Fatty acid methyl ester (FAME) profiles as measures of soil microbial community structure

Analysis of fatty acid methyl ester (FAME) profiles extracted from soils is a rapid and inexpensive procedure that holds great promise in describing soil microbial community structure without traditional reliance on selective culturing, which seems to severely underestimate community diversity. Interpretation of FAME profiles from environmental samples can be difficult because many fatty acids are common to different microorganisms and many fatty acids are extracted from each soil sample. We used principal components (PCA) and cluster analyses to identify similarities and differences among soil microbial communities described using FAME profiles. We also used PCA to identify particular FAMEs that characterized soil sample clusters. Fatty acids that are found only or primarily in particular microbial taxa-marker fatty acids-were used in conjunction with these analyses. We found that the majority of 162 soil samples taken from a conventionally-tilled corn field had similar FAME profiles but that about 20% of samples seemed to have relatively low, and that about 10% had relatively high, bacterial:fungal ratios. Using semivariance analysis we identified 21:0 iso as a new marker fatty acid. Concurrent use of geostatistical and FAME analyses may be a powerful means of revealing other potential marker FAMEs. When microbial communities from the same samples were cultured on R2A agar and their FAME profiles analyzed, there were many differences between FAME profiles of soil and plated communities, indicating that profiles of FAMEs extracted from soil reveal portions of the microbial community not culturable on R2A. When subjected to PCA, however, a small number of plated communities were found to be distinct due to some of the same profile characteristics (high in 12:0 iso, 15:0 and 17:1 ante A) that identified soil community FAME profiles as distinct. Semivariance analysis indicated that spatial distributions of soil microbial populations are maintained in a portion of the microbial community that is selected on laboratory media. These similarities between whole soil and plated community FAME profiles suggest that plated communities are not solely the result of selection by the growth medium, but reflect the distribution, in situ, of the dominant, culturable soil microbial populations.     

Fidelity of select restriction endonucleases in determining microbial diversity by terminal-restriction fragment length polymorphism

An evaluation of 18 DNA restriction endonucleases for use in terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed by using richness and density indices in conjunction with computer simulations for 4,603 bacterial small-subunit rRNA gene sequences. T-RFLP analysis has become a commonly used method for screening environmental samples for precursory identification and community comparison studies due to its precision and high-throughput capability. The accuracy of T-RFLP analysis for describing a community has not yet been thoroughly evaluated. In this study, we attempted to classify restriction endonucleases based upon the ability to resolve unique terminal-restriction fragments (T-RFs) or operational taxonomic units (OTUs) from a database of gene sequences. Furthermore, we assessed the predictive accuracy of T-RFLP at fixed values of community richness (n = 1, 5, 10, 50, and 100). Classification of restriction endonuclease fidelity was performed by measuring richness and density for the entire database of T-RFs. Further analysis of T-RFLP accuracy for determining richness was performed by iterative, random sampling from the derived database of T-RFs. It became apparent that two constraints were influential for measuring the fidelity of a given restriction endonuclease: (i) the ability to resolve unique sequence variants and (ii) the number of unique T-RFs that fell within a measurable size range. The latter constraint was found to be more significant for estimating restriction endonuclease fidelity. Of the 18 restriction endonucleases examined, BstUI, DdeI, Sau96I, and MspI had the highest frequency of resolving single populations in model communities. All restriction endonucleases used in this study detected < or =70% of the OTUs at richness values greater than 50 OTUs per modeled community. Based on the results of our in silico experiments, the most efficacious uses of T-RFLP for microbial diversity studies are those that address situations where there is low to intermediate species richness (e.g., colonization, early successional stages, biofilm formation).     

Evaluating the effect of fertilizer application on soil microbial community structure in rice based cropping system using fatty acid methyl esters (FAME) analysis

The present study was undertaken to observe the effects of chemical fertilizers and compost amendments on microbial community structure in rice based cropping system under long term fertilizer application experiment. Changes in microbial community structure assessed by the relative abundance of fatty acid methyl ester (FAME) profiles revealed that compost amendments led to promoting the abundance of Gram-positive bacterial FAMEs. Multivariate analysis of FAME profiles separated the treatments primarily on the basis of the amount of added compost. A link between different fertilizer application treatment and soil microbial components was tentatively established, but it needs to be verified in further studies.     

Use of the T-RFLP technique to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants

The aim of this study was to examine whether the terminal restriction fragment length polymorphism (T-RFLP) analysis represents an appropriate technique for monitoring highly diverse soil bacterial communities, i.e. to assess spatial and/or temporal effects on bacterial community structure. The T-RFLP method, a recently described fingerprinting technique, is based on terminal restriction fragment length polymorphisms between distinct small-subunit rRNA gene sequence types. This technique permits an automated quantification of the fluorescence signal intensities of the individual terminal restriction fragments (T-RFs) in a given community fingerprint pattern. The indigenous bacterial communities of three soil plots located within an agricultural field of 110 m(2) were compared. The first site was planted with non-transgenic potato plants, while the other two were planted with transgenic GUS and Barnase/Barstar potato plants, respectively. Once prior to planting and three times after planting, seven parallel samples were taken from each of the three soil plots. The T-RFLP analysis resulted in very complex but highly reproducible community fingerprint patterns. The percentage abundance values of defined T-RFs were calculated for the seven parallel samples of the respective soil plot. A multivariate analysis of variance was used to test T-RFLP data sets for significant differences. The statistical treatments clearly revealed spatial and temporal effects, as well as spacextime interaction effects, on the structural composition of the bacterial communities. T-RFs which showed the highest correlations to the discriminant factors were not those T-RFs which showed the largest single variations between the seven-sample means of individual plots. In summary, the T-RFLP technique, although a polymerase chain reaction-based method, proved to be a suitable technique for monitoring highly diverse soil microbial communities for changes over space and/or time.     

持续干旱对樱桃根际土壤细菌数量及结构多样性影响

以1年生吉塞拉实生容器苗为试材,采用绿色荧光蛋白基因标记技术,研究了干旱胁迫(连续干旱0、7、14、21、28 d和35 d)对樱桃根际促生细菌YT3的标记菌YT3-gfp数量的影响,同时结合平板计数法和末端限制性片段长度多态性分析(terminal restriction fragment length polymorphism,T-RFLP)技术,研究了干旱对樱桃土壤中的微生物数量及细菌群落结构多样性影响。结果表明:樱桃根际土壤中的YT3-gfp数量是非根际土壤中的8.75—28.77倍,随着持续干旱强度的增加,YT3-gfp的数量先增加后减小。干旱对根际土壤中YT3-gfp数量的影响大于对非根际土壤的影响,分别在持续干旱至第21天和28天时,YT3-gfp的数量达到最大值。随着持续干旱强度的增加,根际土壤中细菌和放线菌数量先增加后减小,而真菌的数量一直减少。此外,持续干旱至第21天或28天时,樱桃根际土壤具有最高的丰富度指数、多样性指数和最低的优势度指数。基于T-RFLP的主成分分析结果显示持续干旱14—35 d时,其细菌群落结构成为一个相对独立的群,群落结构趋于多样性;而持续干旱7 d和42 d构成另外两个相对独立的群,群落结构趋于简单。以上分析可知,干旱对土壤微生物影响显著,一定强度的干旱可提高细菌和放线菌数量,提高细菌群落结构多样性,适当干旱对维持根际土壤细菌群落结构多样性是有益的。     

脂肪酸甲酯法检测空心莲子草入侵影响土壤微生物群落结构的初步研究

空心莲子草是我国2003年公布的第一批外来入侵物种。为了进一步了解该植物的入侵机制,采集湖北咸宁、仙桃和武汉三地的土样,采用土壤脂肪酸甲酯谱图分析的方法探讨该植物入侵对土壤微生物的影响。结果显示：空心莲子草入侵后土壤可培养细菌、真菌的数量显著增加,而放线菌的数量显著下降。脂肪酸分析表明土壤微生物群落结构发生一定程度的改变,但其变化因土壤的不同而有差异。     

长期施用化肥及秸秆还田对砂姜黑土细菌群落的影响

【目的】在施用化肥的基础上进行秸秆还田是提高砂姜黑土肥力的有效措施,以往的研究只注重秸秆还田对土壤结构、肥力等物理化学性状方面的研究,缺少施肥对砂姜黑土微生物群落影响的研究。本研究以安徽蒙城典型的砂姜黑土为研究对象,以期揭示长期施用化肥和秸秆还田对砂姜黑土细菌群落的影响。【方法】采用454高通量测序对砂姜黑土不同农业施肥措施下的细菌群落进行分析研究,并通过生物信息学的分析方法揭示影响砂姜黑土细菌群落的主要因素。【结果】通过对454高通量测序数据的分析,发现砂姜黑土主要的细菌门类为放线菌、变形菌、酸杆菌、绿弯菌和拟杆菌。长期施用化肥显著提高了砂姜黑土肥力和作物产量,但导致了细菌群落结构的显著变化和多样性的显著降低。秸秆还田有利于土壤肥力的进一步提高,但是并没有缓解长期施用化肥对土壤细菌群落产生的不利影响。分析发现土壤pH的变化是导致土壤细菌群落变异的主要因素。【结论】在施用化肥的基础上进行秸秆还田有利于砂姜黑土肥力的提升,然而并没有缓解由施肥导致的土壤酸化对土壤细菌群落组成和多样性产生的不利影响。这暗示秸秆还田可能并未对砂姜黑土微生物生态产生根本性的有益影响,对于秸秆农田的利用方式还需要进一步研究,以达到农业生产效益和生态效益的并重。     

Analysis of T-RFLP data using analysis of variance and ordination methods: a comparative study

The analysis of T-RFLP data has developed considerably over the last decade, but there remains a lack of consensus about which statistical analyses offer the best means for finding trends in these data. In this study, we empirically tested and theoretically compared ten diverse T-RFLP datasets derived from soil microbial communities using the more common ordination methods in the literature: principal component analysis (PCA), nonmetric multidimensional scaling (NMS) with Sørensen, Jaccard and Euclidean distance measures, correspondence analysis (CA), detrended correspondence analysis (DCA) and a technique new to T-RFLP data analysis, the Additive Main Effects and Multiplicative Interaction (AMMI) model. Our objectives were i) to determine the distribution of variation in T-RFLP datasets using analysis of variance (ANOVA), ii) to determine the more robust and informative multivariate ordination methods for analyzing T-RFLP data, and iii) to compare the methods based on theoretical considerations. For the 10 datasets examined in this study, ANOVA revealed that the variation from Environment main effects was always small, variation from T-RFs main effects was large, and variation from T-RF × Environment (T × E) interactions was intermediate. Larger variation due to T × E indicated larger differences in microbial communities between environments/treatments and thus demonstrated the utility of ANOVA to provide an objective assessment of community dissimilarity. The comparison of statistical methods typically yielded similar empirical results. AMMI, T-RF-centered PCA, and DCA were the most robust methods in terms of producing ordinations that consistently reached a consensus with other methods. In datasets with high sample heterogeneity, NMS analyses with Sørensen and Jaccard distance were the most sensitive for recovery of complex gradients. The theoretical comparison showed that some methods hold distinct advantages for T-RFLP analysis, such as estimations of variation captured, realistic or minimal assumptions about the data, reduced weight placed on rare T-RFs, and uniqueness of solutions. Our results lead us to recommend that method selection be guided by T-RFLP dataset complexity and the outlined theoretical criteria. Finally, we recommend using binary or relativized peak height data with soil-based T-RFLP data for ordination-based exploratory microbial analyses.     

A novel strategy to extract specific phylogenetic sequence information from community T-RFLP

Cultivation-independent analyses of soil microbial community structures are frequently used to describe microbiological soil characteristics. Semi-automated terminal restriction fragment length polymorphism (T-RFLP) analyses yield high-resolution genetic profiles of highly diverse soil microbial communities and hold great potential for use in routine soil quality monitoring. A serious limitation of T-RFLP analyses has been the inability to reliably affiliate observed terminal restriction fragments (T-RF) to phylogenetic groups. In the study presented here, we were able to overcome this limitation of T-RFLP. With a combination of adapter ligation, fragment size selection, and re-amplification with adapter site specific PCR, we were able to isolate a T-RF-fraction of a narrow size-range containing a T-RF that was significantly more abundant in heavy metal amended soils. Cloning the size-selected T-RF fraction allowed for the efficient isolation of clones containing this specific T-RF. Sequence determination and phylogenetic inference in RDP-II affiliated the sequence to unclassified cyanobacteria. Specific primer design and PCR amplification from bulk soil DNA allowed for independent confirmation of the results from bacterial T-RFLP and T-RF cloning. Our results show that specific T-RFs can be efficiently isolated and identified, and that the adapter ligation approach holds great potential for genetic profiling and for identification of community components of interest.     

Molecular analysis of microbial population transition associated with the start of denitrification in a wastewater treatment process

AIMS: The objective of this study is to determine the bacteria playing an important role in denitrification by monitoring the molecular dynamics accompanying the start of denitrification. METHODS AND RESULTS: cDNA reverse-transcribed from 16S rRNA was amplified with fluorescent labelled primer for terminal restriction fragment length polymorphism (T-RFLP) analysis and an unlabelled primer for cloning analysis. The terminal restriction fragments (T-RFs) that increased in association with the start of denitrification were determined. These T-RFs were identified by in silico analysis of 16S rRNA sequences obtained from cloning. As a result, it was clearly observed that the bacteria belonging to the genera Hydrogenophaga and Acidovorax increased in number after the start of denitrification. CONCLUSIONS: It was demonstrated that T-RFLP analysis targeting 16S rRNA is appropriate for the daily monitoring of a bacterial community to control wastewater treatment processes. Combination of the results of T-RFLP analysis and 16S rRNA clone library indicated that the bacteria belonging to the genera Hydrogenophaga and Acidovorax play an important role in denitrification. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide new insight to the 16S rRNA level of active denitrifying bacteria in wastewater treatment processes.     

The impact of crop plant cultivation and peat amendment on soil microbial activity and structure

A rapid means for restoring soil fertility could be addition of peat to the plough layer. The impact of cultivation of eight different crops (the joint impact of plant and the management tailored for each plant), with and without soil amendment by peat treatment on soil microbiological, physical and chemical properties was assessed for two consecutive growing seasons. As a measure of the functional diversity of soil microbial community we estimated the activity of several different extracellular soil enzymes using the ZymProfiler® test kit. ATP content was measured to yield information on the amount of the active microbial biomass, and phospholipid fatty acid (PLFA) profiles were analysed to reveal the microbial community structure. The enzyme activity patterns of the soil samples indicated several differences due to the different crops and years but ATP content and PLFA profiles were rather stable. However, microbial biomass as total amount of PLFAs depended on the plant and peat treatment and ATP content varied between the years. The effects of the peat treatments were less clearly indicated by the biological parameters one or two years after the amendment, as only arylsulphatase and β-xylosidase activities were affected in both the years. Soil moisture, affecting enzyme activities, depended on the year and crop plant and peat addition increased it. Abbreviations: AMC – 7-amino-4-methylcoumarin; AP – aminopeptidase; ATP – adenosine triphosphate; C_mic– microbial biomass carbon; DNA – deoxyribonucleic acid; EC – electrical conductivity; FAME – fatty acid methyl ester; fw – fresh weight; MUF – 4-methylumbelliferyl; na – not added; N_mic– microbial biomass nitrogen; PDE – phosphodiesterase; PLFA – phospholipid fatty acid; PME – phosphomonoesterase; SOM – soil organic matter     

The relationship between microbiota and polyamine concentration in the human intestine: a pilot study

The fecal microbiota of 10 hospitalized elderly subjects and 14 healthy adults were analyzed by terminal-restriction fragment length polymorphism (T-RFLP) analysis using Hha I, Msp I, Hae III, and Alu I, as well as fecal polyamine (PA) concentration. The T-RFLP profiles of the fecal microbiota of the subjects were roughly divided into 2 clusters-I (9 out of 11 were derived from hospitalized elderly subjects) and II (12 out of 13 were derived from healthy adults). The average concentration of putrescine in Cluster II was 5.8 times higher than that of putrescine in Cluster I (P=0.0015). Using a phylogenetic assignment database for T-RFLP analysis of human colonic microbiota, the terminal-restriction fragments (T-RFs) characteristically detected in the case of subjects with high fecal PA concentration were predicted to be derived from bacterial species and phylotypes belonging to Clostridium subcluster XIVa, particularly including Clostridium xylanolyticum, Clostridium saccharolyticum, the uncultured human intestinal bacterium clone JW1H4 (a relative of Desulfotomaculum guttoideum), Roseburia intestinalis, the uncultured bacterium clone 41F10 (a relative of Eubacterium ramulus), Roseburia cecicola, Ruminococcus obeum and its relatives. From these results, we concluded that fecal microbiota may be linked with fecal PA concentration and that some bacterial species belonging to Clostridium subcluster XIVa may play a major role in the control of intestinal PA concentration in humans.     

生态有机肥对番茄青枯病及土壤微生物多样性的影响

采用田间小区试验和FAMEs分析研究了不同用量生态有机肥对番茄青枯病的抑制效果和土壤微生物的影响。结果表明,连作地番茄青枯病发生率为100％,施生态有机肥显著地降低了连作地番茄青枯病的发生。用量为1kg·m^-2和0.5kg·m^-2时,番茄青枯病发生率分别为39％和50％,两种施肥量间差异不显著。施生态有机肥后土壤微生物群落脂肪酸指纹发生明显改变,革兰氏阴性菌生物标记物cyc17：0的含量增加,真菌生物标记物c18:2w6的含量增加,作为AM菌根的生物标记物c16:111c呈上升趋势。聚类分析表明,对照和施生态有机肥处理土壤微生物脂肪酸指纹之间的最短距离是1.08,不同施用量处理之间的最短距离是0.70。施用生态有机肥能调控土壤微生物群落结构,促进有益微生物的生长,增强土壤生态系统的稳定性和抑病性,从而提高了土壤质量。     

施肥对设施菜地nirK型反硝化细菌群落结构和丰度的影响

采用末端限制性片段多态性分析(T-RFLP)和实时荧光定量PCR(real-time PCR)方法,研究了甘肃武威设施菜地不同施肥条件下0～20 cm、20～40 cm土层中土壤nirK型反硝化细菌群落结构和丰度的变化.结果表明： 施肥对土壤中nirK型反硝化细菌的群落结构具有明显影响,且对70、156、190 bp片段所代表设施菜地土壤优势种群影响最显著.施肥对0～20 cm土层nirK型反硝化细菌丰度有明显影响,其最大值出现在全有机肥(M)处理、为每克干土2.16×10⁷个拷贝数,分别是对照(CK)和全化肥(NPK)处理的2.04和2.02倍.设施菜地土壤0～20 cm与20～40 cm土层nirK型反硝化细菌的优势种群及其基因丰度均存在显著差异,且设施菜地土壤中nirK型反硝化细菌的群落结构和丰度与大田差异明显.土壤pH值、有机质及硝酸盐含量均影响nirK型反硝化细菌的群落结构和丰度.系统发育分析结果表明,土壤中除存在与厌氧反硝化细菌亲缘相近的nirK型反硝化微生物外,还存在与好氧反硝化菌亲缘关系相近的nirK型反硝化微生物,如根瘤菌属、苍白杆菌属、土壤杆菌属等.
     

基于T-RFLP方法的连栽杉木根际土壤细菌群落变化研究

运用末端限制性片段长度多态性技术(T-RFLP),研究福建省尤溪国有林场的多代连栽杉木根际土壤细菌群落变化特征。结果表明:杉木多代连栽后根际微生态失衡,表现为碳循环菌和氮循环菌等有益菌数量减少,病原菌数量增加。在不同代数杉木土壤中,4种内切酶消化后产生的末端限制性片段(T-RFs)数量具有显著差异,第一代杉木(first Chinese fir rotation plantation,FCP)最高,第二代杉木(second Chinese fir rotation plantation,SCP)次之,第三代杉木(third Chinese fir rotation plantation,TCP)最少。土壤理化性质结果表明连栽导致土壤养分逐代降低,除土壤碳氮比与细菌群落多样性呈显著负相关外,土壤养分含量指标与细菌群落多样性呈显著正相关。T-RFLP实验共鉴定出细菌门类12门,其中厚壁菌门、蓝藻门、放线菌门和变形菌门是主要优势菌群。随栽植代数的增加,变形菌门细菌比例递增,而厚壁菌门细菌比例递减。土壤细菌群落多样性指数,表现为随栽植代数增长而减少,呈现FCPSCPTCP趋势,与T-RFs片段数量变化趋势基本一致,表明多代连栽降低了杉木根际土壤细菌群落多样性。依据功能性不同,可将鉴定出的细菌划分为5类:碳循环功能菌、硫循环功能菌、氮循环功能菌、纤维降解菌及病原菌。连栽导致碳循环菌和氮循环菌相对含量降低,硫循环菌和纤维降解菌相对含量提高,病原菌相对含量随栽植代数增加而上升。     

牛场肥水灌溉对土壤nirK、nirS型反硝化微生物群落结构的影响

以徐水县梁家营长期定位施肥试验田为研究对象,利用末端限制性片段长度多态性(T-RFLP)分析和克隆文库构建,研究了5种施肥处理(清水灌溉CK、无机肥灌溉CF、牛场肥水不同浓度、不同次数灌溉T4、T5和T11)下土壤中nirK、nirS型反硝化细菌群落多样性及其群落结构的演变。结果表明,不同施肥处理下nirK、nirS型反硝化细菌群落多样性无显著差异,但群落结构却有明显变化:nirK型反硝化细菌群落结构既受施肥种类又受施肥量影响,优势种群尤其对施肥种类和施肥量响应显著;nirS型反硝化细菌则主要受施肥种类影响,施肥量影响微弱。牛场肥水处理和无机肥处理分别促进和抑制不同的nirS型反硝化细菌,群落主成分受无机肥促进、牛场肥水抑制。系统发育分析结果表明,土壤中nirK型反硝化细菌主要与假单胞菌属(Pseudomonas)、产碱杆菌属(Alcaligenes)和根瘤菌属(Rhizobium)的反硝化细菌具有较近的亲缘关系;nirS型反硝化细菌主要与劳尔氏菌(Ralstonia)和红长命菌属(Rubrivivax)有较近的亲缘关系。试验土壤中反硝化微生物多与目前已报道的好氧反硝化细菌亲缘关系较近,这可能与微生物分析取自表层土有关。     

Changes in bacterial community structure in the colon of pigs fed different experimental diets and after infection with Brachyspira hyodysenteriae

Bacterial communities in the large intestines of pigs were compared using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the 16S ribosomal DNA. The pigs were fed different experimental diets based on either modified standard feed or cooked rice supplemented with dietary fibers. After feeding of the animals with the experimental diets for 2 weeks, differences in the bacterial community structure in the spiral colon were detected in the form of different profiles of terminal restriction fragments (T-RFs). Some of the T-RFs were universally distributed, i.e., they were found in all samples, while others varied in distribution and were related to specific diets. The reproducibility of the T-RFLP profiles between individual animals within the diet groups was high. In the control group, the profiles remained unchanged throughout the experiment and were similar between two independent but identical experiments. When the animals were experimentally infected with Brachyspira hyodysenteriae, causing swine dysentery, many of the T-RFs fluctuated, suggesting a destabilization of the microbial community.     

Effects of fertilization on bacterial community structure and function in a black soil of Dehui region estimated by Biolog and PCR-DGGE methods

Soil microbial community structure and function are commonly used as indicators for soil quality and fertility. In this paper, the bacterial community structure and function in a black soil of Dehui region influenced by fertilization were investigated by Biolog and PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) methods. Biolog examination showed that substrate richness and catabolic diversities of bacterial communities were the highest in the treatment of farm yard manure, and the lowest in the chemical fertilizer treatment. DGGE fingerprint showed that the majority of bands were similar among all treatments, suggesting that microbial communities with those bands were stable, and not influenced by fertilization. In general, chemical fertilizer decreased the diversity of soil bacterial communities. The PCA (principal component analysis) plots of Biolog and DGGE revealed that the structure and function of bacterial communities were similar in the non-fertilized control and the treatment of farm yard manure alone, which inferred that the application of farm yard manure increased the quantity of soil microbes but had less effect on the changes of community structure. The catabolic function was similar, but the composition structure differed between the treatments of chemical fertilizer alone and combined application of farm yard manure with chemical fertilizer. These results suggest that the use of chemical fertilizer mainly decreased the catabolic activity of the fast growth bacteria or eutrophic bacteria.     

Effect of N-fertilization, plant genotype and environmental conditions on nifH gene pools in roots of rice

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified nitrogenase gene (nifH) fragments is a rapid technique for profiling of diazotrophic microbial communities without the necessity of cultures for study. Here, we examined the impact of N-fertilization, plant genotype and environmental conditions on diazotrophic microbial populations in association with roots of rice (Oryza species) by T-RFLP community profiling and found marked effects on the composition of the microbial community. We found a rapid change of the diazotrophic population structure within 15 days after application of nitrogen fertilizer and a strong effect of environmental conditions and plant genotype. Control experiments revealed that phylogenetically distantly related nifH genes were proportionately amplified, and that signal strength reflected the relative abundance of nifH genes in the sample within a 10-fold range of template concentrations. These results clearly demonstrated that our T-RFLP method was suitable to reflect compositional differences in the diazotrophic community in a semiquantitative manner and that the diazotrophic rhizosphere communities of rice are not static but presumably rather highly dynamic.     

Changes in Bacterial Community Structure in the Colon of Pigs Fed Different Experimental Diets and after Infection with Brachyspira hyodysenteriae

Bacterial communities in the large intestines of pigs were compared using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the 16S ribosomal DNA. The pigs were fed different experimental diets based on either modified standard feed or cooked rice supplemented with dietary fibers. After feeding of the animals with the experimental diets for 2 weeks, differences in the bacterial community structure in the spiral colon were detected in the form of different profiles of terminal restriction fragments (T-RFs). Some of the T-RFs were universally distributed, i.e., they were found in all samples, while others varied in distribution and were related to specific diets. The reproducibility of the T-RFLP profiles between individual animals within the diet groups was high. In the control group, the profiles remained unchanged throughout the experiment and were similar between two independent but identical experiments. When the animals were experimentally infected with Brachyspira hyodysenteriae, causing swine dysentery, many of the T-RFs fluctuated, suggesting a destabilization of the microbial community.