模拟干湿交替对水稻土古菌群落结构的影响

干湿交替是自然界普遍存在的现象,但长期以来由于技术的限制,复杂土壤中微生物对水分变化的响应规律仍不清楚。针对我国江苏常熟湖泊底泥发育的典型水稻土,在室内开展湿润-风干以及风干-湿润各三次循环,每次循环中湿润、风干状态各维持7d,利用微生物核糖体rRNA的通用引物进行PCR扩增,通过高通量测序分析土壤古菌多样性变化,同时结合实时荧光定量PCR技术,在DNA和RNA水平研究古菌数量对干湿交替过程的响应规律。结果表明:水稻土湿润-风干过程中,在DNA水平土壤古菌数量降幅约为149倍~468倍,而在RNA水平降幅最高仅为2.06倍;水稻土风干-湿润过程中,在DNA水平古菌数量增幅在147倍~360倍之间,而在RNA水平增幅最高仅为2.95倍。表明在干湿交替过程中,DNA水平的古菌16S rRNA基因数量变化远高于RNA水平。基于高通量测序多样性的结果表明,在DNA和RNA水平,湿润土壤3次风干、以及风干土壤3次加水湿润7d恢复后,土壤古菌群落结构均发生统计显著性改变。在微生物门、纲、目、科和属的不同分类水平下,水稻土古菌主要包括3、10、13、14、10种不同的类群,在RNA和DNA水平的结果基本一致。干湿交替导致部分古菌类群发生显著变化,其中在微生物分类学目水平发生显著变化的古菌最高达到6种,主要包括产甲烷古菌和氨氧化古菌,如Methanobacteriales、Methanosarcinales、Methanomicrobiales和Nitrososphaerales等。这些研究结果表明,反复的干湿交替并未显著改变水稻土中古菌的主要类群组成,古菌类群的绝对数量和相对丰度发生了一定程度的变化,但这些变化与微生物生理作用的联系仍需进一步研究;风干土壤中古菌RNA序列极可能来自于完整的古菌细胞,暗示了这些古菌细胞能够较好地适应水稻土中水分的剧烈变化,风干状态的土壤在一定程度也可用于土壤古菌群落组成研究。     

DNA- versus RNA-based denaturing gradient gel electrophoresis profiles of a bacterial community during replenishment after soil fumigation

We compared the responsiveness and sensitivity to soil fumigation of DNA- and RNA-based analyses of a bacterial community. We first established an improved RNA extraction method using DNA as an adsorption competitor, because it is extremely difficult to extract nucleic acids from clay-rich volcanic ash soil (Andisol), which adsorbs nucleic acids. This novel method facilitated RNA extraction from 500 mg of Andisol for molecular analyses. Then we monitored 16S rDNA PCR and 16S rRNA RT-PCR denaturing gradient gel electrophoresis (DGGE) profiles of samples collected from a chloropicrin (CP)-treated field over 2 months. The difference between untreated control and CP-treated plots was detected clearly both in DNA- and RNA-based DGGE profiles after treatment. The temporal changes in DGGE profiles, however, differed between DNA- and RNA-based analyses in CP-treated plots. RNA-based DGGE showed quicker and greater changes in the bacterial community after CP treatment than did DNA-based DGGE, which showed similar trends to RNA-based DGGE but with a time lag. The extent of decrease in the diversity index (H′) and the change in principal response curves was larger in RNA-based analyses. These results indicate that the rDNA PCR-DGGE method also detects DNA of microbes no longer alive after fumigation, and that rRNA provides a more responsive biomarker than rDNA.     

Soil bacterial communities under slash and burn in Mozambique as revealed by a metataxonomic approach

The slash-and-burn system is a subsistence agronomical practice widespread in tropical areas worldwide. This system has been extensively studied,especially for its impacts on agronomical aspects and soil physicochemical properties; however, knowledge of soil microbial diversity under slash and bum is scarce. In this study, for the first time, soil bacterial diversity of three locations from Central Mozambique, where slash and burn has been practiced for different durations of the forest fallow p...     

根际微生物研究进展

根际是土壤-植物生态系统物质交换的活跃界面。植物作为第一生产者同化大气CO2,将部分光合产物转运至地下,激发土壤微生物的生长和新陈代谢;土壤微生物则将有机态养分转化成无机形态,利于植物吸收利用。这个植物-微生物的互作关系维系和主宰着陆地生态系统的功能。自从100年前德国科学家LorenzHiltner提出根际概念以来,根际研究方兴未艾,研究内容不断得以丰富和发展。近年来,随着分子生物技术在土壤环境领域的应用,根际微生物研究出现快速发展的趋势。本文根据2004年在慕尼黑召开的第一届国际根际大会交流内容、结合近年来国际上报道的研究动向,对根际微生物研究方法、根际微生物生物多样性和生态功能、转基因生物的环境安全和根际微生物生物修复技术等内容作一综述。期望我国的根际微生物研究能在基础和应用领域得到快速发展。     

Characterization of nitrifying bacteria communities of soils from different ecological regions of China by molecular and conventional methods

Soil nitrification rate is very different among soil types, as a result of differences in physical and chemical properties. Little is known about the composition of the nitrifying bacteria community. In this investigation, three soils (fluvo-aquic soil, permeable paddy soil and red earth) from different geo-ecological regions in China were characterized for their nitrification activities and their nitrifying bacteria communities determined either by molecular approaches or by conventional culture methods. A 28-day long-term soil incubation showed that the maximum nitrification potential was found in the fluvo-aquic soil with almost 100% of inorganic N present as NO₃^–-N, while the minimum nitrification potential was in red earth with only a 4.9% conversion rate from ammonium into nitrate. There was no relationship between nitrification potential and numbers of nitrifiers in the soil. The conventional most probable number (MPN) method could enumerate ammonia oxidizers, but failed in enumerating nitrite oxidizers. Therefore, we used an MPN-PCR procedure which gave a convincing nitrite oxidizer count result, instead of MPN-diphylamine. Soils were characterized by denaturing gradient gel electrophoresis (DGGE) of DNA extracted from soils and amplified using a primer specific for the 16S rRNA gene and/or for the amoA gene. The DGGE columns of the three soils differed from each other. There were two similar bands present in DGGE columns of the fluvo-aquic and permeable paddy soils, but no similar band was found in DGGE columns of the red earth. The sequence of amoA indicated that all ammonia oxidizers in these soils were grouped into Nitrosospira clusters 1 and 3, and each soil had a common band similar to the other soils and a special band which differed from the other 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.     

Establishment of macro-aggregates and organic matter turnover by microbial communities in long-term incubated artificial soils

The study of interactions between minerals, organic matter (OM) and microorganisms is essential for the understanding of soil functions such as OM turnover. Here, we present an interdisciplinary approach using artificial soils to study the establishment of the microbial community and the formation of macro-aggregates as a function of the mineral composition by using artificial soils. The defined composition of a model system enables to directly relate the development of microbial communities and soil structure to the presence of specific constituents. Five different artificial soil compositions were produced with two types of clay minerals (illite, montmorillonite), metal oxides (ferrihydrite, boehmite) and charcoal incubated with sterile manure and a microbial community derived from a natural soil. We used the artificial soils to analyse the response of these model soil systems to additional sterile manure supply (after 562 days). The artificial soils were subjected to a prolonged incubation period of more than two years (842 days) in order to take temporally dynamic processes into account. In our model systems with varying mineralogy, we expected a changing microbial community composition and an effect on macro-aggregation after OM addition, as the input of fresh substrate will re-activate the artificial soils. The abundance and structure of 16S rRNA gene and internal transcribed spacer (ITS) fragments amplified from total community DNA were studied by quantitative real-time PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE), respectively. The formation of macro-aggregates (>2 mm), the total organic carbon (OC) and nitrogen (N) contents, the OC and N contents in particle size fractions and the CO₂ respiration were determined. The second manure input resulted in higher CO₂ respiration rates, 16S rRNA gene and ITS copy numbers, indicating a stronger response of the microbial community in the matured soil-like system. The type of clay minerals was identified as the most important factor determining the composition of the bacterial communities established. The additional OM and longer incubation time led to a re-formation of macro-aggregates which was significantly higher when montmorillonite was present. Thus, the type of clay mineral was decisive for both microbial community composition as well as macro-aggregation, whereas the addition of other components had a minor effect. Even though different bacterial communities were established depending on the artificial soil composition, the amount and quality of the OM did not show significant differences supporting the concept of functional redundancy.     

Microbial communities in rice rhizosphere altered by intermittent and continuous flooding in fields with long-term arsenic application

Rice cultivated on arsenic (As)-contaminated soils can, under some conditions, accumulate high concentrations of As in grain, mostly as a result of the continuous flooding practices commonly used for rice cultivation. Intermittent flooding, as opposed to continuous flooding, might reduce soluble As concentrations in the rice rhizosphere, but it might also alter soil microbial populations that may impact As chemistry. A field-scale study was conducted to analyze As concentrations and microbial populations in the rice rhizosphere, in response to intermittent and continuous flooding in plots that were historically amended with “As-containing” pesticide and unamended soil. Rhizosphere, pore-water and grain As concentrations were quantified, and microbial populations in the rhizosphere were characterized using community quantitative-PCR and 16S rRNA gene sequencing. Pore-water As concentrations decreased by 41-81% and grain As by 31-48% in the intermittently flooded plots relative to the continuously flooded plots. The relative abundance of bacteria increased over the course of the growing season, while archaeal and fungal gene abundances decreased. Bacterial community structure and composition were significantly different between As amended and unamended plots, as well as between the flooding treatments. Proteobacteria was the predominant phylum detected in most treatments with relative abundance of 24-29%. The relative abundance of iron-reducing bacteria was higher with the continuous flood compared to the intermittent-flood treatment, implying greater relative iron reduction and possibly As release from the iron oxides under the continuously flooded conditions. These differences in rhizosphere-microbial communities may have contributed to the lower pore-water arsenic concentrations in the intermittently flooded conditions.     

Genetic diversity of Rhizobium present in nodules of Phaseolus vulgaris L. cultivated in two soils of the central region in Chile

Although Phaseolus vulgaris L. is native from the Americas and is currently cultured in diverse areas, very little is known about the diversity of symbiotic nitrogen fixing Rhizobium (mycrosymbiont) in many of those cultures. Therefore, the aim of this study was to assess the genetic diversity of Rhizobium present in nodules of P. vulgaris in the central region of Chile. A method to extract DNA from surface-sterilized nodules was applied to two populations of the same seed variety grown in different fields. The 16S rRNA and nifH genes were amplified directly from the DNA extracted. DGGE analysis and clone libraries showed a restricted genetic diversity of the microsymbiotic populations that nodulate P. vulgaris. Both molecular markers revealed the presence of a microsymbiont closely related to Rhizobium etli in all the plants from the soils studied, indicating that the populations of Rhizobium sp. nodulating P. vulgaris in the central region of Chile displayed an extremely low genetic diversity. The level of genetic diversity in microsymbiont populations in plants grown in soils with different origin suggested that other factors rather than the indigenous soil rhizobial populations play a major role in the selection of the symbiotic partner in P. vulgaris.     

Long-term inorganic fertilizer use influences bacterial communities in Mollisols of Northeast China based on high-throughput sequencing and network analyses

The effects of chemical fertilizers on soil microbial communities have been well studied in a variety of terrestrial systems. However, the individual effects of N, P, and K fertilizers and their combinations (NP, NK, PK and NPK) on bacterial community structure in black soils have not been fully understood. In this study, we applied Illumina MiSeq sequencing and network analysis targeting 16S rRNA gene to comprehensively characterize the effects of 35 years of these inorganic fertilizers on community diversity and composition in the black soil. The results of α-diversity indices and principal coordinates analysis (PCoA) indicated that the N-addition treatments had larger effects than the others. The network analysis showed that the N fertilizer shifted the network structure, topological roles of individual OTUs and key bacterial populations, contributing to a simple network under the N-fertilizer treatments compared with non-N fertilizers treatments. These findings suggested that N fertilizer reduced the bacterial diversity and simplified the interactions among key bacterial members in Mollisol. The results of this study could provide basis for further researches balancing different N application levels, the bacterial community network and crop yield to maintain the ecosystem function and stability.

Abbreviations: N: nitrogen fertilizer; P: phosphorus fertilizer; K: potassium fertilizer; NP: nitrogen and phosphorus fertilizers; NK: nitrogen and potassium fertilizers; PK: phosphorus and potassium fertilizers; NPK: phosphorus and potassium fertilizers; TC: total carbon; TN: total nitrogen; TP: total phosphorus; TK: total potassium; AP: available phosphorus; AK: available potassium; OTUs: operational taxonomic Units; ACE: abundance-based coverage estimator; Chao1: estimated OTU richness; PCoA: principal coordinates analysis     

Common and distinguishing features of the bacterial and fungal communities in biological soil crusts and shrub root zone soils

Soil microbial communities in dryland ecosystems play important roles as root associates of the widely spaced plants and as the dominant members of biological soil crusts (biocrusts) colonizing the plant interspaces. We employed rRNA gene sequencing (bacterial 16S/fungal large subunit) and shotgun metagenomic sequencing to compare the microbial communities inhabiting the root zones of the dominant shrub, Larrea tridentata (creosote bush), and the interspace biocrusts in a Mojave desert shrubland within the Nevada Free Air CO₂ Enrichment (FACE) experiment. Most of the numerically abundant bacteria and fungi were present in both the biocrusts and root zones, although the proportional abundance of those members differed significantly between habitats. Biocrust bacteria were predominantly Cyanobacteria while root zones harbored significantly more Actinobacteria and Proteobacteria. Pezizomycetes fungi dominated the biocrusts while Dothideomycetes were highest in root zones. Functional gene abundances in metagenome sequence datasets reflected the taxonomic differences noted in the 16S rRNA datasets. For example, functional categories related to photosynthesis, circadian clock proteins, and heterocyst-associated genes were enriched in the biocrusts, where populations of Cyanobacteria were larger. Genes related to potassium metabolism were also more abundant in the biocrusts, suggesting differences in nutrient cycling between biocrusts and root zones. Finally, ten years of elevated atmospheric CO₂ did not result in large shifts in taxonomic composition of the bacterial or fungal communities or the functional gene inventories in the shotgun metagenomes.     

Molecular and functional characterization of bacteria isolated from straw and goat manure based vermicompost

A total of 193 vermicompost bacteria that exhibit antagonistic and biofertilizing potential were isolated from straw and goat manure based vermicompost produced by earthworm Eisenia foetida and taxonomically designated on the basis of their 16S rRNA sequence homology and subsequent molecular phylogeny analysis. Bacteria belonged to three major genera viz., Pseudomonas (15%), Bacillus (57%), Microbacterium (12%) and remaining bacteria comprised of genus Acinetobacter (5%), Chryseobacterium (3%), Arthrobacter, Pseudoxanthomonas, Stenotrophomonas, Paenibacillus, Rhodococcus, Enterobacter, Rheinheimera and Cellulomonas. Of 193 bacteria, 96 strains (49%) showed antagonistic potential against phytopathogenic fungus. Functional characterization of the bacteria was assessed by the production of protease, cellulase, lipase, xylanase, chitinase, amylase, gelatinase, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, indole-3-acetic acid (IAA), phosphate solubilization, nitrate reduction, and assimilation of different carbon sources. The bacterial strains showed a varying degree of carbon utilization profiles and majority of them were known to utilize citrate, malonate. A total of 86 strains produced protease (44%) and 99 strains (51%) produced cellulase. 51 strains (26%) produced IAA, 99 strains (51%) produced siderophores and 124 strains (64%) produced ACC deaminase. 52 strains (27%) showed phosphate solubilization while 24 strains (12%) produced HCN. 43 strains (22%) showed antibacterial activity against human pathogens under in vitro conditions. None of the strains tested positive for lipase and chitinase. A total of 31 strains (16%) produced DNase, 80 strains (41%) produced xylanase and 93 strains (48%) produced amylase. Among the vermicompost bacteria reported in the present study, Bacillus was found to be the predominant bacteria followed by Pseudomonas and Microbacterium. Present study, reports the molecular and functional characterization of vermicompost bacteria.     

Impact of urine and the application of the nitrification inhibitor DCD on microbial communities in dairy-grazed pasture soils

Tools to manage the emission of the greenhouse gas nitrous oxide (N₂O), an intermediate of both nitrification and denitrification, from soils are limited. To date, the nitrification inhibitor dicyandiamide (DCD) is one of the most effective tools available to livestock farmers for reducing N₂O emissions and minimizing leaching of nitrogen in response to increased urine deposition in grazed pasture systems. Despite its effectiveness in decreasing N losses from animal urine by inhibiting N processes in soils, the effect of DCD on the population structure of denitrifiers and overall bacterial community composition is still uncertain. Here we use three New Zealand dairy-grazed pasture soils to determine the effects of DCD application on microbial community richness and composition at both functional (genes involved in the denitrification process) and phylogenetic (overall bacterial community composition based on 16S rRNA profiling) levels. Results further confirm that the effects on microbial populations are minimal and transient in nature. The impact of DCD on microbial community structure was soil dependent, and a greater effect was attributed to intrinsic soil properties like soil texture, with community response to DCD in combination with urine being comparable to that under urine alone. Addition of DCD to cattle urine also reduced N₂O emission between 23 and 67%.     

一株富含类胡萝卜素光合细菌菌株GP-7的分离鉴定

从珠海横琴岛近海海水中筛选到一株光合细菌GP-7,测定了其类胡萝卜素含量,通过形态观察及16S rRNA编码序列同源性比较分析确认其种属,并对该菌的生长条件和生物学特征进行了初步的研究。结果表明：GP-7的类胡萝卜素质量分数为4.87 mg/g（干菌体）,该菌符合光合细菌的生物学特征,其最适pH为7.0,盐度为1.0%;16S rRNA基因分子系统学分析表明,该菌株与紫色非硫菌（Rhodoplanes）相似性为99.5%。     

Biogeography and biophysicochemical traits link N2O emissions,N2O emission potential and microbial communities across New Zealand pasture soils

The process of denitrification has been studied for decades, with current evidence suggesting that an ecosystem's ability to produce and emit N₂O is controlled both by transient ‘proximal’ regulators (e.g. temperature, moisture, N availability) as well as distal regulators (e.g. soil type, microbial functional diversity, geography). In this study we use New Zealand soils as a model system to test the impact of distal regulators (i.e. geography) on microbial communities and their N₂O emission potential. Using gas chromatography, soil chemical analyses, 16S amplicon sequencing, terminal restriction fragment length polymorphism (T-RFLP) and quantitative PCR (qPCR) on three denitrifier functional genes (nirS, nirK and nosZ), we assessed the factors linked to N₂O emissions across a latitudinal gradient. Results show that soil drainage class, soil texture class, and latitude were powerful regulators of both emissions and emission end products (N₂ vs. N₂O). Mixed models demonstrate that a few variables (including latitude, texture class, drainage class and denitrifier community data [abundance and diversity] amongst others) were enough to predict both the amount and type of gas emitted. In addition we show that microbial community composition (based on 16S rRNA gene sequencing) can also be used to predict both the gas species and quantity emitted.     

天山云杉林下土壤放线菌的分离与DNA指纹分析方法的初步研究

新疆天山土壤放线菌的分离和鉴定工作,对全面了解该地区放线菌分布状况具有重要意义。使用7种典型的放线菌分离培养基对土样中的放线菌进行分离,共得到8个属的放线菌分离株,并对分离培养基的分离效果进行比较。使用MseI、AluI、PstI和HhaI 4种限制性内切酶对20株链霉菌分离株的16S rRNA基因进行了PCR-RFLP分析。使用BOX-PCR方法构建了9株稀有放线菌菌株的指纹图谱。PCR-RFLP和BOX-PCR聚类分析结果分别与16S rRNA基因全序列聚类分析结果具有一致性。因此,PCR-RFLP和BOX-PCR指纹分析技术可在一定程度上代替16S rRNA基因全序列分析,用于新疆天山土壤放线菌的快速鉴定和多样性研究。     

印度西部卡奇沼泽地沉积物中微生物活性和人工培养细菌多样性的研究

The Great Rann of Kachchh, a vast expanse of salt desert in Western India is a unique hostile ecosystem posing an extreme environment to life forms due to high salt content, hyper-axid climate, seasonal water logging and extremes of temperature. In the virtual absence of natural vegetation, soils and sediments of Rann of Kachchh axe microbially dominated ecosystems. In the present study microbial activity and the diversity of cultivated heterotrophic bacteria were investigated in the sediments collected along a 5-m exposed section at Khadir Island in the Great Rann of Kachchh. Microbial activity （as an index of sediment enzymes） was found to be high in the middle of the section （200-280 cm）. Dehydrogenase （DHA）, substrate-induced DHA and alkaline phosphatase activities revealed the oligotrophic nature of the basal portion （320-480 cm）. Abundant bacterial isolates obtained from different depths were found to be clustered in 12 different phylogenetic groups by amplified ribosomal DNA restriction analysis. 16S rRNA gene sequencing revealed the dominant bacterial ribotypes to be affiliated to Firmicutes （Families Bacillaceae and Staphyloeoccaeeae） and Aetinobaeteria （Family Brevibaeteriaceae） with minor contribution of Proteobacteria （Families Phyllobacteriaeeae and Bartonellaceae）, pointing their endurance and adaptability to environmental stresses. Statistical analysis indicated that sediment organic carbon, salinity, total available nitrogen and total available phosphorous are most likely critical determinants of microbial activity in the Khadir Island sediments.