Differences in the activity and bacterial community structure of drained grassland and forest peat soils

The microbial activity and bacterial community structure were investigated in two types of peat soil in a temperate marsh. The first, a drained grassland fen soil, has a neutral pH with partially degraded peat in the upper oxic soil horizons (16% soil organic carbon). The second, a bog soil, was sampled in a swampy forest and has a very high soil organic carbon content (45%), a low pH (4.5), and has occasional anoxic conditions in the upper soil horizons due to the high water table level. The microbial activity in the two soils was measured as the basal and substrate-induced respiration (SIR). Unexpectedly, the SIR (μl CO₂ g⁻¹ dry soil) was higher in the bog than in the fen soil, but lower when CO₂ production was expressed per volume of soil. This may be explained by the notable difference in the bulk densities of the two soils. The bacterial communities were assessed by terminal restriction fragment length polymorphism (T-RFLP) profiling of 16S rRNA genes and indicated differences between the two soils. The differences were determined by the soil characteristics rather than the season in which the soil was sampled. The 16S rRNA gene libraries, constructed from the two soils, revealed high proportions of sequences assigned to the Acidobacteria phylum. Each library contained a distinct set of phylogenetic subgroups of this important group of bacteria.     

Archaeal populations in biological soil crusts from arid lands in North America

Archaea are common and abundant members of biological soil crust communities across large-scale biogeographic provinces of arid North America. Regardless of microbial community development, archaeal populations averaged 2 × 10⁷ 16S rRNA gene copies per gram of soil, representing around 5% of the prokaryotic (total calculated bacterial and archaeal) numbers assessed by quantitative-PCR. In contrast, archaeal diversity, determined by denaturing gradient gel electrophoresis fingerprinting and clone libraries of 16S rRNA genes, was very restricted. Only six different phylotypes (all Crenarchaea) were detected, three of which were very dominant. Some phylotypes were widespread, while others were typical of Southern desert areas.     

Microbial Diversity and Bioactive Substances in Disease Suppressive Composts from India

The present study aimed to investigate microbial communities in seven Indian composts and their potential for biocontrol of Fusarium oxysporum f. sp. lycopersici. In addition, identification of bioactive substances in disease suppressive composts was also attempted. Composts were chosen based on disease suppressiveness and subjected to molecular microbial analyses. Total genomic DNA from the composts was extracted and amplified with polymerase chain reaction using primers targeting the 18S rRNA and 16S rRNA genes of fungi and bacteria, respectively. Denaturing gradient gel electrophoresis (DGGE) fingerprinting and DNA sequencing were used to identify the fungal and bacterial targets. Phylogenetic analysis of the fungal 18S rRNA ITS gene sequences showed that phylum Ascomycota was dominant in all composts, while in the bacterial 16S rRNA gene sequences, the phylum Proteobacteria was dominant. Some fungi in disease suppressive composts grouped phylogenetically close to F. oxysporum. Bacterial sequences with close similarity (>95% identity) with Actinobacterium showed a strong presence only in disease suppressive composts. Disease suppressive composts formed a separate group in the cluster analysis of 18S rRNA ITS and 16S rRNA gene sequences. Gas chromatography-time of flight-mass spectrometry was performed with compost extracts to determine if bioactive substances were present in disease suppressive composts. The analysis of compost organic matter showed a negative association of disease suppressiveness with phloroglucinol, sitosterol, and monoenoic fatty acid, while cholesterol and certain organic acids were positively associated with suppressiveness.     

Characterization of redox-related soil microbial communities along a river floodplain continuum by fatty acid methyl ester (FAME) and 16S rRNA genes

Redox states affect substrate availability and energy transformation, and, thus, play a crucial role in regulating soil microbial abundance, diversity, and community structure. We evaluated microbial communities in soils under oxic, intermittent, and anoxic conditions along a river floodplain continuum using fatty acid methyl ester (FAME) and 16S rRNA genes-based terminal-restriction fragment length polymorphism (T-RFLP) bacterial fingerprints. In all the soils tested, microbial communities clustered according to soil redox state by both evaluation techniques. Bacteria were dominant components of soil microbial communities, while mycorrhizal fungi composed about 12% of the microbial community in the oxic soils. Gram-positive bacteria consisted >10% of the community in all soils tested and their abundance increased with increasing soil depth when shifting from oxic to anoxic conditions. In the anoxic soils, Gram-positive bacteria composed about 16% of the total community, suggesting that their growth and maintenance were not as sensitive to oxygen supply as for other microbes. In general, microorganisms were more abundant and diverse, and distributed more evenly in the oxic layers than the anoxic layers. The decrease in abundance with increasing oxygen and substrate limitation, however, was considerably more drastic than the decrease in diversity, suggesting that growth of soil microorganisms is more energy demanding than maintenance. The lower diversity in the anoxic than the oxic soils was attributed primarily to the differences in oxygen availability in these soils.     

指纹图谱技术在动物肠道微生物多样性研究中的应用

随着分子生物技术的发展,不可培养微生物多样性研究的难题得到了解决。肠道微生物处于特殊的生态环境条件下,分子生物学技术的应用使得肠道微生物多样性的研究进入了一个崭新的阶段。本文主要介绍了基于16S rRNA基因片段的一些肠道微生物研究工作中常用的分子生物学分析方法,主要包括变性梯度凝胶电泳(DGGE),温度梯度凝胶电泳(TGGE),单链构象多态性(SSCP),限制性片段长度多态性(RFLP),放大片断长度多态性(AFLP)和随机扩增多态性DNA(RAPD)等指纹图谱技术。     

Reduction processes in forest wetlands: Tracking down heterogeneity of source/sink functions with a combination of methods

Wetlands are considered to be the biggest unknowns regarding the influence of global climate change on element dynamics, so knowledge of processes and conditions controlling sink and source functions of redox processes is crucial. The aim of this study was to investigate the sink/source function of nitrate, Fe, sulfate reduction and methanogenesis of an upland and a lowland fen within a boreal spruce catchment, southern Germany. We used suction cups and anaerobic dialysis chambers for soil solution sampling, FeS probes for the determination of S oxidation potential and stability of anoxic conditions and analysis of the soil solid phase (contents of C, S and Fe species). Both fens had high rates of nitrate reduction and potentially high rates of CH₄ production. The upper few cm of all profiles were oxic with low CH₄ concentrations, suggesting low CH₄ emission rates from the soil, though emission by vascular plants cannot be excluded. Sulfate and Fe reduction processes differed significantly in the fens. The upland fen was characterized by relatively stable anoxic conditions, low Fe contents but high contents of organic S and low C/S ratios. We concluded that the upland fen is an effective sink for sulfate with long-term S storage. In contrast, the lowland fen was characterized by alternating oxidation-reduction cycles with high Fe contents, lower contents of organic S and higher C/S ratios. Thus, even though low sulfate and high Fe concentrations in soil solutions indicated high reduction rates in the lowland fen, long-term storage of S is not likely in this fen. Differences in biogeochemical processes between sites are most likely not associated with hydrology but rather with the role of vascular plants.     

Molecular analysis of β-proteobacterial ammonia oxidizer populations in surface layers of a submerged paddy soil microcosm

The structure of the β-proteobacterial autotrophic ammonia-oxidizing bacterial (AOB) communities in a microcosm of submerged paddy soil was determined by denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments amplified using AOB-selective primers. Shift in the community composition was observed 4 weeks after submergence. The communities from the surface layers (0–1, 2–3 mm) of the soil microcosm were different from those of the subsurface layers (6–9, > 15 mm) and DGGE bands specific to each layer were detected. The majority of the retrieved sequences were Nitrosospira-like, whereas no Nitrosomonas-like sequences were obtained. The 16S rDNA primer set also amplified sequences that were not related to the known Nitrosospira-Nitrosomonas group, although they showed a close relationship with other groups of β-proteobacteria. The results suggest that Nitrosospira-like populations are dominant AOB populations in the submerged paddy soil, and that the oxic layer of submerged paddy soil harbours the specific AOB.     

SSCP intron marker system is a convenient tool for clonal fingerprinting of poplar (<Emphasis Type="Italic">Populus</Emphasis>) cultivars of different species and interspecific hybrids

In this study, we evaluate the effectiveness of SSCP analysis of intron markers, as a routine tool for fingerprinting of clones of different poplar species and interspecific hybrids. Exon primed intron crossing primers were designed on two catalase genes. Analysis of these fragments on SSCP gels revealed that catalase introns exhibit substantial sequence variation within poplar species and cultivars. SSCP analysis resulted effective in detecting intra and interspecific polymorphisms among the 119 poplar cultivars obtaining 110 different individual profiles. A hierarchical clustering process showed the high level of divergence among clones reflecting in most cases the section and the species the clones belongs to. This marker system was also proved with high transferability within Salicaceae. SSCP intron analysis represents a powerful tool in detecting DNA sequence polymorphism, in screening diversity and an effective method for clonal fingerprinting.     

Demethylation of Dimethylarsinic Acid and Arsenobetaine in Different Organic Soils

Methylation and demethylation of arsenic may change substantially the toxicity and mobility of arsenic in soils. Little is known about demethylation of organic arsenic species in organic soils. We incubated dimethylarsinic acid (DMA) and arsenobetaine (AsB) in soils and aqueous soil extracts from a forest floor and fen, in order to investigate demethylation processes. Incubations were conducted at 5°C in the dark under oxic or anoxic conditions. Arsenobetaine demethylated rapidly in all soil extracts with half-lives of 3.6–12 days, estimated from first order kinetic. Demethylation of DMA was relatively slow with half-lives of 187 and 46 days in the forest floor extracts and oxic fen extracts, respectively. In comparison, DMA was stable for 100 days in anoxic fen extracts. The apparent half-lives were much shorter in soils for DMA (1.3–12.6 days) and AsB (0.5–1.9 days) than in soil extracts, suggesting also irreversible AsB and DMA adsorption to soils beside demethylation. An unknown arsenic species and DMA were detected as metabolites of AsB demethylation. The results indicate rapid demethylation of AsB probably via the pathway AsB → Dimethylarsenoylacetate → DMA, followed up by slow demethylation of DMA → monomethylarsonic acid → inorganic As species.     

Microbial responses to fluctuation of soil aeration status and redox conditions

 The response of the microbial community to changes in aeration status, from oxic to anoxic and from anoxic to oxic, was determined in arable soil incubated in a continuous flow incubation apparatus. Soil incubated in permanently oxic (air) and/or anoxic (O₂-free N₂) conditions was used as the control. Before experiments soil was preincubated for 6 days, then aeration status was changed and glucose added. Glucose concentration, extractable C, CO₂ production, microbial biomass, pH and redox potential were determined 0, 4, 8, 12, 16, 24, 36 and 48 h after change of aeration status. If oxic conditions were changed to anoxic, the amount of glucose consumed was reduced by about 60%, and CO₂ production was 10 times lower at the end of incubation compared to the control (permanently oxic conditions). Microbial biomass increased by 114% in glucose-amended soil but did not change in unamended soil. C immobilization prevailed over C mineralization. Redox potential decreased from +627 mV to –306 mV. If anoxic conditions were changed to oxic, consumption of glucose and CO₂ evolution significantly increased, compared to permanently anoxic conditions. Microbial biomass did not change in glucose-amended soil, but decreased by 78% in unamended soil. C mineralization was accelerated. Redox potential increased from +238 to +541 mV. The rate of glucose consumption was low in anoxic conditions if soil was incubated in pure N₂ but increased significantly when incubation was carried out in a CO₂/N₂ mixture. Received: 6 January 1999     

Active community structure of microeukaryotes in a rice (Oryza sativa L.) rhizosphere revealed by RNA-based PCR-DGGE

The rhizosphere is one of the hot spots in soil ecosystems for a variety of microorganisms. In this study, we explored the seasonal change of the microeukaryotic community of a rice rhizosphere focusing on the active members through an RNA-based molecular approach. Rice plants (Oryza sativa L.) were grown in a pot where the rhizosphere was compartmented from bulk soil with a nylon gauze. The Eh in the rhizosphere compartment indicated that the rhizosphere was under oxic conditions in the initial stage of plant growth and then suddenly became anoxic or suboxic. Denaturing gradient gel electrophoresis targeting 18S rRNA-transcribed cDNA demonstrated that the active community of microeukaryotes in the rice rhizosphere was different from that in the bulk soil. The rhizosphere community showed a temporal shift in accordance with the shift of the redox conditions having three stages: the oxic before maximum tillering stage, anoxic/suboxic stage before maximum tillering stage, and anoxic/suboxic stage in the panicle initiation stage and thereafter. Active members specific to the rhizosphere at either the oxic or anoxic/suboxic stage were found: Heterolobosea amoeba, ciliates, and Chytridiomycota fungi for the oxic stage and oomycetes, ciliates and Ascomycota fungi in the anoxic/suboxic stage. The present results demonstrate that a specific group of microeukaryotes inhabit the rice rhizosphere even under anoxic/suboxic conditions and play various ecological roles as plant parasites, microbial grazers and organic decomposers.     

Search of environmental descriptors to explain the variability of the bacterial diversity from maize rhizospheres across a regional scale

The regional scale variability of the bacterial community inhabiting the rhizosphere was studied with soil collected from maize fields located in the Santo Domingo Valley (SDV; Baja California Sur, Mexico), a semi-arid agricultural ecosystem of approximately 200 km². The bacterial community structure was visualized by single-strand conformation polymorphism (SSCP) profiles of PCR-amplified partial 16S rRNA genes of directly extracted rhizosphere soil DNA. SSCP profiles of different SDV sites and an external field site in Germany were evaluated for their similarities and the contributing bacteria were characterized by DNA sequence analyses. SSCP profiles from each site were significantly different from the others, as revealed by permutation of pairwise similarities (P < 0.05). In comparison to the German site, SSCP profiles from SDV were more similar to each other despite contrasting soil salinity levels. Correspondence analysis revealed that among SDV sites, salinity levels, soil organic carbon and calcium (Ca²⁺) were most influential on the bacterial community structure. Depending on the phylogenetic group analyzed (Bacteria, Alphaproteobacteria, Pseudomonas), the importance of these soil variables varied. Interestingly, the East–West direction also revealed an effect, suggesting that future explorations of bacterial diversity patterns should also consider landscape topography in search of explaining patterns of bacterial diversity in soils.     

NaCl盐度对A2/O工艺去除废水污染物和系统微生物的影响

为了提高含盐废水的有机物去除率和脱氮效率,考察NaCl盐度对A~2/O工艺污染物去除和微生物群落的影响,采用高通量测序技术分析了厌氧区、缺氧区和好氧区的微生物群落结构,结合有机物去除和脱氮效率的变化探讨不同盐度下A~2/O工艺优势种群的演替规律,以期揭示含盐废水生物脱氮机理。结果表明:1)随着NaCl盐度的增大,A~2/O工艺污染物去除率下降,当盐度由0增大至40 g/L时,A~2/O反应器厌氧、缺氧和好氧区域COD去除率分别由52%、80%和56%下降至30%、50%和40%;厌氧区和好氧区NH4+-N去除率分别由33%和61%下降至11%和39%;缺氧区NO3--N去除率由63%下降至47%。2)与无NaCl废水相比,加入NaCl后,微生物的多样性降低;高盐度(40 g/L)与低盐度(0、10 g/L)处理的微生物群落结构差异较大;缺氧区陶氏菌属和副球菌属、好氧区梭菌属和硝化螺旋菌相对丰度的降低是导致A~2/O工艺脱氮效率下降的主要原因;厚壁菌门中的部分菌属(如Lactobacillus、Streptococcus、Tepidibacterium、Veillonella、Lachnoclostridium、Zoogloea)相对丰度增大,具有较强的耐盐性;随着盐度的增大,与脱氮相关的微生物(如变形菌门、拟杆菌门、厚壁菌门等)一直是A~2/O工艺厌氧区、缺氧区和好氧区的优势菌门,保证了不同盐度下A~2/O工艺始终具有一定的脱氮效能。     

Bacterial community incorporating carbon derived from plant residue in an anoxic non-rhizosphere soil estimated by DNA-SIP analysis

Purpose

Plant residues are one of the main sources of soil organic matter in paddy fields, and elucidation of the bacterial communities decomposing plant residues was important to understand their function and roles, as the microbial decomposition of plant residues is linked to soil fertility. We conducted a DNA stable isotope probing (SIP) experiment to elucidate the bacterial community assimilating 13-carbon (¹³C) derived from plant residue under an anoxic soil condition. In addition, we compared the bacterial community with that under the oxic soil condition, which was elucidated in our previous study (Lee et al. in Soil Biol Biochem 43:814–822, 2011).

Materials and methods

We used the ¹³C-labeled dried rice callus cells as a model of rice plant residue. A paddy field soil was incubated with unlabeled and ¹³C-labeled callus cells. DNA extracted from the soils was subjected to buoyant density gradient centrifugation to fractionate ¹³C-enriched DNA. Then, polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis of bacterial 16S rDNA band patterns and band sequencing method were used to evaluate bacterial community.

Results and discussion

DGGE analysis showed that the band patterns in the ¹³C-enriched fractions were distinctly changed over time, while the changes in the community structure before fractionation were minor. Sequencing of the ¹³C-labeled DGGE bands revealed that Clostridia were a major group in the bacterial communities incorporating the callus-derived carbon although Gram-negative bacteria, and Actinobacteria also participated in the carbon flow from the callus under the anoxic condition. The proportion of Gram-negative bacteria and Actinobacteria increased on 14 days after the onset of incubation, suggesting that the callus was decomposed by diverse bacterial members on this phase. When the bacterial groups incorporating the ¹³C were compared between under anoxic and oxic soil conditions, the composition was largely different under the two opposite conditions. However, some members of Gram-negative bacteria were commonly found under the anoxic and oxic soil conditions.

Conclusions

The majority of bacterial members assimilating the callus carbon was Clostridia in the soil under anoxic conditions. However, several Gram-negative bacterial members, such as Acidobacteria, Bacteroidetes, and Proteobacteria, also participated in the decomposition of callus under anoxic soil conditions. Our study showed that carbon flow into the diverse bacterial members during the callus decomposition and the distinctiveness of the bacterial communities was formed under the anoxic and oxic soil conditions.

     

A novel approach to the quantification of bovine milk in ovine cheeses using a duplex polymerase chain reaction method

A duplex Polymerase Chain Reaction (PCR) method able to detect bovine milk in ovine cheeses was developed. This method is based on the mitochondrial 12S and 16S rRNA genes to generate fragments of different lengths. The proposed methodology presents an alternative DNA extraction procedure faster and more economical than the kits commercially available. A linear normalized calibration curve was obtained between the log of the ratio of the bovine band intensity and the sum of bovine and ovine band intensities versus the log of cow's milk percentage. The method was applied successfully to the detection and quantification of raw, pasteurized, and powdered bovine milk in different cheeses. The proposed duplex PCR provides a simple, sensitive, and accurate approach to detect as low as 0.1% bovine milk in cheeses and to quantify bovine milk in ovine cheeses in the range of 1-50%.     

An improved method for purifying genomic DNA from forest leaf litters and soil suitable for PCR

Background, aim and scope  An improving knowledge of bacterial community within natural environments including forest soils and leaf litters requires extraction of nucleic acids directly from environmental samples since molecular approaches provide less biased access to a larger portion of uncultivable microorganisms. However, when DNA was extracted successfully from these samples, it might still have been difficult to apply it as a template for polymerase chain reaction (PCR) amplifications due to the effect of PCR inhibitors. Various compounds from plant tissues including polysaccharides, phenolic compounds and especially humic acids can inhibit PCR amplification. Some of these inhibitors could inhibit PCR amplification by chelating the Mg²⁺ (cofactor for Taq polymerase), or by binding to target DNA, and PCR amplification would consequently be interfered with. Therefore, eliminating the effects of these PCR inhibitors is one of the most important steps for PCR-based molecular techniques. Four different methods were assessed in this study to purify the genomic DNA extracted from F, L layer leaf litters and forest soil in an exotic pine plantation of southeast Queensland, Australia. Materials and methods  Three samples including two leaf litters and one forest soil were collected with a core (25 × 40 cm) from a 22-year-old slash pine plantation in southeast Queensland, Australia. The DNA fragments were extracted directly using the Ultra Clean™ Mega Prep Soil DNA kit (Mo Bio Labs, Solana Beach, CA). Then, four different purification methods were applied and compared to purify the DNA for PCR amplification, which include PVPP, Sephadex ^TM spin column, low-melting agarose gel and a new modified gel purification method. The purified DNA from these four purification methods was detected by agarose gel electrophoresis, and the purity and usefulness of DNA samples were ultimately determined by successful PCR amplifications. Results and discussion  The DNA was extracted from each sample using the Ultra Clean™ Mega Prep Soil DNA kit, and the DNA eluents were dark in colour and sometimes formed compact aggregates. Subsequently, PCR amplification from such samples failed, although a series of dilutions had been made from neat to 1:10³. The DNA purification step could not, therefore, be avoided. It was observed that both the colour of eluent and the DNA concentration decreased gradually after elution. Considering the difficulties of removing PCR inhibitors and the possibility of high DNA losses, 50–200 μl of sample DNA was used for purification. Four DNA purification methods (the PVPP spin column, Sephadex™ spin column, low-melting agarose gel and the modified gel purification method) were applied and compared on leaf litter and soil samples. The DNA purified by the modified gel purification method provided the best PCR products for 16S rRNA gene amplification, but the other methods, PVPP, Sephadex™ spin column and low-melting agarose gel, produced very weak or no products. Thus, in this study, DNA fragments which were purified by the modified gel purification method were amplified efficiently. This may be attributed to running the low-melting agrose gel for a longer time, which could remove substantial humic substances and also some other compounds from the samples and, thus, prevent them from being involved in PCR amplification. Conclusions  A new modified gel purification method which can improve DNA purification and PCR amplification of environmental DNA is first introduced in this study. Comparing PVPP, Sephadex ™ spin column, low-melting agarose gel and modified gel purification method for the effect of DNA purification, the modified gel purification method is more successful in removing the PCR amplification inhibitors and obtaining the highly purified PCR amplifiable high-molecular-weight DNA. The method described here is cheap, fast and easy to operate. It suggests in this study that the method containing less and easier following steps should be widely used to relieve the heavy working load of molecular-biological researchers. Recommendations and perspectives  This study introduces a new modified DNA purification method, and it is found that this modified gel purification method is effective in removing the PCR inhibitors and obtains highly purified DNA from leaf litters for PCR amplification. The modified gel purification method may have wider applications, although it was only assessed on leaf litter and soil samples. The effect of the modified gel purification method on the DNA purification would need to be further investigated on a variety of samples which suffered from PCR inhibitors, such as clinical samples, plant tissues and environmental samples.     

Wood ash fertilization alters the forest humus Archaea community

The combined and separate effects of Cd and wood ash on Archaea from coniferous forest humus were studied in a microcosm experiment. Nonmetric multidimensional scaling of the denaturing gradient gel analysis of polymerase chain reaction amplified 0.9 kb 16S ribosomal DNA fragments revealed changes in archaeal communities due to the ash treatments. Cd with or without ash did not further influence the result. Representatives of the ash and control communities were cloned, grouped by restriction fragment length polymorphism analysis and finally sequenced. All sequences belonged to non-thermophilic Crenarchaea.     

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.