Bacterial and fungal communities of wilted Italian ryegrass silage inoculated with and without Lactobacillus rhamnosus or Lactobacillus buchneri

Aims: To understand the effects of lactic acid bacteria (LAB) inoculation on fermentation products, aerobic stability and microbial communities of silage. Methods and Results: Wilted Italian ryegrass was stored in laboratory silos with and without inoculation of Lactobacillus rhamnosus and Lactobacillus buchneri. The silos were opened after 14, 56 and 120 days and then subjected to aerobic deterioration for 7 days. Intensive alcoholic fermentation was found in untreated silage; the sum of ethanol and 2,3‐butanediol content at day 14 was about 7 times higher than that of lactic and volatile fatty acids. Alcoholic fermentation was suppressed by L. rhamnosus and L. buchneri inoculation and lactic acid and acetic acid became the dominant fermentation products, respectively. Silages were deteriorated in untreated and L. rhamnosus‐inoculated silages, whereas no spoilage was found in L. buchneri‐inoculated silage. Enterobacteria such as Erwinia persicina, Pantoea agglomerans and Rahnella aquatilis were detected in untreated silage, whereas some of these bacteria disappeared or became faint with L. rhamnosus treatment. When silage was deteriorated, Lactobacillus brevis and Bacillus pumilus were observed in untreated and L. rhamnosus‐inoculated communities, respectively. The inoculated LAB species was detectable in addition to untreated bacterial communities. Saccharomyces cerevisiae and Pichia anomala were the main fungi in untreated and L. rhamnosus‐inoculated silages; however, P. anomala was not visibly seen in L. buchneri‐inoculated silage either at silo opening or after exposure to air. Conclusion: Inoculation with L. rhamnosus can suppress alcoholic fermentation of wilted grass silage with elimination of enterobacteria at the beginning of fermentation. Addition of L. buchneri may improve aerobic stability, with distinct inhibitory effect observed on P. anomala after silo opening. Significance and Impact of the Study: Bacterial and fungal community analyses help us to understand how inoculated LAB can function to improve the fermentation and aerobic stability of silage.     

Analysis of fungal diversity of grapes by application of temporal temperature gradient gel electrophoresis - potentialities and limits of the method

AIMS: Fungi could be responsible for several problems in wines but the fungal ecosystem of grapes remains little known. The use of traditional methods does not allow to describe quickly this ecosystem. Therefore, we need to improve the knowledge about these fungi to prevent defects in wine. This study aims at evaluating the potentialities of the temporal temperature gradient gel electrophoresis (TTGE) method to describe the fungal ecosystem of grapes. METHODS AND RESULTS: The internal transcribed spacer (ITS) region was amplified and analysed using TTGE. A reference database of 56 fungal species was set up to evaluate the discrimination power of the method. The database was used for the direct identification of the fungal species present in complex samples. The sensitivity of the method is below 10(4) spores per ml. CONCLUSIONS: This method allows to describe the fungal diversity of grapes, but does not always allow to directly identify all fungal species, because of the taxonomic resolution of the ITS sequences. However, this identification strategy is less time consuming than traditional analysis by cloning and sequencing the bands. SIGNIFICANCE AND IMPACT OF THE STUDY: With this method, it will be possible to compare the fungal species present in different vineyards and to connect the presence of some fungi with particular defects in wine.     

洪湖沉积物中真菌群落结构的垂直分布

湖泊沉积物中真菌的类群及其作用所知甚少。以长江中上游代表性湖泊洪湖为研究对象,从湖心截取了一个72cm长的沉积柱,通过rDNA内转录间隔区（ITS）的变性梯度凝胶电泳（DGGE）,对洪湖沉积物中真菌群落结构的垂直变化进行了分析。结果显示,DGGE条带以14－18cm层次为过渡,在2－14cm和14－72cm之间存在一定差异。Shannon-Weaver index（H′）在上下层之间有波动。聚类分析上层2－18cm和中下层18－72cm分别以不同的小类聚在一起。表明14－18cm以上和以下的类群存在差异,     

Effect of continuous olive mill wastewater applications, in the presence and absence of nitrogen fertilization, on the structure of rhizosphere–soil fungal communities

Olive mill wastewater (OMW) is rich in potentially toxic organics precluding its disposal into water receptors. However, land application of diluted OMW may result in safe disposal and fertilization. In order to investigate the effects of OMW on the structure of soil fungal groups, OMW was applied daily to pepper plants growing in a loamy sand and a sandy loam at two doses for a period of 3 months (total OMW equivalents 900 and 1800 m³ ha⁻¹). Nitrogen (N) fertilization alleviated N scarcity and considerably enhanced plant biomass production; however, when applied in combination with the high OMW dose, it induced plant stress. OMW applications resulted in marked changes in the denaturing gradient gel electrophoresis patterns of soil basidiomycete communities, while concurrent N fertilization reduced these effects. In contrast, the ascomycete communities required N fertilization to respond to OMW addition. Cloning libraries for the basidiomycete communities showed that Cryptococcus yeasts and Ceratobasidium spp. dominated in the samples treated with OMW. In contrast, certain plant pathogenic basidiomycetes such as Thanatephorus cucumeris and Athelia rolfsii were suppressed. The observed changes may be reasonably explained by the capacity of OMW to enrich soils in organic substrates, to induce N immobilization and to directly introduce OMW-derived basidiomycetous yeasts.     

Assessment of sample handling practices on microbial activity in sputum samples from patients with cystic fibrosis

Aim: The aim of this study was to quantitatively and qualitatively assess the effect of sample storage on the metabolically active microbial community found in sputum samples from patients with cystic fibrosis (CF). Methods: Sputum samples were collected and split in two equal aliquots one of which was immersed in RNAlater and refrigerated immediately, the second stored at room temperature for 24 h and RNAlater was subsequently added. mRNA was extracted, and RT‐PCR‐DGGE and qPCR analysis of the bacterial and fungal communities was carried out. Results: Significant differences in the bacterial communities between the two protocols were observed but there were no significant difference seen in the fungal community analyses. Analysis by qPCR demonstrated that room temperature storage gave statistically significant increases in eubacteria and Pseudomonas spp. and a statistically significant decrease in those of Haemophilus influenzae. Conclusions: The analysis of metabolically active microbial communities from CF sputum using molecular techniques indicated that samples should be stored at 4°C upon addition of RNAlater to obtain an accurate depiction of the CF lung microbiota. Also, storing respiratory samples at room temperature may cause an over representation of Pseudomonas aeruginosa and mask the presence of other clinically significant organisms.     

PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。     

Influence of long-term repeated prescribed burning on mycelial communities of ectomycorrhizal fungi

To demonstrate the efficacy of direct DNA extraction from hyphal ingrowth bags for community profiling of ectomycorrhizal (ECM) mycelia in soil, we applied the method to investigate the influence of long-term repeated prescribed burning on an ECM fungal community. DNA was extracted from hyphal ingrowth bags buried in forest plots that received different prescribed burning treatments for 30 yr, and denaturing gradient gel electrophoresis (DGGE) profiles of partial fungal rDNA internal transcribed spacer (ITS) regions were compared. Restriction fragment length polymorphism (RFLP) and sequence analyses were also used to compare clone assemblages between the treatments. The majority of sequences derived from the ingrowth bags were apparently those of ECM fungi. DGGE profiles for biennially burned plots were significantly different from those of quadrennially burned and unburned control plots. Analysis of clone assemblages indicated that this reflected altered ECM fungal community composition. The results indicate that hyphal ingrowth bags represent a useful method for investigation of ECM mycelial communities, and that frequent long-term prescribed burning can influence below-ground ECM fungal communities.     

贵州烟草根围AM真菌多样性的初步研究

从贵州省内烟区不同土壤生态环境下采集烟草根际土样,湿筛离心法分离丛枝菌根（AM）真菌孢子,鉴定出烟草AM真菌4属20种,其中球囊霉属9种,无梗囊霉属7种,巨孢囊霉属3种,盾巨孢囊霉属1种。从土壤样品DNA中扩增AM真菌特异性片段并采用DGGE技术对AM真菌多样性进行分析。测序结果显示烟草根际土壤中菌根真菌主要菌群为球囊霉属,与湿筛离心法的鉴定结果一致。为进一步研究贵州地区AM真菌多样性以及开发应用提供了依据。     

Variations in bacterial communities in laboratory-scale and big bale silos assessed by fermentation products, colony counts and denaturing gradient gel electrophoresis profiles

Aims: To assess the variation in bacterial communities in laboratory‐scale and big bale silos. Methods and Results: Wilted Italian ryegrass (628 g dry matter kg^?1) was ensiled in vacuum‐packed plastic pouches and big bales. Silos were opened after 3 months, and the fermentation products, colony counts and denaturing gradient gel electrophoresis (DGGE) profiles were determined. Eight samples were collected separately from a big bale, while one representative sample was taken from a plastic pouch. Significant variation was found between big bales in dry matter, ethanol, lactic acid, acetic acid and ammonia‐N contents. No differences were shown between plastic pouches and big bales, except that more ethanol was produced in the former air‐tight silos. Plastic pouches could resemble a specific silo and outer sampling sites of big bales based on fermentation products and DGGE profiles respectively. Conclusions: Considerable variation in fermentation products may exist between big bale silos. Plastic pouches can serve as a model of big bale silos, although they do not provide information on the heterogeneity within and between bales. Significance and Impact of the Study: Assessment of bacterial communities associated with ensiling can differ according to the criteria of fermentation products, colony counts and DGGE profiles.     

Microbial diversity in lake sediments detected by PCR-DGGE

In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was applied to analyze the microbial communities in lake sediments from Lake Xuanwu, Lake Mochou in Nanjing and Lake Taihu in Wuxi. Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted. The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high (10 μg/g), while that of sediments in Lake Taihu was relatively low. After DNA purification, the 16S rDNA genes (V3 to V5 region) were amplified and the amplified DNA fragments were separated by parallel DGGE. The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments. The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of microorganisms were identified in the sediment samples of both lakes. These results suggest that the sediment samples of these two city lakes (Xuanwu, Mochou) have similar microbial communities. However, the DGGE profiles of sediment samples in Lake Taihu were significantly different from these two lakes. Furthermore, the DGGE profiles of sediment samples in different locations in Lake Taihu were also different, suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou. The differences in microbial diversity may be caused by the different environmental conditions, such as redox potential, pH, and the concentrations of organic matters. Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further re-amplified and sequenced. The results of sequencing analysis indicate that five sequences shared 99%–100% homology with known sequences (Bacillus and Brevibacillus, uncultured bacteria), while the other two sequences shared 93%–96% homology with known sequences (Acinetobacter, and Bacillus). The study shows that the PCR-DGGE technique combined with sequence analysis is a feasible and efficient method for the determination of microbial communities in sediment samples. __________ Translated from Acta Ecologica Sinica, 2006, 26(11): 3610–3616 [译自: 生态学报]     

PCR-DGGE方法分析原油储层微生物群落结构及种群多样性

使用基于 16 S r DNA的 PCR- DGGE(变性梯度凝胶电泳 )图谱分析结合条带割胶回收 DNA进行序列分析 ,对新疆克拉玛依油田一中区注水井 (12 # 9- 11)和与该注水井相应的两个采油井 (12 # 9- 9S、13# 11- 8)井口样品微生物群落的多样性进行了比较并鉴定了部分群落成员。 DGGE图谱聚类分析表明注水井与两油井微生物群落的相似性分别为 30 %和 2 0 % ,而两油井间微生物群落结构的相似性为 5 4 %。DGGE图谱中优势条带序列分析表明注水井样品和油井样品中的优势菌群为未培养的环境微生物 ,它们与数据库中 α、γ、δ、ε变形杆菌 (Proteobacteria)和拟杆菌 (Bacteroidetes)有很近的亲缘关系。 DGGE与分子克隆相结合的分子生物学方法在研究微生物提高原油采收率 (MEOR)机理 ,以及指导 MEOR在油田生产中的应用有着重要的意义     

Screening of bacterial contamination during gelatine production by means of denaturing gradient gel electrophoresis, focussed on Bacillus and related endospore-forming genera

AIMS: To screen for bacterial contamination during gelatine production by means of denaturing gradient gel electrophoresis (DGGE). As members of Bacillus and related genera were found to persist in the final product, this study focussed on these taxa. METHODS AND RESULTS: Template DNA was extracted from gelatine samples at five crucial points of a gelatine production process. A primer specific for Bacillus and related genera was designed and used in a selective PCR, followed by a nested DGGE-PCR targeting the V9 region of the 16S rDNA. DGGE analysis of the resulting amplicons, and sequence analysis of selected bands, showed high sequence similarities of these bands with Bacillus fumarioli, B. licheniformis, B. coagulans and Clostridium perfringens. When the selective PCR was omitted, primarily Lactobacillus bands were retrieved. CONCLUSIONS: PCR-DGGE analysis of gelatine extracts can be used for tracing and screening of bacterial contamination during gelatine production. A selective PCR, nested with DGGE-PCR, gave much more accurate information about endospore-forming contaminants than did the direct DGGE procedure alone. Significance AND IMPACT OF THE STUDY: Use of this nested DGGE-PCR protocol may provide important information about possible hazards to the final microbiological quality and/or safety of gelatine, so allowing production parameters and/or remediation procedures may be adjusted on-line.     

Microbial diversity in lake sediments detected by PCR-DGGE

In this study,PCR-denaturing gradient gel electrophoresis (DGGE) was applied to analyze the microbial communities in lake sediments from Lake Xuanwu,Lake Mochou in Nanjing and Lake Taihu in Wuxi.Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted.The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high (10 μg/g),while that of sediments in Lake Taihu was relatively low.After DNA purification,the 16S rDNA genes (V3 to V5 region) were amplified and the amplified DNA fragments were separated by parallel DGGE.The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments.The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of micro-organisms were identified in the sediment samples of both lakes.These results suggest that the sediment samples of these two city lakes (Xuanwu,Mochou) have similar microbial communities.However,the DGGE profiles of sediment samples in Lake Taihu were significantly differ-ent from these two lakes.Furthermore,the DGGE pro-files of sediment samples in different locations in Lake Taihu were also different,suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou.The differences in microbial diversity may be caused by the different environmental conditions,such as redox potential,pH,and the concentrations of organic matters.Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further re-amplified and sequenced.The results of sequencing analysis indicate that five sequences shared 99%-100% homology with known sequences (Bacillus and Brevibacillus,uncultured bacteria),while the other two sequences shared 93%-96% homology with known sequences (Acinetobacter,and Bacillus).The study shows that the PCR-DGGE tech-nique combined with sequence analysis is a feasible and efficient method for the determination of microbial com-munities in sediment samples.     

变性梯度凝胶电泳法研究我国不同土壤氨氧化细菌群落组成及活性

实验选用了我国3种不同土壤研究土壤硝化活性、硝化细菌数量,并使用变性梯度凝胶电泳(DGGE)的方法研究了不同土壤中氨氧化细菌(AOB)区系变化。通过28d的土壤培养实验研究发现,潮土具有最强的硝化势,几乎100%的铵态氮转化为硝态氮;而红壤中的硝化势最弱,只有4.9%的铵态氮转化为硝态氮。对这3种土壤硝化细菌进行计数发现,3种土壤氨氧化菌数量差异显著,而3种土壤亚硝酸氧化菌(NOB)处于一个数量级。采用氨氧化菌功能基因amoA(氨单加氧酶ammoniamonooxygenase)特异PCR结合DGGE的方法对土壤氨氧化菌区系进行分析。红壤有4个氨氧化菌种属,与潮土和黄泥土没有共同的氨氧化菌种属。4个种属中两个是与潮土和黄泥土亲源性比较远的,特有的氨氧化菌种属,这两个种属与已知的Nitrosospira属的cluster3bz97838和Nitrosospira属的cluster3aAF353263亲源性比较近。潮土有5个氨氧化菌种属,潮土与黄泥土有两个共同的氨氧化菌种属,这两个种属中的一个是潮土和黄泥土特有的,与其他氨氧化菌种属亲源性比较远的氨氧化菌种属,这个种属与已知的Nitrosospira属的cluster3bZ97849亲源性比较近。黄泥土有4个氨氧化菌种属,除了与潮土共有的一个种属是两种土壤特有的氨氧化菌种属外,黄泥土还有一个与其他氨氧化菌种属亲源性比较远的,黄泥土特有的种属,与Nitrosospira属的cluster3aAF353263亲源性很近。3种土壤中分离到的硝化细菌表现出不同的硝化能力。实验结果表明,以amoA基因为目标的PCR-DGGE是比以16SrDNA为目标的PCR-DGGE更有效的研究氨氧化菌种群的方法;3种土壤的氨氧化菌种群差异显著,尤其是红壤的氨氧化菌种群与另外两种土壤差异明显,这种差异可能与红壤的低pH条件对氨氧化菌种群的长期选择有关;3种土壤中的硝化活性与土壤中的硝化细菌数量没有显著相关,可能由于3种土壤差异显著的土壤环境对硝化活性的影响造成。因此在对不同土壤硝化细菌进行研究时不仅需要对硝化细菌数量进行研究,还需要研究不同土壤中硝化细菌的种属及不同土壤环境条件对硝化细菌硝化活性的影响。     

中国中部太白山不同海拔杜鹃兰根部内生真菌多样性

本研究以太白山自然保护区蒿坪站杜鹃兰Cremastra appendiculata为材料,采用菌丝团和根组织分离法进行真菌分离,并用ITS序列分子鉴定;用变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)分析根部内生真菌多样性,研究海拔和根际土理化性质对真菌多样...     

Analysing diversity among beta-lactamase encoding genes in aquatic environments

The most common mechanism of resistance to beta-lactam antibiotics is the production of beta-lactamases. These enzymes are encoded by genes that evolve rapidly, thus constituting a group characterized by high levels of molecular diversity. Most of the genetic determinants of resistance to beta-lactam antibiotics characterized until now were obtained from clinical isolates. This study was designed in order to exploit the presence of beta-lactamase gene sequences in an aquatic environment, and to get information on the distinctive features of those sequences when compared to others available on databases. DNA sequences potentially encoding proteins of three different families of clinically relevant beta-lactamases were assessed: TEM, IMP and OXA-2 derivatives. The presence of bla sequences in DNA extracted from water samples from the lagoon Ria de Aveiro was checked by PCR and hybridization. Sequences representing the three families of beta-lactamases studied were detected. The molecular diversity of the amplicons was assessed by cloning and sequence analysis, and denaturing gradient gel electrophoresis (PCR-DGGE) separation. Most of the retrieved sequences (particularly sequences representing bla(TEM)and bla(OXA-2)) were identical or very similar to beta-lactamase gene sequences previously characterized from clinical isolates. Phylogenetic analysis suggests that this aquatic ecosystem is a reservoir of molecular diverse putative bla sequences. The patterns of molecular diversity found within the beta-lactamase gene families studied do not correspond to those reported in studies focussing on clinical isolates.     

Evaluation of the impact of DNA extraction methods on BAC bacterial community composition measured by denaturing gradient gel electrophoresis

Aims: The impact of DNA extraction methods on biological activated carbon (BAC) DNA yield and bacterial community was evaluated. Methods and Results: Three different DNA extraction methods were compared: method a, method b and method c. Method c with ultrasonic pretreatment improved cell lysis efficiency (from 34% to 87%) and DNA yield [from 10·58 μg g^?1 (dry wt) of carbon to 21·42 μg g^?1 (dry wt) of carbon]. denaturing gradient gel electrophoresis profiles obtained by method c recovered the five seeded bacteria (Bacillus subtilis Strain WSO 6, Pseudomonas putida Strain WSO 7, Acinetobacter lwoffii WSO 10, Pseudomonas pertucinogena WSO 11 and Brevibacterium mcbrellneri WSO 13). Conclusions: The results showed method c with ultrasonic pretreatment was the most successful for the analysis of BAC bacterial community because it was effective in the detachment of bacteria and cell lysis, thereby resulting in good yields. Significance and Impact of Study: These results must be taken into consideration when extracting DNA for analysing BAC bacterial community.     

Effects of food on bacterial community composition associated with the copepod Acartia tonsa Dana

The estuarine copepod Acartia tonsa naturally carried diverse strains of bacteria on its body. The bacterial community composition (BCC) remained very conservative even when the copepod was fed different axenic algal species, indicating that the food per se did not much affect BCC associated with the copepod. In xenic algal treatments, however, copepod-associated BCC differed with each alga fed, even though the same bacterial source was used to inoculate the algae. In addition, starved copepods taken at the same location but at different times significantly differed in their BCC. Algal species composition and copepod life history therefore serve to regulate BCC associated with copepods, and spatial and temporal variations in algal species composition and copepod origin would alter bacteria–copepod interactions.