转几丁质酶和葡聚糖酶双价基因棉花对土壤细菌种群多样性的影响

以转几丁质酶和葡聚糖酶双价基因棉花为研究对象,非转基因受体棉花为对照,通过比较可培养细菌数量和基于16S rRNA克隆文库细菌种群分析,评价外源双价基因的导入在苗期、蕾期、花铃期和吐絮期对棉花根际细菌群落多样性的影响。结果表明,可培养细菌的数量不受外源双价基因的影响,随着棉花生育期的交替而变化,以代谢旺盛的花铃期最多。构建的转基因和非转基因不同生育期根际土壤细菌16S rRNA文库容量为2400个克隆,涵盖了细菌的283个属。其中,Acidobacterium是最大优势类群,共包括624个克隆,其次为未知细菌种群和Flavisolibacter。比较转基因和非转基因棉花根际土壤细菌的种群结构,结果显示,同一生育期内前者种群的多样性显著低于后者,二者的共有类群随着生长发育的进行而增多。研究结果说明几丁质酶基因和葡聚糖酶基因对棉花根际细菌种群多样性有着不同程度的削减作用,但是随着种植时间的延长,该差异呈现逐渐缩小的趋势。     

Comparison of culturable Gram-negative bacterial community structures in the rhizosphere of three fruit plants

This research work was oriented to outlining the diversity of Gram-negative culturable portion of the bacterial community in three fruit plants rhizosphere. Rhizosphere samples were taken from European chestnut (Castanea sativa Mill), true service tree (Sorbus domestica L.) and cornelian cherry (Cornus mas L.) plants. Experiments were conducted for three years during the vegetation period, and the bacterial community structure was assessed with cultivation-dependent approach. Many Gram-negative isolates (n = 251) from the rhizosphere survived sub culturing and were identified by biochemical tests. A total of 57 species belonging to 29 genera were identified and assigned to four broad taxonomic groups (Bacteroidetes, Alpha-, Beta- and Gamma-proteobacteria). Several specific bacterial cluster communities were identified inside all the three rhizospheres. Most of the species belonged to the genera Moraxella, Pseudomonas, Pantoea, Enterobacter and Acinetobacter. In addition, while, using the plate count analysis, large discrepancies in numbers among physiological groups of bacteria cultured from three rhizosphere samples have not been revealed, more expressive distinctions among bacterial populations were obtained concerning the relative abundance of different genera, different taxonomic groups as well as different diversity indices. Furthermore, the number of cultured bacteria and their taxonomic distribution in the rhizosphere of all three plants changed not only explicitly during vegetation period but continually during the three years of investigation. It seems that rhizosphere bacterial populations of each plant are under the influence of the specific root-released materials.     

Cultivation-independent detection of autotrophic hydrogen-oxidizing bacteria by DNA stable-isotope probing

Knallgas bacteria are a physiologically defined group that is primarily studied using cultivation-dependent techniques. Given that current cultivation techniques fail to grow most bacteria, cultivation-independent techniques that selectively detect and identify knallgas bacteria will improve our ability to study their diversity and distribution. We used stable-isotope probing (SIP) to identify knallgas bacteria in rhizosphere soil of legumes and in a microbial mat from Obsidian Pool in Yellowstone National Park. When samples were incubated in the dark, incorporation of (13)CO(2) was H(2) dependent. SIP enabled the detection of knallgas bacteria that were not detected by cultivation, and the majority of bacteria identified in the rhizosphere soils were betaproteobacteria predominantly related to genera previously known to oxidize hydrogen. Bacteria in soil grew on hydrogen at concentrations as low as 100 ppm. A hydB homolog encoding a putative high-affinity NiFe hydrogenase was amplified from (13)C-labeled DNA from both vetch and clover rhizosphere soil. The results indicate that knallgas bacteria can be detected by SIP and populations that respond to different H(2) concentrations can be distinguished. The methods described here should be applicable to a variety of ecosystems and will enable the discovery of additional knallgas bacteria that are resistant to cultivation.     

Characterization of Bacterial Community Structure in Rhizosphere Soil of Grain Legumes

Molecular techniques were used to characterize bacterial community structure, diversity (16S rDNA), and activity (16S rRNA) in rhizospheres of three grain legumes: faba beans (Vicia faba L., cv. Scirocco), peas (Pisum sativum L., cv. Duel) and white lupin (Lupinus albus L., cv. Amiga). All plants were grown in the same soil under controlled conditions in a greenhouse and sampled after fruiting. Amplified 16S rDNA and rRNA products (using universal bacterial primers) were resolved by denaturing gradient gel electrophoresis (DGGE). Distinct profiles were observed for the three legumes with most of the bands derived from RNA being a subset of those derived from DNA. Comparing the total bacterial profiles with actinomycete-specific ones (using actinomycete-specific primers) highlighted the dominance of this group in the three rhizospheres. 16S PCR and RT-PCR products were cloned to construct libraries and 100 clones from each library were sequenced. Actinomycetes and proteobacteria dominated the clone libraries with differences in the groups of proteobacteria. Absence of β-subdivision members in pea and γ-subdivision members of proteobacteria in faba bean rhizosphere was observed. Plant-dependent rhizosphere effects were evident from significant differences in the bacterial community structure of the legume rhizospheres under study. The study gives a detailed picture of both residing and „active” bacterial community in the three rhizospheres. The high abundance of actinomycetes in the rhizospheres of mature legumes indicates their possible role in soil enrichment after the legumes are plowed into the soil as biofertilizers.     

Characterization of Ni-resistant bacteria in the rhizosphere of the hyperaccumulator <Emphasis Type="Italic">Alyssum murale</Emphasis> by 16S rRNA gene sequence analysis

The diversity of 184 isolates from rhizosphere and bulk soil samples taken from the Ni hyperaccumulator Alyssum murale, grown in a Ni-rich serpentine soil, was determined by 16S rRNA gene analysis. Restriction digestion of the 16S rRNA gene was used to identify 44 groups. Representatives of each of these groups were placed within the phyla Proteobacteria, Firmicutes and Actinobacteria by 16S rRNA gene sequence analysis. By combining the 16S rRNA gene restriction data with the gene sequence analysis it was concluded that 44.6% (82/184) of the isolates were placed within the phylum Proteobacteria, among these 35.9% (66/184) were placed within the class α-Proteobacteria, and 20.7% (38/184) had 16S rRNA gene sequences indicative of bacteria within genera that form symbioses with legumes (rhizobia). Of the remaining isolates, 44.6% (82/184) and 5.4% (10/184) were placed within the phyla Actinobacteria and Firmicutes, respectively. No placement was obtained for a small number (10/184) of the isolates. Bacteria of the phyla Proteobacteria and Actinobacteria were the most numerous within the rhizosphere of A. murale and represented 32.1% (59/184) and 42.9% (79/184) of all isolates, respectively. The approach of using 16S rRNA gene sequence analysis in this study has enabled a comprehensive characterization of bacteria that predominate in the rhizosphere of A. murale growing in Ni-contaminated soil.     

Diversity of predominant endophytic bacteria in European deciduous and coniferous trees

The diversity of endophytic bacteria residing in root, stem, and leaf tissues was examined in coniferous and deciduous tree species, Scots pine (Pinus sylvestris L.), silver birch (Betula pendula Roth), and rowan (Sorbus aucuparia L.). Using cultivation-dependent and -independent analyses, the bacterial communities were observed to be significantly different in the belowground (roots and rhizosphere) and aboveground (leaves and stems) samples of the respective host trees. No significant differences, with respect to the different tree species, were observed in the associated communities. Predominant cultivable endophytes isolated included bacteria closely related to Bacillus subtilis, Bacillus licheniformis, Paenibacillus spp., and Acinetobacter calcoaceticus. Comparisons of the most abundant cultivable bacteria in the rhizosphere and root samples suggested that root endophytic bacteria may be in residence through processes of selection or active colonization rather than by passive diffusion from the rhizosphere.     

Phylogenies of symbiotic genes of Bradyrhizobium symbionts of legumes of economic and environmental importance in Brazil support the definition of the new symbiovars pachyrhizi and sojae

Bradyrhizobium comprises most tropical symbiotic nitrogen-fixing strains, but the correlation between symbiotic and core genes with host specificity is still unclear. In this study, the phylogenies of the nodY/K and nifH genes of 45 Bradyrhizobium strains isolated from legumes of economic and environmental importance in Brazil (Arachis hypogaea, Acacia auriculiformis, Glycine max, Lespedeza striata, Lupinus albus, Stylosanthes sp. and Vigna unguiculata) were compared to 16S rRNA gene phylogeny and genetic diversity by rep-PCR. In the 16S rRNA tree, strains were distributed into two superclades—B. japonicum and B. elkanii—with several strains being very similar within each clade. The rep-PCR analysis also revealed high intra-species diversity. Clustering of strains in the nodY/K and nifH trees was identical: 39 strains isolated from soybean grouped with Bradyrhizobium type species symbionts of soybean, whereas five others occupied isolated positions. Only one strain isolated from Stylosanthes sp. showed similar nodY/K and nifH sequences to soybean strains, and it also nodulated soybean. Twenty-one representative strains of the 16S rRNA phylogram were selected and taxonomically classified using a concatenated glnII-recA phylogeny; nodC sequences were also compared and revealed the same clusters as observed in the nodY/K and nifH phylograms. The analyses of symbiotic genes indicated that a large group of strains from the B. elkanii superclade comprised the novel symbiovar sojae, whereas for another group, including B. pachyrhizi, the symbiovar pachyrhizi could be proposed. Other potential new symbiovars were also detected. The co-evolution hypotheses is discussed and it is suggested that nodY/K analysis would be useful for investigating the symbiotic diversity of the genus Bradyrhizobium.     

Effect of transgenic alfalfa plants with introduced gene for Alfalfa Mosaic Virus coat protein on rhizosphere microbial community composition and physiological profile

The aim of this study was to evaluate the effect of transgenic alfalfa (Medicago sativa L.) plants, in comparison to their non-transgenic counterpart, on the density and physiological profiles of aerobic bacteria in the rhizosphere. Plants of transgenic alfalfa expressing the AMVcp-s gene coding for Alfalfa Mosaic Virus coat protein were cultivated in a climatic chamber. Two methods were used to determine the microbial diversity in rhizospheres of transgenic plants. First, the cultivation-dependent plating method, based on the determination of the density of colony-forming bacteria, and second, a biochemical method using the Biolog™ system, based on the utilization of different carbon sources by soil microorganisms. Statistically significant differences in densities of rhizospheric bacteria between transgenic and non-transgenic alfalfa clones were observed in ammonifying bacteria (GTL4/404-1), cellulolytic bacteria (GTL4/404-1, GTL4/402-2, A5-3-3), rhizobial bacteria (GTL4/402-2), denitrifying bacteria (A5-3-3) and Azotobacter spp. (GTL4/402-2). The highest values of substrate utilization by microbial communities and average respiration of C-sources were determined in non-transgenic alfalfa plants of the isogenic line SE/22-GT2. Carbohydrates, carboxylic acids and amino-acids were the most utilized carbon substrates by both Gram-negative and Gram-positive bacteria. Both, the community metabolic diversity and the utilization of C-sources increased in all alfalfa lines with culture time and regardless of transgenic or non-transgenic nature of lines.     

Bacterial communities associated with the rhizosphere of pioneer plants (<Emphasis Type="Italic">Bahia xylopoda</Emphasis> and <Emphasis Type="Italic">Viguiera linearis</Emphasis>) growing on heavy metals-contaminated soils

In this study, the bacterial communities associated with the rhizospheres of pioneer plants Bahia xylopoda and Viguiera linearis were explored. These plants grow on silver mine tailings with high concentration of heavy metals in Zacatecas, Mexico. Metagenomic DNAs from rhizosphere and bulk soil were extracted to perform a denaturing gradient gel electrophoresis analysis (DGGE) and to construct 16S rRNA gene libraries. A moderate bacterial diversity and twelve major phylogenetic groups including Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes, Chloroflexi, Firmicutes, Verrucomicrobia, Nitrospirae and Actinobacteria phyla, and divisions TM7, OP10 and OD1 were recognized in the rhizospheres. Only 25.5% from the phylotypes were common in the rhizosphere libraries and the most abundant groups were members of the phyla Acidobacteria and Betaproteobacteria (Thiobacillus spp., Nitrosomonadaceae). The most abundant groups in bulk soil library were Acidobacteria and Actinobacteria, and no common phylotypes were shared with the rhizosphere libraries. Many of the clones detected were related with chemolithotrophic and sulfur-oxidizing bacteria, characteristic of an environment with a high concentration of heavy metal-sulfur complexes, and lacking carbon and organic energy sources.     

黄河三角洲盐碱地花生根层土壤菌群结构多样性

花生属豆科固氮作物,具较强的抗旱耐盐性,土壤微生物在盐碱土生态系统中具有重要的生态功能。以花生平作、花生/棉花间作为对象,通过16S rRNA基因克隆文库技术分析了黄河三角洲滨海盐碱地花生旺盛生长期不同含盐量盐碱地和非盐碱地0—40cm根层非培养土壤微生物群落组成及其多样性,分析了盐碱地花生根层土壤细菌群落与非盐碱地花生根层土壤细菌群落的差异,为揭示盐碱地花生根层土壤微生物的多样性以及土地利用变化与生态环境效应间的关系奠定基础。利用免培养技术直接从土壤样品提取总DNA,针对细菌基因组16S rRNA基因的V3高变区进行PCR扩增;利用焦磷酸测序的方法对V3高变区PCR产物进行高通量测序,并对测序数据进行生物信息学分析。结果表明,(1)黄河三角洲滨海盐碱土较高含盐量土壤中根层土壤微生物种类、优势种群数量和群落功能多样性较非盐碱土壤较为丰富。(2)盐碱土花生平作或花生//棉花间作两种种植方式基本不影响二者0—40cm根层土壤微生物优势类群;不同土壤类型和种植模式下,花生和棉花根层土壤中优势菌群均为变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、绿弯菌门(Chloroflexi)和酸杆菌门(Acidobacteria) 4种菌群,其总丰度为80%—90%。非盐碱土壤中花生根层的酸杆菌门(Acidobacteria)丰度是盐碱土壤中的3倍以上,嗜热油菌纲(Thermoleophilia)和放线菌纲(Actinomycetales)丰度远高于各种盐碱土壤花生平作和花生//棉花间作两种植模式下的花生根层土壤;非盐碱土平作花生0—40cm土层中Rubellimicrobium、Pontibacter和Lamia细菌则显著缺失。(3)土壤类型对土壤微生物菌群类型影响较大,聚类分析表明,10个土壤样本依据土壤含盐量高低和根系分布深度聚为3类,即非盐碱土壤归为1类,盐碱土壤根系密集分布层0—20cm、20—40cm各归为1类。     

Molecular characterization of novel Bradyrhizobium strains nodulating Eriosema chinense and Flemingia vestita,important unexplored native legumes of the sub-Himalayan region (Meghalaya) of India

Root nodule bacterial strains were isolated from the little-studied legumes Eriosema chinense and Flemingia vestita (both in tribe Phaseoleae, Papilionoideae) growing in acidic soil of the sub-Himalayan region of the Indian state of Meghalaya (ME), and were identified as novel strains of Bradyrhizobium on the basis of their 16S rRNA sequences. Seven isolates selected on the basis of phenotypic characters and assessment of ARDRA and RAPD patterns were subjected to multilocus sequence analysis (MLSA) using four protein-coding housekeeping genes (glnII, recA, dnaK and gyrB). On the basis of 16S rRNA phylogeny as well as a concatenated MLSA five strains clustered in a single separate clade and two strains formed novel lineages within the genus Bradyrhizobium. The phylogenies of the symbiotic genes (nodA and nifH) were in agreement with the core gene phylogenies. It appears that genetically diverse Bradyrhizobium strains are the principal microsymbionts of these two important native legumes. The novel genotypes of Bradyrhizobium strains isolated in the present study efficiently nodulate the Phaseoloid crop species Glycine max, Vigna radiata and Vigna umbellata. These strains are genetically different from strains of Bradyrhizobium isolated earlier from a different agro-climatic region of India suggesting that the acidic nature of the soil, high precipitation and other local environmental conditions are responsible for the evolution of these newly-described Bradyrhizobium strains. In global terms, the sub-Himalayan region of India is geographically and climatically distinct and the Bradyrhizobium strains nodulating its legumes appear to be novel and potentially unique to the region.     

高寒特境中甸黄芪植物根际微生物物种多样性及其抗生物膜活性成分

滇西北高寒地区分布着丰富的黄芪属植物资源，该属植物“根际效应”明显，其根际微生物极具抗菌药用资源研究价值。【目的】认知滇西北高寒特境中甸黄芪根际微生物的物种多样性，探究其可培养菌株次生代谢产物的化学多样性及抗菌、抗生物膜活性。【方法】采用宏基因组和微生物纯培养方法对中甸黄芪植物根际微生物进行物种多样性分析，同时采用高效液相色谱（high performance liquid chromatography，HPLC）、超高效液相色谱-质谱联用法（ultra-performance liquid chromatography-mass spectrometry，UPLC-MS）结合“微量肉汤稀释法” “孔板法”等多级联合筛选策略综合评估可培养菌株的抗菌活性药源研究价值。【结果】对中甸黄芪根际土壤样本的微生物分类操作单元（operational taxonomic units，OTU）进行分类注释，得到22门54纲105目187科316属856种微生物，其中优势菌群为慢生根瘤菌属。纯培养共获得27属54种95株可培养菌株，包括20属33种54株细菌和7属21种41株真菌，优势属分别为芽孢杆菌属和青霉属。其中，1株细菌Pseudomonas tolaasii ZTB4和3株真菌Aspergillus tabacinus ZNF17、Lecanicillium aphanocladii ZNF15、Umbelopsis nana ZTF31的次生代谢产物具有广谱抗菌活性。同时，菌株ZTB4和ZNF17的次生代谢产物也显示出优秀的抗耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus，MRSA）生物膜活性，并已验证这2株菌株的主要活性成分分别为环脂肽类与黄酮类。【结论】中甸黄芪植物根际微生物物种多样性较为丰富，其可培养菌株次生代谢产物有较好的化学多样性和抗菌、抗生物膜活性。研究结果为我国特境特色微生物药用资源的开发利用提供理论依据。     

Cultivable Bacterial Community from South China Sea Sponge as Revealed by DGGE Fingerprinting and 16S rDNA Phylogenetic Analysis

The cultivable bacterial communities associated with four South China Sea sponges—Stelletta tenuis, Halichondria rugosa, Dysidea avara, and Craniella australiensis in mixed cultures—were investigated by microbial community DNA-based DGGE fingerprinting and 16S rDNA phylogenetic analysis. Diverse bacteria such as α-, γ-, δ-Proteobacteria, Bacteroidetes, and Firmicutes were cultured, some of which were previously uncultivable bacteria, potential novel strains with less than 95% similarity to their closest relatives and sponge symbionts growing only in the medium with the addition of sponge extract. According to 16S rDNA BLAST analysis, most of the bacteria were cultured from sponge for the first time, although similar phyla of bacteria have been previously recognized. The selective pressure of sponge extract on the cultured bacterial species was suggested, although the effect of sponge extract on bacterial community in high nutrient medium is not significant. Although α- and γ-Proteobacteria appeared to form the majority of the dominant cultivable bacterial communities of the four sponges, the composition of the cultivable bacterial community in the mixed culture was different, depending on the medium and sponge species. Greater bacterial diversity was observed in media C and CS for Stelletta tenuis, in media F and FS for Halichondria rugosa and Craniella australiensis. S. tenuis was found to have the highest cultivable bacterial diversity including α-, γ-, δ-Proteobacteria, Bacteroidetes, and Firmicutes, followed by sponge Dysidea avara without δ-Proteobacteria, sponge Halichondria rugosa with only α-, γ-Proteobacteria and Bacteroidetes, and sponge C. australiensis with only α-, γ-Proteobacteria and Firmicutes. Based on this study, by the strategy of mixed cultivation integrated with microbial community DNA-based DGGE fingerprinting and phylogenetic analysis, the cultivable bacterial community of sponge could be revealed effectively.     

Application of targeted metagenomics to explore abundance and diversity of CO2-fixing bacterial community using cbbL gene from the rhizosphere of Arachis hypogaea

Sequestration of CO₂ by autotrophic bacteria is a key process of biogeochemical carbon cycling in soil ecosystem. Rhizosphere is a rich niche of microbial activity and diversity, influenced by change in atmospheric CO₂. Structural changes in rhizosphere composition influence microbial communities and the nutrient cycling. In the present study, the bacterial diversity and population dynamics were established using cbbL and 16S rRNA gene targeted metagenomics approach from the rhizosphere of Arachis hypogaea. A total of 108 cbbL clones were obtained from the rhizospheric soil which revealed predominance of cbbL sequences affiliated to Rhizobium leguminosarum, Bradyrhizobium sp., Sinorhizobium meliloti, Ochrobactrum anthropi and a variety of uncultured cbbL harboring bacteria. The 16S rRNA gene clone library exhibited the dominance of Firmicutes (34.4%), Proteobacteria (18.3%), Actinobacteria (17.2%) and Bacteroidetes (16.1%). About 43% nucleotide sequences of 16S rRNA gene clone library were novel genera which showed < 95% homology with published sequences. Gene copy number of cbbL and 16S rRNA genes, determined by quantitative real‐time PCR (qRT PCR), was 9.38 ± 0.75 × 10⁷ and 5.43 ± 0.79 × 10⁸ (per g dry soil), respectively. The results exhibited bacterial community structure with high bacterial diversity and abundance of CO₂‐fixing bacteria, which can be explored further for their role in carbon cycling, sustainable agriculture and environment management.     

Indirect effects of polycyclic aromatic hydrocarbon contamination on microbial communities in legume and grass rhizospheres

Background and aims

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) is accelerated in the presence of plants, due to the stimulation of rhizosphere microbes by plant exudates (nonspecific enhancement). However, plants may also recruit specific microbial groups in response to PAH stress (specific enhancement). In this study, plant effects on the development of rhizosphere microbial communities in heterogeneously contaminated soils were assessed for three grasses (ryegrass, red fescue and Yorkshire fog) and four legumes (white clover, chickpea, subterranean clover and red lentil).

Methods

Plants were cultivated using a split-root model with their roots divided between two independent pots containing either uncontaminated soil or PAH-contaminated soil (pyrene or phenanthrene). Microbial community development in the two halves of the rhizosphere was assessed by T-RFLP (bacterial and fungal community) or DGGE (bacterial community), and by 16S rRNA gene tag-pyrosequencing.

Results

In legume rhizospheres, the microbial community structure in the uncontaminated part of the split-root model was significantly influenced by the presence of PAH-contamination in the other part of the root system (indirect effect), but this effect was not seen for grasses. In the contaminated rhizospheres, Verrucomicrobia and Actinobacteria showed increased populations, and there was a dramatic increase in Denitratisoma numbers, suggesting that this genus may be important in rhizoremediation processes.

Conclusion

Our results show that Trifolium and other legumes respond to PAH-contamination stress in a systemic manner, to influence the microbial diversity in their rhizospheres.     

伊犁河流域春季可培养细菌多样性及其胞外酶检测

利用可培养法对新疆伊犁河流域水体和沉积物中细菌多样性进行分析，以期初步阐明流域河流可培养细菌群落结构。采用5种琼脂培养基分离纯化可培养细菌，依据其16S rRNA基因序列进行系统发育分析，并运用平板法对纯化菌株的胞外酶产生情况进行检测。序列分析结果表明，225株细菌分别属于变形菌门γ亚群(Gamma-pseudomonadota, 56.44%)、放线菌门(Actinomycetota, 18.22%)、厚壁菌门(Firmicutes, 14.22%)、变形菌门α亚群(Alpha-pseudomonadota, 4.89%)、变形菌门β亚群(Beta-pseudomonadota, 4%)、拟杆菌门(Bacteroidota, 0.44%)和异常球菌-栖热菌门(Deinococcota, 0.44%)等7个大的系统发育类群，41个属84个种。其中假单胞菌属(Pseudomonas,42.22%)、不动杆菌属(Acinetobacter,9.33%)和芽胞杆菌属(Bacillus,9.33%)为优势菌属。菌种分布结果显示，伊犁河流域主要支流和干流中可培养细菌地域分布性强。分离菌株产胞外酶...     

白马雪山云南黄芪与灰毛康定黄芪根际微生物物种多样性及抗生物膜活性菌株筛选

【背景】细菌生物膜是造成病原菌耐药性增强和持续感染的主要因素,但目前尚无针对抗菌膜的特效药物。特境植物根际微生物可产生大量具有提高宿主免疫功能的活性成分,极具抗生物膜药源开发潜力。【目的】了解滇西北高寒特境白马雪山分布的云南黄芪与灰毛康定黄芪植物根际微生物的物种多样性,并对可培养菌株进行抑菌与抗生物膜活性筛选。【方法】采用宏基因组技术结合传统微生物培养方法,对采自我国云南迪庆藏族自治州德钦县白马雪山的云南黄芪与灰毛康定黄芪的根际微生物进行物种多样性研究,并通过“孔板法”测定其可培养菌株发酵液乙酸乙酯粗浸膏的抗菌、抗生物膜活性。【结果】宏基因组测序结果显示,云南黄芪根际土壤样本中的微生物来自6门7纲8目8科9属10种,其中栖热菌属为优势菌群;灰毛康定黄芪根际土壤样本中的微生物来自6门8纲10目11科14属15种,其中慢生根瘤菌属为优势菌群。通过纯培养共获得145株可培养菌株,包括112株细菌和33株真菌。其中,云南黄芪根际细菌59株,共计16属35种,优势属为假单胞菌属和链霉菌属;根际真菌19株,共计4属5种,优势属为曲霉属;灰毛康定黄芪根际细菌53株,归属于16属29种,优势属为芽孢杆菌属与寡养单胞菌属;根际真菌14株,归属于3属4种,优势属为曲霉属。从不同种水平上选择51株细菌和7株真菌为代表菌株进行抗生素药源评估,发现5株细菌及1株真菌发酵液的乙酸乙酯粗浸膏具有中等至较强的抗革兰阳性菌活性,而且其中4株具有抗MRSA生物膜活性,最终确定了链霉属放线菌Streptomyces fulvissimus KTA1和曲霉属真菌Aspergillus fumigatus YNF5为潜力活性菌株。【结论】首次报道了滇西北地区高寒特境黄芪属植物根际微生物具有较好的物种多样性,而且具有一定的抗生素药用资源开发潜力。本研究对滇西北高寒特境特色植物来源的微生物资源开发利用与保护具有重要的借鉴意义。     

Two Invasive Plants Alter Soil Microbial Community Composition in Serpentine Grasslands

Plant invasions pose a serious threat to native ecosystem structure and function. However, little is known about the potential role that rhizosphere soil microbial communities play in facilitating or resisting the spread of invasive species into native plant communities. The objective of this study was to compare the microbial communities of invasive and native plant rhizospheres in serpentine soils. We compared rhizosphere microbial communities, of two invasive species, Centaurea solstitialis (yellow starthistle) and Aegilops triuncialis (barb goatgrass), with those of five native species that may be competitively affected by these invasive species in the field (Lotus wrangelianus, Hemizonia congesta, Holocarpha virgata, Plantago erecta, and Lasthenia californica). Phospholipid fatty acid analysis (PLFA) was used to compare the rhizosphere microbial communities of invasive and native plants. Correspondence analyses (CA) of PLFA data indicated that despite yearly variation, both starthistle and goatgrass appear to change microbial communities in areas they invade, and that invaded and native microbial communities significantly differ. Additionally, rhizosphere microbial communities in newly invaded areas are more similar to the original native soil communities than are microbial communities in areas that have been invaded for several years. Compared to native plant rhizospheres, starthistle and goatgrass rhizospheres have higher levels of PLFA biomarkers for sulfate reducing bacteria, and goatgrass rhizospheres have higher fatty acid diversity and higher levels of biomarkers for sulfur-oxidizing bacteria, and arbuscular mycorrhizal fungi. Changes in soil microbial community composition induced by plant invasion may affect native plant fitness and/or ecosystem function.     

Effect of Field Inoculation with Sinorhizobium meliloti L33 on the Composition of Bacterial Communities in Rhizospheres of a Target Plant (Medicago sativa) and a Non-Target Plant (Chenopodium album)—Linking of 16S rRNA Gene-Based Single-Strand Conformation Polymorphism Community Profiles to the Diversity of Cultivated Bacteria

Fourteen weeks after field release of luciferase gene-tagged Sinorhizobium meliloti L33 in field plots seeded with Medicago sativa, we found that the inoculant also occurred in bulk soil from noninoculated control plots. In rhizospheres of M. sativa plants, S. meliloti L33 could be detected in noninoculated plots 12 weeks after inoculation, indicating that growth in the rhizosphere preceded spread into bulk soil. To determine whether inoculation affected bacterial diversity, 1,119 bacteria were isolated from the rhizospheres of M. sativa and Chenopodium album, which was the dominant weed in the field plots. Amplified ribosomal DNA restriction analysis (ARDRA) revealed plant-specific fragment size frequencies. Dominant ARDRA groups were identified by 16S rRNA gene nucleotide sequencing. Database comparisons indicated that the rhizospheres contained members of the Proteobacteria (α, β, and γ subgroups), members of the Cytophaga-Flavobacterium group, and gram-positive bacteria with high G+C DNA contents. The levels of many groups were affected by the plant species and, in the case of M. sativa, by inoculation. The most abundant isolates were related to Variovorax sp., Arthrobacter ramosus, and Acinetobacter calcoaceticus. In the rhizosphere of M. sativa, inoculation reduced the numbers of cells of A. calcoaceticus and members of the genus Pseudomonas and increased the number of rhizobia. Cultivation-independent PCR–single-strand conformation polymorphism (SSCP) profiles of a 16S rRNA gene region confirmed the existence of plant-specific rhizosphere communities and the effect of the inoculant. All dominant ARDRA groups except Variovorax species could be detected. On the other hand, the SSCP profiles revealed products which could not be assigned to the dominant cultured isolates, indicating that the bacterial diversity was greater than the diversity suggested by cultivation.     

Rhizosphere-Bacterial Community in <Emphasis Type="Italic">Eperua falcata</Emphasis> (Caesalpiniaceae) a Putative Nitrogen-Fixing Tree from French Guiana Rainforest

The rainforest of French Guiana is still largely unaffected by human activity. Various pristine sites like the Paracou Research Station are devoted to study this tropical ecosystem. We used culture-independent techniques, like polymerase chain reaction-temperature gradient gel electrophoresis, and construction of clone libraries of partial 16S rRNA and nifH genes, to analyze the composition of the bacterial community in the rhizosphere of mature trees of Eperua falcata and Dicorynia guianensis, both species within the Caesalpiniaceae family. E. falcata is one of the more abundant pioneer tree species in this ecosystem and so far, no root nodules have ever been found. However, its nitrogen-fixing status is regarded as “uncertain”, whereas D. guianensis is clearly considered a non-nitrogen-fixing plant. The rhizospheres of these mature trees contain specific bacterial communities, including several currently found uncultured microorganisms. In these communities, there are putative nitrogen-fixing bacteria specifically associated to each tree: D. guianensis harbors several Rhizobium spp. and E. falcata members of the genera Burkholderia and Bradyrhizobium. In addition, nifH sequences in the rhizosphere of the latter tree were very diverse. Retrieved sequences were related to bacteria belonging to the α-, β-, and γ-Proteobacteria in the E. falcata rhizoplane, whereas only two sequences related to γ-Proteobacteria were found in D. guianensis. Differences in the bacterial communities and the abundance and diversity of nifH sequences in E. falcata rhizosphere suggest that this tree could obtain nitrogen through a nonnodulating bacterial interaction.