from culture-independent clone libraries (CB DGGE profiles) 34% of the bands were unique to the culture-dependent profiles, 32% were unique to the culture-independent profiles and 34% were found in both communities. These data demonstrate that culture-independent DGGE profiles are supplemented by the distinct bands detected in culture-dependent profiles. CD DGGE can be a useful technique to follow the dynamics of distinct culturable fractions of the soil bacterial community.     

不同筛分方式对土壤团聚体及土壤微生物群落的影响

探究土壤团聚体组成、稳定性、有机碳含量及微生物数量在不同土壤筛分方式下的变化,对团聚体筛分方式进行优化。以黑土为研究对象,设置不同筛分方式处理,比较干土干筛、干土湿筛、润土干筛、润土湿筛4种筛分方式的优缺点。结果表明,润土干筛比干土湿筛处理≥2 mm粒径的土壤团聚体高出约85.97%,而在2～0.25、≤0.25 mm粒级中团聚体所占比例最高的干土湿筛比最低的润土干筛分别高出约80.90%、91.82%。>0.25 mm水稳性团聚体含量(R_0.25)在润土干筛下达到最高,为99.35%,该指标与土壤团聚体稳定性成正相关,其值越高则表示土壤抗蚀能力越强。干土湿筛处理下各粒级团聚体土壤有机碳分布平均。干土湿筛处理下,土壤细菌和放线菌的数量最高。而润土干筛处理下土壤真菌数量较高,与润土湿筛、干土干筛、干土湿筛3个处理方式相比有显著性差异。综上发现,干土湿筛处理下土壤团聚体稳定性更好,土壤微生物数量较高,在一定程度上对土壤结构、土壤质量起到优化的作用。     

Effect of fungicide thiram on microbial communities of rice seed surface estimated by culture-dependent and culture-independent (PCR-RFLP) methods

Coating of rice seeds with fungicide Thiram improved the seed germination capability over a long period of time (11 weeks) under low temperature conditions (4 and 8°C), which simulated the sowing of rice seeds in the winter season (the farmer's slack season). To analyze the effect of Thiram on the community structure of microorganisms on the rice seed surface, culture-dependent and culture-independent (PCR-RFLP) methods were applied. PCR-RFLP patterns of 16S rDNA showed that the bacterial communities on the rice seed surface were different between coated and uncoated treatments under 8°C conditions, but that they were very similar under 4°C conditions. PCR-RFLP patterns of 18S rDNA revealed the remarkable effect of Thiram on the fungal community structure under both 4 and 8°C conditions. Although the fungal communities were quite different between coated and uncoated seeds at the beginning of incubation, the fungal communities on the coated seed surface became similar to those of uncoated seeds along with the duration of the incubation period. As the dominance percentage of Fusarium spp. among the isolates increased with the duration of the incubation period for both coated and uncoated seeds, Fusarium was considered to be a responsible for the poor germination of rice seeds that were sown in the winter season.     

Analysis of bacterial communities in soil by PCR-DGGE targeting protease genes

We studied the effects of the application of organic (OM) and inorganic fertilizer (CF) on soil protease activity and proteolytic bacterial communities in rhizosphere and bulk soil on an experimental lettuce field in Hokkaido, Japan. The protease activity always was higher in soils of the OM than with the CF treatment, and also higher in the rhizosphere than in the bulk soil. We analyzed proteolytic bacterial communities by denaturing gradient gel electrophoresis (DGGE) of the alkaline metalloprotease (apr) and neutral metalloprotease (npr) genes. Most apr forms detected were closely related to apr of Pseudomonas fluorescens, and all npr variants closely resembled the gene of Bacillus megaterium. These results were consistent with findings from tests using cultured bacterial communities, indicating a high specificity of our PCR-DGGE for amplifying apr and npr genes. The community compositions of proteolytic bacteria were assessed by principal component analysis of the DGGE profiles. There were significant differences in the effects of CF and OM on the community compositions of apr- and npr-expressing bacteria, and the communities of the two types of bacteria played different roles in rhizosphere and bulk soil. We found significant correlations between the protease activity and the communities of the two types of bacteria. The results indicate that different proteolytic bacteria release different amounts or activities of protease, and that the composition of proteolytic bacterial communities may play a major role in determining overall soil protease activity.     

Simultaneous estimates of the diversity and the degradative capability of heavy-metal-affected soil bacterial communities

Summary A method for simultaneous estimation of the impact of heavy metal stress on the diversity and the degradative capability of soil bacteria was developed and tested. It is based on the ability of soil bacteria to use aromatic substances as C sources. Though these characters were selected to indicate specific biochemical potential, they were sufficiently capable of differentiating the isolated strains into biochemical types. Using these characters implied that only organisms capable of growing at the expense of aromatics were tested. However, this made it possible to restrict the number of assays to 20 and to test up to 200 isolates per soil sample. In three out of five experiments, we found that heavy metal stress definitely decreased the diversity of bacteria in a flora. In two other experiments, an unchanged or even higher diversity in the metal-contaminated soils was observed. These unexpected results may have been caused by a high soil pH rendering metals unavailable or by selection of fast-growing strains in the control soil (decrease in evenness). The relative scoring of all characters in a community (also the average number of substrates used per isolate) was not a reliable indicator of changes in the degradative capability of bacterial communities. However, the ratio of the 10 lowest-scoring to the 10 highest-scoring out of the 20 characters tested was capable of indicating these changes. In all heavy-metal-affected bacterial communities so far tested, this ratio was lower than in the corresponding unaffected communities. These data suggest that some of the rare biochemical capabilities of bacterial flora were lost following contamination of soils by heavy metals.     

Members of soil bacterial communities sensitive to tillage and crop rotation

Pyrosequencing was used to study the effect of rotation and tillage on total bacterial communities. We designed primers to the bacterial 16s rDNA and amplified DNA from soil samples from a long-term tillage/rotation trial in Kansas for two seasons. The 2 × 2 factorial trial had two rotation treatments (wheat-wheat and wheat-soybean) and two tillage treatments (conventional and no-till). A total of 20,180 16s rDNA sequences were generated and 2337 operational taxonomic units (OTUs) were assembled using a 97% similarity cut-off. The phylum Proteobacteria represented 38% of 299 identified taxa. The second most abundant phylum was Acidobacteria, making up 20% of the sequences, the majority of which were Acidobacteria Group 1. The phyla Actinobacteria and Gemmatimonadetes comprised 12% and 3.5% of the sequences. Other groups detected included TM7, Nitrospira, Verrucomicrobia, and Bacteroidetes. Some clusters of Acidobacteria Group 1 were more frequent in continuous wheat versus wheat-soybean rotation, some Acidobacteria Group 2 were more frequent in no-till, and some Acidobacteria Group 4 were more frequent in wheat-soybean rotation. These results were validated by quantitative real-time PCR. Pyrosequencing provided taxonomic information about the overall bacterial community, and detected community shifts resulting from different cropping practices.     

土地资源评价方法及评价因素权重的确定探析

综述了目前土地资源评价方法和评价系数权重的确定研究成果,评价了主观赋权法(德尔菲、AHP法)和客观赋权法(主成分分析法、因子分析法、灰色关联法和熵值法)的优、缺点,并认为运用主观赋权法掺杂了决策者的主观随意性,而运用客观赋权法却缺乏决策者的意愿。指出主、客观信息综合集成的新方法是土地资源评价方法及评价因素权重确定的新途径。     

Fluazifop-P-butyl (herbicide) affects richness and structure of soil bacterial communities

Fusilade (fluazifop-P-butyl) is an extensively used herbicide in legumes cropping systems; nevertheless, the extent of its ecological risk remains unknown. The aim of this work is to estimate the impact of this herbicide on some key enzyme activities, namely acid phosphatase, urease and FDA, and the effect on soil bacterial communities in the rhizosphere of pea (Pisum sativum). Acid phosphatase, urease and FDA showed a high decrease following the application of fluazifop-P-butyl mainly at ten-fold field rate. Grain yield was also severely affected at this concentration (−50%). T-RFLP analysis of 16S rDNA revealed a significant effect of fluazifop-P-butyl application on richness and structure of soil bacterial communities. Putative taxonomic assignation of induced TRFs revealed a marked pattern of acidogenic bacteria like as Staphylococcus, Escherichia, Desulfobacter, Clostridium, Paludibacter and other sulfate-reducing bacteria like as Desulfocella halophila and Desulfocurvus vexinensis. These bacteria may participate in the acidogenesis and the production of propionic acid from the fluazifop-P-butyl after hydrolytic cleavage. They are also known to play important roles in the global biogeochemical sulfur cycles. Unfortunately, some potential human pathogens were also stimulated. Moreover, many putative plant-growth-promoting bacteria belonging to the genera Streptomyces and Bacillus were found to be inhibited in the bulk soil and rhizosphere. The fluazifop-P-butyl effect on bacterial diversity was more pronounced in the rhizosphere compared to bulk soil or uncultivated soil which may suggest that a major component of this effect is mediated by the root system.     

Effects of rhamnolipids on bacterial communities in a dioxin-contaminated soil and the gut of earthworms added to the soil

The biosurfactants rhamnolipids and the “soil ecosystem engineers” earthworms are often used to remediate contaminated soils. However, the effects of rhamnolipids on earthworm intestinal flora and microbial community in soil containing earthworms are not clearly understood. In our study, a 21-d microcosm experiment was carried out to reveal the effects of rhamnolipids on microbial abundance, composition, and metabolism, as well as contaminant degradation capacity. Both rhamnolipids and earthworm...