应用PCR-DGGE技术比较绵羊瘤胃、网胃、瓣胃和皱胃菌群的多样性

本研究采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术结合DGGE图谱条带的克隆和测序比较了绵羊瘤胃、网胃、瓣胃和皱胃菌群的多样性,同时对菌群进行了聚类分析和主成分分析。结果表明:绵羊瘤胃、网胃、瓣胃和皱胃内容物DGGE图谱的平均条带数分别为18、13、16和15条;瘤胃和瓣胃内菌群的多样性指数、均匀度和丰富度均较高,分别为2.83、0.79、17.00和2.82、0.79、16.80,而网胃和皱胃内容物较低,分别为2.52、0.70、12.60和2.73、0.76、15.40;聚类分析结果显示不同胃的内容物菌群具有一定差异,但不同动物个体相同胃的内容物相似性系数均高于0.63;PCR-DGGE图谱中测序的条带大多数归为拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes),优势菌群为厚壁菌门(Firmicutes)细菌、拟杆菌门(Bacteroidetes)细菌、未培养瘤胃细菌(uncultured rumen bacterium)、未培养细菌(uncultured bacterium)和韦荣球菌科(Veillonellaceae)细菌,特异性细菌为不动杆菌属(Acinetobacter sp.)细菌和瘤胃球菌属(Ruminococcaceae)细菌。结果提示,绵羊4个胃中均含有种类丰富和数量巨大的细菌,且随着消化道部位由前往后的顺序,菌群的多样性呈现先高后降低再升高的趋势。     

复合益生菌菌液对奶牛产奶量和肠道菌群的影响

为了研究复合益生菌菌液对奶牛产奶量、乳品质量、牛群健康和肠道菌群的影响,选择20头条件相近的健康奶牛随机分为试验组和对照组,采用菌液与饲料混合的方法饲喂牛群,考察其对奶牛产奶量,肠道菌群乳品质量和牛群健康的影响。结果表明,食用复合益生菌液的每头牛比对照组平均每天可以多产2.9kg牛奶,提高产奶量14.8%,差异极显著(P0.01),经济效益显著;复合益生菌菌液可以促进肠道菌群调整,使有益菌-主要是乳酸菌成为优势菌群,并可以抑制霉菌和降解霉菌毒素,降低牛乳中体细胞数,预防隐性乳腺(房)炎。     

Analysis of Inter-Individual Bacterial Variation in Gut of Cicada Meimuna mongolica (Hemiptera: Cicadidae)

Intestinal bacterial community plays a crucial role in the nutrition, development, survival, and reproduction of insects. When compared with other insects with piercing-sucking mouthparts, the habitats of cicada nymphs and adults are totally different. However, little is known about the differences in the gut bacterial communities in the nymphs and adults within any cicada species. The diversity of bacteria in the gut of nymphs and adults of both genders of Meimuna mongolica (Distant) was studied using the denaturing gradient gel electrophoresis (DGGE) method. Few inter-individual variations among gut microbiota were observed, suggesting that M. mongolica typically harbors a limited and consistent suite of bacterial species. Bacteria in the genera Pseudomonas and Enterobacter were the predominant components of the gut microflora of M. mongolica at all life stages. Bacteria of Pantoea, Streptococcus, and Uruburuella were also widespread in the cicada samples but at relatively lower concentrations. The relative stability and similarity of the PCR-DGGE patterns indicate that all individuals of this cicada species harbor a characteristic bacterial community which is independent from developmental stages and genders. Related endosymbionts that could be harbored in bacteromes of cicadas were not detected in any gut samples, which could be related to the cicada species and the distribution of these endosymbionts in the cicada cavity, or due to some of the possible limitations of PCR-DGGE community profiling. It is worthwhile to further address if related cicada endosymbiont clades distribute in the alimentary canals and other internal organs through diagnostic PCR using group-specific primer sets.     

罗非鱼-鱼腥草共生养殖池塘沉积物菌群结构与功能特征

为探究鱼腥草浮床对罗非鱼池塘沉积物中细菌群落的影响,采用Biolog-Eco和PCR-DGGE技术,对比分析了2013年9—11月鱼腥草浮床(试验塘)和常规养殖(对照塘)2种模式下,罗非鱼养殖池塘沉积物中菌群的结构和功能多样性特征。Biolog-Eco结果显示:9月份试验塘沉积物微生物代谢强度和菌群多样性指数均低于对照塘;但10—11月份则明显高于对照塘,且不同月份丰富度指数与对照塘相比均差异显著(P0.05),说明鱼腥草浮床在养殖中、后期能有效增加细菌生物量和提高种群丰富度。主成分分析(PCA)显示:试验塘和对照塘不同月份沉积物中菌群对羧酸类碳源的相对利用率差异较大,且不同月份利用碳源的差异程度为10月11月9月;DGGE结果显示:试验塘和对照塘沉积物主要的菌群结构组成相似,分属于6个门,包括变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、酸杆菌门(Acidobacteria)、绿弯菌门(Chloroflexi)、蓝细菌门(Cyanobacteria)和梭杆菌门(Fusobacteria),其中变形菌门(54.17%)均为优势菌群,但同一月份试验塘菌群多样性均明显多于对照塘。结果表明,鱼腥草浮床具有增加罗非鱼池塘沉积物菌群功能多样性和优化菌群结构的作用。     

接种溶磷真菌对玉米和大豆根际细菌群落结构多样性的影响

Application of phosphate-solubilizing microorganisms （PSMs） has been reported to increase P uptake and plant growth. However, no information is available regarding the ecological consequences of the inoculation with PSMs. The effect of inoculation with phosphate-solubilizing fungal （PSF） isolates Aspergillus niger P39 and Penicillium oxalicum P66 on the bacterial communities in the rhizospheres of maize （Zea mays L. ‘Haiyu 6＇） and soybean （Glycine max Merr. ‘Heinong 35＇） was examined using culture-dependent methods as well as a culture-independent method, polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE）. Compared with the control, the number of culturable microbes for soybean was significantly greater with P39, whereas for maize, the same was significantly greater with P66. In addition, a greater number of microbes were found in the rhizosphere of maize compared with soybean. The fingerprint of DGGE for 16S rDNA indicated that inoculation with PSF also increased bacterial communities, with the P66 treatment having higher numbers of DGGE bands and a higher Shannon-Weaver diversity index compared with P39; the composition of the microbial community was also more complex with the P66 treatment. Overall, complex interactions between plant species and exotic PSMs affected the structure of the bacterial community in the rhizosphere, but plant species were more important in determining the bacterial community structure than the introduction of exotic microorganisms.     

Soil fumigation and compost amendment alter soil microbial community composition but do not improve tree growth or yield in an apple replant site

Apple replant disease (ARD) is a disease complex that reduces survival, growth and yield of replanted trees, and is often encountered in establishing new orchards on old sites. Methyl bromide (MB) has been the fumigant used most widely to control ARD, but alternatives to MB and cultural methods of control are needed. In this experiment, we evaluated the response of soil microbial communities and tree growth and yield to three pre-plant soil treatments (compost amendment, soil treatment with a broad-spectrum fumigant, and untreated controls), and use of five clonal rootstock genotypes (M.7, M.26, CG.6210, G.30 and G.16), in an apple replant site in Ithaca, New York. Polymerase chain reaction (PCR)—denaturing gradient gel electrophoresis (DGGE) analysis was used to assess changes in the community composition of bacteria and fungi in the bulk soil 8, 10, 18 and 22 months after trees were replanted. PCR-DGGE was also used to compare the community composition of bacteria, fungi and pseudomonads in untreated rhizosphere soil of the five rootstock genotypes 31 months after planting. Tree caliper and extension growth were measured annually in November from 2002 to 2004. Apple yield data were recorded in 2004, the first fruiting year after planting. Trees on CG.6210 rootstocks had the most growth and highest yield, while trees on M.26 rootstocks had the least growth and lowest yield. Tree growth and yield were not affected by pre-plant soil treatment except for lateral extension growth, which was longer in trees growing in compost-treated soil in 2003 as compared to those in the fumigation treatment. Bulk soil bacterial PCR-DGGE fingerprints differed strongly among the different soil treatments 1 year after their application, with the fingerprints derived from each pre-plant soil treatment clustering separately in a hierarchical cluster analysis. However, the differences in bacterial communities between the soil treatments diminished during the second year after planting. Soil fungal communities converged more rapidly than bacterial communities, with no discernable pattern related to pre-plant soil treatments 10 months after replanting. Changes in bulk soil bacterial and fungal communities in response to soil treatments had no obvious correlation with tree performance. On the other hand, rootstock genotypes modified their rhizosphere environments which differed significantly in their bacterial, pseudomonad, fungal and oomycete communities. Cluster analysis of PCR-DGGE fingerprints of fungal and pseudomonad rhizosphere community DNA revealed two distinct clusters. For both analyses, soil sampled from the rhizosphere of the two higher yielding rootstock genotypes clustered together, while the lower yielding rootstock genotypes also clustered together. These results suggest that the fungal and pseudomonad communities that have developed in the rhizosphere of the different rootstock genotypes may be one factor influencing tree growth and yield at this apple replant site.     

Nitrogen availability decreases prokaryotic diversity in sandy soils

In this study, the interrelation between nitrogen availability and prokaryotic diversity are studied using a well-characterised system from a long-term field experiment on a loamy sandy soil. The prokaryotic potential functional diversity and community composition were assessed using community-level physiological profiling (CLPP), and their phylogenetic diversity was analysed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) in combination with sequencing analysis. Highest prokaryotic potential functional diversity was measured in the control soil receiving no N fertilisation, indicating an efficient as well as versatile utilisation of the substrates in this soil. Both substrate utilisation richness and substrate utilisation evenness, the two constituents of the functional diversity, were decreased with increasing N supply. Furthermore, distinct prokaryotic community compositions were generated in N-enriched soils compared to unfertilised control soils. These differences suggest a dominance of populations adapted to utilising readily available substrates. We demonstrated that the shift in prokaryotic functional diversity was connected to a shift in the phylogenetic structure of the bacterial and archaeal communities. Taken together, our data clearly show that, for the sandy soil system, prokaryotic diversity and N availability were interrelated.     

Community composition of ammonia-oxidizing bacteria in the rhizosphere of intercropped wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.), maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.), and faba bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.)

Cereal/cereal and cereal/legume intercropping systems are popular in the north, northwest, and southwest of China and often result in yield increases compared to monocropping. Rhizosphere interactions may play a significant role in the yield increases, particularly with respect to nutrient availability. The aim of this study was to investigate the effects of intercropping on N availability and community composition of ammonia-oxidizing bacteria in the rhizosphere of wheat, maize, and faba bean at different growth stages. Denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA genes was used to analyze the community composition of bacterial ammonia oxidizers belonging to β-proteobacteria. The results showed that intercropping with faba bean significantly increased nitrate concentrations in the rhizosphere of wheat and maize at the second sampling time (20 June) compared to monocropping or intercropping between maize and wheat. Intercropping significantly affected the community composition of ammonia-oxidizing bacteria in the rhizosphere compared to monocropping, and the effects were most pronounced in the maize/faba bean and wheat/maize intercropping systems when faba bean and wheat were at anthesis and maize was in seedling stage. In wheat/faba bean intercropping, the effects of intercropping on community composition of ammonia-oxidizing bacteria were less pronounced at the seedling stage of the two species but were significant at anthesis.     

Effect of Azospirillum brasilense inoculation on rhizobacterial communities analyzed by denaturing gradient gel electrophoresis and automated ribosomal intergenic spacer analysis

Nucleic acid-based techniques allow the exploration of microbial communities in the environments such as the rhizosphere. Azospirillumbrasilense, a plant growth promoting rhizobacterium (PGPR), causes morphological changes in the plant root system. These changes in root physiology may indirectly affect the microbial diversity of the rhizosphere. In this study, the changes in the rhizobacterial structure following A. brasilense inoculation of maize (Zea mays) plants was examined by PCR-denaturating gradient gel electrophoresis (DGGE) and automated ribosomal intergenic spacer analysis (ARISA), using two universal primers sets for the 16S rRNA gene, and an intergenic 16S-23S rDNA primer set, respectively. Similar results were obtained when using either ARISA or DGGE performed with these different primer sets, and analyzed by different statistical methods: no prominent effect of A. brasilense inoculation was observed on the bacterial communities of plant roots grown in two different soils and in different growth systems. In contrast, plant age caused significant shifts in the bacterial populations.