不同年限毛竹林土壤固氮菌群落结构和丰度的演变

应用变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)和荧光定量PCR(real time fluorescent quantitative PCR,q PCR)方法研究了不同年限毛竹林土壤固氮菌群落结构和丰度的变化。结果表明:土壤p H、有机质、有效磷、速效钾和铵态氮含量在马尾松林改造成毛竹林5 a后明显提高,而后逐渐降低,并趋于稳定;土壤固氮菌多样性和nif H基因丰度也呈现相似的趋势。条带测序分析表明,毛竹林土壤固氮菌均为不可培养的固氮菌,与慢生根瘤菌(Bradyrhizobium sp.)具有较高的相似度。冗余分析结果表明,不同栽培年限毛竹林地土壤固氮菌群落组成发生了明显变化,长期栽培毛竹林引起的土壤养分变化对土壤固氮菌多样性具有重要影响。     

Survival of bacterial DNA and culturable bacteria in archived soils from the Rothamsted Broadbalk experiment

Dried soil samples from many sources have been stored in archives world-wide over the years, but there has been little research on their value for studying microbial populations. Samples collected since 1843 from the Broadbalk field experiment on crop nutrition at Rothamsted have been used to document changes in the structure and composition of soils as agricultural practices evolve, also offering an invaluable record of environmental changes from the pre- to post-industrial era in the UK. To date, the microbial communities of these soils have not been studied, in part due to the well-documented drop in bacterial culturability in dried soils. However, modern molecular methods based on PCR amplification of DNA extracted directly from soil do not require bacterial cells to be viable or intact and may allow investigations into the legacy of bacteria that were present at the time of sample collection.

In a preliminary study, to establish if dried soils can provide a historical record of bacterial communities, samples from the Broadbalk soil archive dating back to 1868 were investigated and plots treated with either farmyard manure (FYM) or inorganic fertilizer (NPK) were compared. As anticipated, the processes of air-drying and milling greatly reduced bacterial viability whilst DNA yields declined less and may be preserved by desiccation. A higher proportion of culturable bacteria survived the archiving process in the FYM soil, possibly protected by the increased soil organic matter. The majority of surviving bacteria were firmicutes, whether collected in 2003 or in 1914, but a wide range of genera was detected in DNA extracted from the samples using PCR and DGGE of 16S rRNA genes. Analysis of DGGE band profiles indicated that the two plots maintained divergent populations. Sequence analysis of bands excised from DGGE gels, from a sample collected in 1914, revealed DNA from - and β-proteobacteria as well as firmicutes. PCR using primers specific for ammonia oxidizing bacteria showed similar band profiles across the two treatments in recently collected samples, however older samples from the NPK plot showed greater divergence. Primers specific for the genus Pseudomonas were designed and used in real-time quantitative PCR to indicate that archived soil collected in 1868 contained 10-fold less pseudomonad DNA than fresh soil, representing around 10⁵ genomes g⁻¹ soil. Prior to milling, dramatically less pseudomonad DNA was extracted from recently collected air-dried soil from the NPK compared to the FYM plot; otherwise, the two plots followed similar trends. Overall bacterial abundance, diversity and survival during the archiving process differed in the two soils, possibly due to differences in clay and soil organic matter content. Nevertheless, the results demonstrate that air-dried soils can protect microbial DNA for more than 150 years and offer an invaluable resource for future research.     

Intestinal Microbiota in Large Yellow Croaker,Larimichthys crocea,at Different Ages

The polymerase chain reaction–denaturing gradient gel electrophoresis (PCR‐DGGE) of 16S ribosomal RNA gene was used to investigate bacterial communities in the intestines of large yellow croaker at six different ages (12 d, 18 d, 26 d, 40 d, 3 mo, and 1 yr old) as well as within the corresponding feed and culture water. In addition, Illumina Miseq sequencing was utilized to compare intestinal microbiota between 12‐d‐old and 1‐yr‐old individuals. PCR‐DGGE results revealed that the culture water had the highest bacterial diversity, followed by the feed, while the intestines had the lowest diversity. The intestinal microbiota at six ages changed severely; however, the change did not follow any trend. The large yellow croaker intestines harbored specific bacterial communities that differed from those in both feed and water. Illumina Miseq sequencing results revealed that the diversity of intestinal bacteria in 12‐d‐old fish was higher than that in 1‐yr‐old fish, and the bacterial composition differed significantly between them. γ‐Proteobacteria and Pseudoalteromonas supplied the most abundant phylum and genus in the 12‐d‐old fish intestine. However, in the 1‐yr‐old fish intestine, Firmicutes and Clostridium were the most dominant, respectively. The study may contribute to a better understanding of gut microbiota and dynamics of the large yellow croaker and the relationship with their surrounding environment.     

木聚糖酶对肉仔鸡后肠道微生物的影响

主要研究了小麦基础口粮添加木聚糖酶对肉仔鸡后肠道微生物的影响.将120只7日龄肉仔鸡分成2组,日粮分别添加0,0.1%木聚糖酶,饲喂至21日龄,研究木聚糖酶对肉仔鸡后肠道菌群数量变化的影响.结果表明,加酶未使同肠和盲肠乳酸杆菌、双歧杆菌和大肠杆菌数量发生显著变化.利用变性梯度凝胶电泳技术研究了木聚糖酶对肉仔鸡后肠细菌群体的影响.结果表明,加酶组回肠和盲肠的图谱比对照组条带相对较多,但差异不显著;组内个体间的图谱相似性比组问的相似性相对较大.木聚糖酶影响肉仔鸡后肠微生物数量和种群的作用效果不明显.     

Influences of quorum‐quenching probiotic bacteria on the gut microbial community and immune function in weaning pigs

The aim of this study is to investigate the dynamic gut microbial diversity in weaning swine after administering feed supplemented with probiotic bacteria that specifically inhibit the activity of quorum molecules. Initially, the universal quorum molecule autoinducer‐2 (AI‐2) bioassay results indicated that AI‐2 activity was profoundly inhibited in enterohemorrhagic Escherichia coli (EHEC) O157:H7 in the presence of Lactobacillus acidophilus strain 30SC cell extract, although the growth of EHEC was not affected. Based on plate counting results, bacterial community analysis revealed a specific reduction in coliforms compared to the control, whereas the population of lactobacilli increased in weaning swine in in vivo trials. Supplementation with L. acidophilus strain 30SC did not affect the counts of other communities, such as total aerobes and yeast/mold. In addition, PCR‐denaturing gradient gel electrophoresis analysis showed a significant difference in the 16S rRNA gene products after administering L. acidophilus strain 30SC. Selected bands were sequenced, and most of them were identified as uncultured bacterium clones or a Lactobacillus‐ and Bifidobacterium‐specific community. Therefore, our results indicate that quorum‐quenching probiotic bacteria can significantly modulate the gut microbiota of swine and these beneficial effects can contribute to the improvement of performance and health in the gastrointestinal tract of weaning pigs.     

Assessment of gut microbiota in different developmental stages of Malaysian Mahseer (Tor tambroides)

Malaysian Mahseer (Tor tambroides) has a good prospect for aquaculture because of its high market demand. However, there is a scarce information on gut microbiota associated with Malaysian Mahseer unlike other fish species. Therefore, we constructed and compared gut microbiota in different developmental stages (larval, juvenile, fingerling, yearling, and adult) using culture dependent and PCR‐DGGE fingerprinting technique for better understanding of gut microbiota composition associated with T. tambroides. Culturable gut microbiota composition in all developmental stages were composed of β‐ and γ‐Proteobacteria, and Bacilli. Biodiversity analysis of culturable gut microbiota showed that larval, juvenile, and adult stages have higher diversity than fingerling and yearling stages. Ward's linkage cluster analysis showed that culturable gut microbiota composition in larval and juvenile stages were close to adult stages, whereas fingerling and yearling stage composed same cluster. PCR‐DGGE fingerprinting technique showed that unculturable gut microbiota were constituted by α‐and γ‐Proteobacteria, and Actinobacteria. Ward's linkage cluster analysis showed that unculturable gut microbiota composition in both larval and juvenile stages were distinct from other developmental stages. Our results revealed that gut microbiota composition were varied in different developmental stages of Malaysian Mahseer and continuous shifts of gut microbiota from larval to adult stages.     

基于16SrDNA比较研究混养三角帆蚌和鲢鳙对池塘养殖水体微生物群落结构的影响

为了研究混养三角帆蚌(Hyriopsis cumingii)和鲢(Hypophthalmichthys molitrix)鳙(Aristichthys nobilis)对池塘养殖水体微生物群落结构的影响,分别在浙江海盐、江西九江、上海浦东开展了罗氏沼虾-三角帆蚌混养、草鱼—三角帆蚌混养、罗氏沼虾-三角帆蚌-鲢鳙鱼混养三组系列实验。利用DGGE技术对养殖水体的16S r DNA进行指纹图谱分析。三组实验共鉴定出57条不同条带,每组实验中混养三角帆蚌或鲢鳙实验组的平均条带数均高于单养水体水样平均条带数。Shannon多样性指数分析结果显示,各养殖水体混养三角帆蚌、鲢鳙后,多样性指数均有所增加。PCA分析和DGGE聚类结果显示,在无鲢鳙的情况下,蚌与主养物种(罗氏沼虾、草鱼)混养实验组的16S r DNA图谱聚为一类,显示出蚌对于微生物群落结构影响的贡献;在同时引入鲢鳙和蚌的情况下,16S r DNA指纹图谱则是按照有无鲢鳙聚为两类,提示在本实验中鲢鳙对于水体微生物的影响要大于蚌。对DGGE图谱其中20条显著条带进行回收、测序和系统发育分析,结果表明,所获序列主要分属于放线菌门(Actinobacteria,25%)、蓝藻门(Cyanobacteria,25%)、α-变形菌亚门(Alphaproteobacteria,15%)、β-变形菌亚门(Betaproteobacteria,15%)、γ-变形菌亚门(Gammaproteobacteria,10%)、ε-变形菌亚门(Epsilonproteobacteria,5%)、裸藻叶绿体(Euglenales,5%)。     

Nitrifier dominance of Arctic soil nitrous oxide emissions arises due to fungal competition with denitrifiers for nitrate

Arctic soils emit nitrous oxide, which is a potent greenhouse gas and also represents an important loss of nitrogen to oligotrophic Arctic ecosystems. However, little is known about the temperature sensitivity of nitrous oxide release in Arctic soils or the organisms mainly responsible for it. We investigated controls on nitrous oxide emissions in an Arctic soil across a typical temperature range (between 4 and 13 °C) on Truelove Lowland, Devon Island, Canada (75°40′N 84°35′W) at two different moisture contents. When fertilized with ammonia or nitrate, nitrous oxide emissions and temperature dependence of nitrous oxide emissions were insensitive to soil moisture content but linked to nitrification rates. Stable isotope analysis revealed that nitrous oxide was predominantly released by nitrifiers. However, nitrous oxide emissions were not linked to nitrifier prevalence with an insignificant (P < 0.219) increase in amoA genes and a (P < 0.01) decrease in archaeal nitrifiers. In contrast, denitrifier nosZ prevalence was 10,000 times greater than that of nitrifiers and was related to nitrous oxide emission potential when soils were fertilized with nitrate. Manipulating water-filled pore space should have changed the pattern of N₂O emissions. We used selective inhibitors to further explore why denitrification did not occur under field conditions when we manipulated water-filled pore space or when we used ¹⁵N analysis. When fungi were inhibited in the soil, nitrous oxide emissions from denitrifiers increased with no change in nitrous oxide released by nitrifiers. When fungi were active in the soil, there was little available nitrate but when fungi were inhibited, available soil nitrate increased over the incubation period. The dominance of nitrifiers in nitrous oxide emissions from Arctic soils under field conditions is linked to the competition for nitrate between fungi and denitrifiers.     

Optimization of plant growth-promoting bacteria-assisted phytostabilization of mine tailings

Recent studies have indicated that plant growth-promoting bacteria (PGPB) can improve revegetation of arid mine tailings as measured by increased biomass production. The goals of the present study were first to evaluate how mode of application of known PGPB affects plant growth, and second to evaluate the effect of this inoculation on rhizosphere microbial community structure. PGPB application strategies investigated include preliminary surface sterilization of seeds (a common practice in phytoremediation trials) followed by a comparison of two application methods; immersion and alginate encapsulation. Results with two native desert plant species, Atriplex lentiformis and Buchloe dactyloides, suggest that seed surface sterilization prior to inoculation is not necessary to achieve beneficial effects of introduced PGPB. Both PGPB application techniques generally enhanced plant growth although results were both plant and PGPB specific. These results demonstrate that alginate encapsulation, which allows for long-term storage and easier application to seeds, is an effective way to inoculate PGPB. In addition, the influence of PGPB application on B. dactyloides rhizosphere community structure was evaluated using PCR-DGGE (denaturing gradient gel electrophoresis) analysis of bacterial DNA extracted from rhizosphere samples collected 75 d following planting. A comparative analysis of DGGE profiles was performed using canonical correspondence analysis (CCA). DGGE-CCA showed that rhizosphere community profiles from PGPB-inoculated treatments are significantly different from both uninoculated tailings rhizosphere profiles and profiles from the compost used to amend the tailings. Further, community profiles from B. dactyloides inoculated with the best performing PGPB (Arthro Mix) were significantly different from two other PGPB tested. These results suggest that introduced PGPB have the potential to influence the development of the rhizosphere community structure found in plants grown in mine tailings.     

天目山毛竹入侵阔叶林后土壤细菌群落16S rDNA V3区片段PCR的DGGE分析

天目山国家级自然保护区是公认的基因库。毛竹入侵天然林并替代原天然林,导致地上植物多样性下降。为了解毛竹入侵天然林后地下土壤微生物多样性的变化,分别采集了毛竹纯林、竹阔混交林和原始天然阔叶林下的土壤样品,应用建立于16S rDNA V3区片段的变性梯度凝胶电泳(DGGE)和克隆测序比对来研究土壤细菌结构的变化。结果表明,3块林地土壤16S rDNA V3区片段高达30条以上,不同林分下土壤16SrDNAV3区片段的DGGE带谱差异不大,但各有特征条带。毛竹林与阔叶林土壤的细菌结构相似度高于其与竹阔混交林的相似度。通过DGGE条带的克隆测序比对发现,调查区土壤细菌主要属于变形菌门(Proteobacterium)、厚壁菌门(Firmicutes)、酸杆菌门(Acidobacteria)、放线细菌属(Actinobacterium)和一些未命名的菌种,并且多数属无法纯培养的物种。本试验结论为:天目山自然保护区内土壤细菌多样性丰富,不同林分下的土壤细菌有各自的特征种,但非优势种;毛竹入侵未导致土壤细菌结构以及多样性发生显著变化。