Seasonal influences on fungal community structure in unimproved and improved upland grassland soils

Seasonal and management influences on the fungal community structure of two upland grassland soils were investigated. An upland site containing both unimproved floristically diverse (U4a) and improved mesotrophic (MG7b) grassland types was selected. Samples from both grassland types were taken at five times in one year. Soil fungal community structure was assessed using fungal automated ribosomal intergenic spacer analysis (ARISA), a DNA-profiling approach. A grassland management regime was found to strongly affect fungal community structure, with fungal ARISA profiles from unimproved and improved grassland soils differing significantly. The number of fungal ribotypes found was higher in unimproved than improved grassland soils, providing evidence that improvement may reduce the suitability of upland soil as a habitat for specific groups of fungi. Seasonal influences on fungal community structure were also noted, with samples taken in autumn (October) more correlated with change in ribotype profiles than samples from other seasons. However, seasonal variation did not obscure the measurement of differences in the fungal community structure that were due to agricultural improvement, with canonical correspondence analysis indicating grassland type had a stronger influence on fungal profiles than did season.     

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.     

Molecular microbiology of gut bacteria: genetic diversity and community structure analysis

Recently developed molecular biology approaches make possible the detailed genetic, taxonomic and ecological examination of microorganisms from various habitats. Animal gut represents one of the most complex microbial ecosystems with a large degree of microbial biodiversity present. Bacteria inhabiting the gut usually play important roles in metabolic transformations of substrates and sometimes, e.g. in ruminants, they make the basis for an obligate symbiosis with the host. Here we discuss molecular microbiology as a strategy for examination of gut bacteria, concentrating on a typical and in such environment dominant group of strictly anaerobic Gram-negative bacteria from the phylogenetic group Cytophaga/Flexibacter/Bacteroides. The bacteria from the genus Prevotella are the most abundant Gram-negative bacteria in the rumen and form a distinctive phylogenetic cluster, clearly separated from prevotellas isolated from other ecological niches. They may represent a good choice for a model organism in genetic manipulation experiments and for studies of gene transfer mechanisms taking place in the gut. The molecular tools for detection and monitoring of ruminal prevotellas are discussed.     

Molecular community analysis of microbial diversity

New technologies that avoid the need for either gene amplification (e.g. microarrays) or nucleic acid extraction (e.g. in situ PCR) have recently been implemented in microbial ecology. Together with new approaches for culturing microorganisms and an increased understanding of the biases of molecular methods, these techniques form the most exciting advances in this field during the past year.     

Seasonal and management influences on bacterial community structure in an upland grassland soil

Floristically diverse Nardo-Galion upland grasslands are common in Ireland and the UK and are valuable in agricultural, environmental and ecological terms. Under improvement (inputs of lime, fertiliser and re-seeding), they convert to mesotrophic grassland containing very few plant species. The effects of upland grassland improvement and seasonality on soil microbial communities were investigated at an upland site. Samples were taken at five times in one year in order to observe seasonal trends, and bacterial community structure was monitored using automated ribosomal intergenic spacer analysis (ARISA), a DNA-fingerprinting approach. Differences in soil chemistry and bacterial community structure between unimproved and improved grassland soils were noted. Season was also found to cause mild fluctuations in bacterial community structure, with soil samples from colder months (October and December) more correlated with change in ribotype profiles than samples from warmer months. However, for the majority of seasons clear differences in bacterial community structures from unimproved and improved soils could be seen, indicating seasonal influences did not obscure effects associated with improvement.     

Composition, diversity, and origin of the bacterial community in grass carp intestine

Gut microbiota has become an integral component of the host, and received increasing attention. However, for many domestic animals, information on the microbiota is insufficient and more effort should be exerted to manage the gastrointestinal bacterial community. Understanding the factors that influence the composition of microbial community in the host alimentary canal is essential to manage or improve the microbial community composition. In the present study, 16S rRNA gene sequence-based comparisons of the bacterial communities in the grass carp (Ctenopharyngodon idellus) intestinal contents and fish culture-associated environments are performed. The results show that the fish intestinal microbiota harbors many cellulose-decomposing bacteria, including sequences related to Anoxybacillus, Leuconostoc, Clostridium, Actinomyces, and Citrobacter. The most abundant bacterial operational taxonomic units (OTUs) in the grass carp intestinal content are those related to feed digestion. In addition, the potential pathogens and probiotics are important members of the intestinal microbiota. Further analyses show that grass carp intestine holds a core microbiota composed of Proteobacteria, Firmicutes, and Actinobacteria. The comparison analyses reveal that the bacterial community in the intestinal contents is most similar to those from the culture water and sediment. However, feed also plays significant influence on the composition of gut microbiota.     

Effects of field-grown genetically modified Zoysia grass on bacterial community structure

Herbicide-tolerant Zoysia grass has been previously developed through Agrobacterium-mediated transformation. We investigated the effects of genetically modified (GM) Zoysia grass and the associated herbicide application on bacterial community structure by using culture-independent approaches. To assess the possible horizontal gene transfer (HGT) of transgenic DNA to soil microorganisms, total soil DNAs were amplified by PCR with two primer sets for the bar and hpt genes, which were introduced into the GM Zoysia grass by a callus-type transformation. The transgenic genes were not detected from the total genomic DNAs extracted from 1.5 g of each rhizosphere soils of GM and non-GM Zoysia grasses. The structures and diversities of the bacterial communities in rhizosphere soils of GM and non-GM Zoysia grasses were investigated by constructing 16S rDNA clone libraries. Classifier, provided in the RDP II, assigned 100 clones in the 16S rRNA gene sequences library into 11 bacterial phyla. The most abundant phyla in both clone libraries were Acidobacteria and Proteobacteria. The bacterial diversity of the GM clone library was lower than that of the non- GM library. The former contained four phyla, whereas the latter had seven phyla. Phylogenetic trees were constructed to confirm these results. Phylogenetic analyses of the two clone libraries revealed considerable difference from each other. The significance of difference between clone libraries was examined with LIBSHUFF statistics. LIBSHUFF analysis revealed that the two clone libraries differed significantly (P?0.025), suggesting alterations in the composition of the microbial community associated with GM Zoysia grass.     

Changes in the bacterial community structure and diversity during bamboo retting

Microbial retting is a critical step in obtaining fiber bundles from bamboo culm using indigenous microorganisms. A cultivation-independent technique for monitoring the changes in bacteria community during bamboo retting was applied in this work. This technique involves genetic profiling of PCR-amplified small-subunit rRNA and the single-strand conformation polymorphism (SSCP) gel analysis of the PCR-amplified 16S rDNA fragments. The study revealed that both the structure and the diversity of investigated communities varied with the incubation periods and sample locations. The bacteria bands from SCCP gel profiles related to Bacillus sp. decreased in intensity, and Phaeospirillum sp. and Azospirillum brasilense completely disappeared during the 4(th) and 5(th) month of incubation, while the bands related to the Sphingomonas japonica, Alphaproteobacterium Ellin335 and Microbacterium sp. increased. The bands closely related to Sphingomonads, Brevundimonas brasilense, Pseudoclavibacter sp., Agrococcus jenensis and Oxalophagus oxalicus remained dominant during the whole incubation period. This study showed that the use of PCR assay targeting 16S rRNA and SCCP profiling provided valuable information on monitoring the bacteria dynamic changes occurring in the bacteria community during bamboo retting, which is crucial for controlling the quality of the retting process and improving the retting efficiency, and thus benefits for fiber recovery.     

Molecular diversity and tools for deciphering the methanogen community structure and diversity in freshwater sediments

Methanogenic archaeal communities existing in freshwater sediments are responsible for approximately 50 % of the total global emission of methane. This process contributes significantly to global warming and, hence, necessitates interventional control measures to limit its emission. Unfortunately, the diversity and functional interactions of methanogenic populations occurring in these habitats are yet to be fully characterized. Considering several disadvantages of conventional culture-based methodologies, in recent years, impetus is given to molecular biology approaches to determine the community structure of freshwater sedimentary methanogenic archaea. 16S rRNA and methyl coenzyme M reductase (mcrA) gene-based cloning techniques are the first choice for this purpose. In addition, electrophoresis-based (denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, and terminal restriction fragment length polymorphism) and quantitative real-time polymerase chain reaction techniques have also found extensive applications. These techniques are highly sensitive, rapid, and reliable as compared to traditional culture-dependent approaches. Molecular diversity studies revealed the dominance of the orders Methanomicrobiales and Methanosarcinales of methanogens in freshwater sediments. The present review discusses in detail the status of the diversity of methanogens and the molecular approaches applied in this area of research.     

Succession of bacterial community structure and diversity in a paddy soil oxygen gradient

Cultivation-independent techniques were applied to assess the succession and phylogenetic composition of bacterial communities in a vertical oxygen gradient in flooded, unplanted paddy soil microcosms. Microsensor measurements showed that within 6 h of flooding, oxygen was depleted from 200 microM at the floodwater-soil interface to undetectable amounts at a depth of approximately 2 mm and below. The gradient was quite stable over time, although the oxygen depletion was less pronounced 84 days than 6 h after flooding. Community fingerprint patterns were obtained by terminal restriction fragment length polymorphism (T-RFLP) analysis from the oxic, transition, and anoxic zones of triplicate soil microcosms at 0, 1 and 6 h, and 1, 2, 7, 21, 30, 42, 84, and 168 days after flooding. Correspondence analyses revealed that T-RFLP patterns obtained using either community DNA or RNA were affected by time and oxygen zone, and that there was a significant interaction between the effects of time and oxygen zone. The temporal dynamics of bacterial populations were resolved more clearly using RNA than using DNA. At the RNA level, successional community dynamics were most pronounced from 1 h to 2 days and less pronounced from 2 to 21 days after flooding, for both oxic and anoxic zones. No effect of time or oxygen zone on the community dynamics was observed from 21 to 168 days after flooding. Dominant early successional populations were identified by cloning and comparative sequence analysis of environmental 16S rRNA and 16S rRNA genes as members of the Betaproteobacteria (oxic zone) and the clostridial cluster I (anoxic zone). Dominant late successional populations belonged to the Verrucomicrobia and Nitrospira (detected mainly in the oxic zone), and to the Myxococcales (detected mainly in the anoxic zone). In conclusion, the bacterial community developed through successional stages, leading at the RNA level to almost stable community patterns within 21 days after flooding. This principal finding, in combination with the phylogenetic identity of early- and late-appearing populations, suggests that the community dynamics can be explained by the principles of r- and K-selection.     

Effects of long-term benzene pollution on bacterial diversity and community structure in groundwater

In this study we analysed the relationship between bacterial community structures and geochemistry of groundwater in a sandstone aquifer (SIReN site) impacted mainly by BTEX hydrocarbons (benzene, toluene, ethylbenzene and xylenes), of which benzene is most abundant. The long-term presence of benzene reduced bacterial diversity: in groundwaters contaminated with more than 1.8 x 10(4) microg l(-1) of benzene, bacterial diversity was half of that in clean groundwaters. Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rDNA revealed that the community structures were very similar in uncontaminated groundwaters, whereas communities subjected to long-term benzene contamination were different, not only from uncontaminated groundwater communities, but also from each other. Canonical correspondence analysis of the community profiles and the geochemical data showed that this divergence in community structure was not primarily caused by the direct toxic or stressful effects of benzene, but by the environmental changes brought about by benzene metabolism, in particular a decrease in redox potential.     

Effect of sheep urine deposition on the bacterial community structure in an acidic upland grassland soil

The effect of the addition of synthetic sheep urine (SSU) and plant species on the bacterial community composition of upland acidic grasslands was studied using a microcosm approach. Low, medium, and high concentrations of SSU were applied to pots containing plant species typical of both unimproved (Agrostis capillaris) and agriculturally improved (Lolium perenne) grasslands, and harvests were carried out 10 days and 50 days after the addition of SSU. SSU application significantly increased both soil pH (P < 0.005), with pH values ranging from pH 5.4 (zero SSU) to pH 6.4 (high SSU), and microbial activity (P < 0.005), with treatment with medium and high levels of SSU displaying significantly higher microbial activity (triphenylformazan dehydrogenase activity) than treatment of soil with zero or low concentrations of SSU. Microbial biomass, however, was not significantly altered by any of the SSU applications. Plant species alone had no effect on microbial biomass or activity. Bacterial community structure was profiled using bacterial automated ribosomal intergenic spacer analysis. Multidimensional scaling plots indicated that applications of high concentrations of SSU significantly altered the bacterial community composition in the presence of plant species but at different times: 10 days after application of high concentrations of SSU, the bacterial community composition of L. perenne-planted soils differed significantly from those of any other soils, whereas in the case of A. capillaris-planted soils, the bacterial community composition was different 50 days after treatment with high concentrations of SSU. Canonical correspondence analysis also highlighted the importance of interactions between SSU addition, plant species, and time in the bacterial community structure. This study has shown that the response of plants and bacterial communities to sheep urine deposition in grasslands is dependent on both the grass species present and the concentration of SSU applied, which may have important ecological consequences for agricultural grasslands.     

青海不同区域农田作物土壤细菌多样性及群落结构分析

【目的】探讨青海省农田土壤细菌多样性及群落结构组成。【方法】采用高通量测序技术分析都兰县、互助县、共和县和大通县种植小麦、油菜、青稞土壤细菌群落结构组成及多样性差异,并解析其与土壤理化特性的关系。【结果】4个地区的土壤pH、水分、有机质、Chao1指数、Shannon指数、线性判别分析效应大小(linear discriminant analysis effect size, LEfSe)组间差异这些指标中部分指标存在极显著差异性(P<0.01),而3种作物的这些指标无显著差异(P>0.05)。主成分分析(principal component analysis, PCA)显示,不同区域细菌群落组成差异性较大,3种作物农田土壤细菌群落、物种组成相似度较高。4个地区共有3 127个可操作性分类单元(operational taxonomic units, OTUs),3种作物农田土壤细菌共有OTUs为3 694个;细菌物种隶属于36门93纲192目276科450属423种;4个地区或3种作物土壤具有相似的优势门、属、种,但相对丰度不同。水分、pH、有机质与Chao1、Shannon存在显著相关性,pH、有机质与未分类种、unclassified RB41、unclassified Sphingomonas具有显著正相关或负相关。Chao1、Shannon与unclassified Sphingomonas显著负相关,与unclassified RB41、unclassified Vicinamibacteraceae显著正相关。【结论】区域差异对土壤的理化性质、细菌群落结构和多样性具有显著影响,相较于作物而言其影响更加明显。     

Molecular tools for deciphering the microbial community structure and diversity in rumen ecosystem

Rumen microbial community comprising of bacteria, archaea, fungi, and protozoa is characterized not only by the high population density but also by the remarkable diversity and the most complex microecological interactions existing in the biological world. This unprecedented biodiversity is quite far from full elucidation as only about 15-20?% of the rumen microbes are identified and characterized till date using conventional culturing and microscopy. However, the last two decades have witnessed a paradigm shift from cumbersome and time-consuming classical methods to nucleic acid-based molecular approaches for deciphering the rumen microbial community. These techniques are rapid, reproducible and allow both the qualitative and quantitative assessment of microbial diversity. This review describes the different molecular methods and their applications in elucidating the rumen microbial community.     

Molecular analysis of the bacterial community in digestive tract of rabbit

This work aimed to study the stability over time of the bacterial community in cæcum and fæces of the rabbit (diversity index and structure) without experimental disturbance and to evaluate its relationships with environmental parameters. Soft and hard fæces of 14 rabbits were sampled for 5 weeks while cæcal content was sampled on the 3rd week (by surgery) and the 5th week (at slaughter). Bacterial communities were assessed by studying CE-SSCP profiles of 16S rRNA genes fragments. Redox potential, pH, NH3-N concentration and volatile fatty acid concentrations were measured in the cæcum. Data showed that bacterial communities of soft and hard fæces barely differed from that of the cæcum (ANOSIM-R < 0.25; p < 0.05). Without disturbance, the bacterial communities of fæces were stable over time (ANOSIM-R < 0.25; p < 0.001). However, the bacterial communities of cæcum and fæces were affected by the surgery (ANOSIM-R = 0.22–0.33; p < 0.001). The cæcal content was an acidic (pH = 6.03 ± 0.33) and an anaerobic environment (redox potential = ?160 ± 43 mV). Only the redox potential was correlated with the diversity index of the bacterial community of the cæcum (R² = 0.35; p < 0.05) and no environmental parameters were correlated to its structure.     

Matrix composition and community structure analysis of a novel bacterial pyrite leaching community

Here we describe a novel bacterial community that is embedded in a matrix of carbohydrates and bio/geochemical products of pyrite (FeS₂) oxidation. This community grows in stalactite-like structures – snottites – on the ceiling of an abandoned pyrite mine at pH values of 2.2–2.6. The aqueous phase in the matrix contains 200 mM of sulfate and total iron concentrations of 60 mM. Micro-X-ray diffraction analysis showed that jarosite [(K,Na,H₃O)Fe₃(SO₄)₂(OH)₆] is the major mineral embedded in the snottites. X-ray absorption near-edge structure experiments revealed three different sulfur species. The major signal can be ascribed to sulfate, and the other two features may correspond to thiols and sulfoxides. Arabinose was detected as the major sugar component in the extracellular polymeric substance. Via restriction fragment length polymorphism analysis, a community was found that mainly consists of iron oxidizing Leptospirillum and Ferrovum species but also of bacteria that could be involved in dissimilatory sulfate and dissimilatory iron reduction. Each snottite can be regarded as a complex, self-contained consortium of bacterial species fuelled by the decomposition of pyrite.     

Pb和Cd对森林土壤细菌功能多样性及群落结构的影响

重金属的毒性系数（The Toxic Factor,TF）是评价重金属潜在生态风险指数（potential ecological risk index,RI）的关键参数。为了探究基于Hakanson提出的TF值是否适用于重金属对土壤微生物的生态风险评估,以TF值为5和30的铅（Pb）和镉（Cd）构建土壤微宇宙试验,构建不同的RI水平（100、200和400）,通过Biolog-ECO板和高通量测序技术分析了Pb和Cd分别对细菌功能多样性及群落结构的影响。结果表明,对照处理（CK）的细菌丰度、功能多样性（Shannon指数,Simpson指数和McIntosh指数）和基因多样性（ACE和Chao1指数）均大于Pb、Cd污染的土壤,随着RI水平的升高,Pb和Cd污染土壤中细菌的丰度、功能多样性（Shannon指数和McIntosh指数）和基因多样性（Chao1指数和ACE指数）呈下降趋势。相同RI水平下,Pb污染土壤中细菌群落的丰度、平均颜色变化率（AWCD）、功能多样性指数、OTUs数和基因多样性指数均显著大于Cd污染（P<0.05）;6大类碳源利用率及主成分（PCA）分析表明,Pb污染土壤中细菌对糖类和羧酸的利用率均显著大于Cd污染（P<0.05）,在不同RI水平和重金属比例下,碳源利用模式而有所不同。同一RI水平下,相对于Pb污染,Cd污染土壤中变形菌门的相对丰度较为丰富,而绿弯菌门的相对丰度稀少;Pb和Cd污染土壤中慢生根瘤菌属、鞘脂单胞菌属、链霉菌属和norank_f__Roseiflexaceae等不同属细菌相对丰度表现出差异性。上述结果表明Hakanson提出的TF值并不适用于评估重金属Pb和Cd对土壤微生物的潜在生态风险。     

生姜根系不同生态位细菌群落多样性特征、组成及结构差异

生姜作为常见的调味品和传统中药材,是我国重要的经济作物之一。作为取食部分的生姜块茎与根系直接相连,其产量、品质与根相关细菌群落密切相关。然而,关于生姜根系微环境中细菌群落的特点仍鲜有报道,土壤环境能否衍生出宿主特异性内生菌群落尚不清楚。以生姜根系不同生态位细菌群落为研究对象,采用高通量测序技术,对非根际、根际及根内细菌进行16S rRNA基因测序。结果表明,不同生态位细菌群落多样性存在显著差异,其中非根际及根际细菌群落多样性(Shannon index, Observed species, Faith′s PD)显著高于内生菌群落。同时,各生态位共现网络稳定性和复杂度表现为非根际>根际>根内细菌群落。而在组成上,细菌群落在不同生态位差异显著(R²=0.57,P=0.001)。其中变形菌门(Proteobacteria)是根内的优势门,该门类下假单胞菌属(Pseudomonas)、短波单胞菌属(Brevundimonas)、寡养单胞菌属(Stenotrophomonas)及泛菌属(Pantoea)在根内显著富集。在根际细菌中,拟杆菌门(Bacteroid...     

滴灌对苜蓿根际土壤细菌多样性和群落结构的影响

【背景】细菌作为土壤微生物中的重要类群,能够有效促进土壤物质循环和能量流动,细菌多样性以及群落结构能够反映土壤的质量状况。【目的】了解滴灌条件下苜蓿根际土壤细菌群落结构及多样性变化,探讨土壤环境因子对细菌群落结构的影响。【方法】基于细菌16Sr RNAV3-V4区高通量测序技术,分析比较滴灌与自然降雨两种模式下生长的苜蓿根际与非根际土壤中细菌多样性和群落分布规律,然后采用冗余分析(Redundancy analysis,RDA)探讨土壤环境因子与细菌多样性的关系。【结果】苜蓿根际土壤中细菌多样性丰富,滴灌根际土壤中细菌多样性显著高于自然降雨根际土壤;土壤样品中共检测到细菌46门53纲116目220科469属,主要的优势菌门为变形菌门(Proteobacteria,25.27%-34.42%),其中α-变形菌纲(Alphaproteobacteria,11.41%-18.97%)为优势亚群,鞘氨醇单胞菌属(Sphingomonas,1.00%-4.54%)为优势属。相较于自然降雨,滴灌条件下苜蓿根际土壤细菌的6个门和16个属的群落结构发生显著变化;此外,RDA分析表明,不同环境因子对微生物群落的影响不同,滴灌根际土壤中9个细菌属的丰度与全磷、全钾、有效磷、碱解氮、有机质、土壤中性磷酸酶以及土壤脲酶的含量显著正相关。【结论】滴灌作为新型节水技术,在促进植物生长、提高产量、节约成本的基础上增加了植物根际土壤中细菌多样性和丰度,该结果为新型灌溉体制的改革以及土壤微生物资源的开发利用提供科学数据。