Effect of Elevated Tropospheric Ozone on the Structure of Bacterial Communities Inhabiting the Rhizosphere of Herbaceous Plants Native to Germany

Current elevated concentrations of ozone in the atmosphere, as they are observed during summer seasons, can cause severe effects on plant vegetation. This study was initiated to analyze whether ozone-stressed plants also transfer signals below ground and thereby alter the bacterial community composition in their rhizospheres. Herbaceous plants, native to Germany, with tolerance (Anthoxanthum odoratum, Achillea millefolium, Poa pratensis, Rumex acetosa, and Veronica chamaedrys) and sensitivity (Matricaria chamomilla, Sonchus asper, and Tanacetum vulgare) to ozone, raised in the greenhouse, were exposed in open-top chambers to two different ozone regimes, i.e., “summer stress” and a normal ozone background. DNA of bacterial cells from the rhizospheres was directly extracted, and partial sequences of the 16S rRNA genes were PCR amplified with primers targeting the following phylogenetic groups: Bacteria, α-Proteobacteria, Actinobacteria, and Pseudomonas, respectively. The diversity of the amplified products was analyzed by genetic profiling based on single-strand conformation polymorphism (SSCP). Neither the tolerant nor the sensitive plants, the latter with visible above-ground damage, showed ozone-induced differences in any of the SSCP profiles, with the single exception of Actinobacteria-targeted profiles from S. asper. To increase the stress, S. asper was germinated and raised in the continuous presence of an elevated level of ozone. SSCP profiles with Bacteria-specific primers combined with gene probe hybridizations indicated an ozone-related increase in a Xanthomonas-related 16S rRNA gene and a decrease in the respective gene from the plant plastids. The fact that only this latter unrealistic scenario caused a detectable effect demonstrated that ozone stress has a surprisingly small effect on the structural diversity of the bacterial community in rhizospheres.     

Succession of Microbial Communities during Hot Composting as Detected by PCR–Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4–V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8–V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of γ-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

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.     

Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of gamma-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

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 (alpha, beta, and gamma 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.     

Effect of primers hybridizing to different evolutionarily conserved regions of the small-subunit rRNA gene in PCR-based microbial community analyses and genetic profiling

Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (Zea mays), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (alpha, beta, and gamma subgroups) and members of the genus Paenibacillus (low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking "universal" primers can affect a PCR-based microbial community analysis.     

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.     

Effect of Primers Hybridizing to Different Evolutionarily Conserved Regions of the Small-Subunit rRNA Gene in PCR-Based Microbial Community Analyses and Genetic Profiling

Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (Zea mays), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (α, β, and γ subgroups) and members of the genus Paenibacillus (low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking “universal” primers can affect a PCR-based microbial community analysis.     

Application of a novel Paenibacillus-specific PCR-DGGE method and sequence analysis to assess the diversity of Paenibacillus spp. in the maize rhizosphere

In this study, a Paenibacillus-specific PCR system, based on the specific primer PAEN515F in combination with bacterial primer R1401, was tested and used to amplify specific fragments of the 16S rRNA gene from rhizosphere DNA. The amplicons were used in a second (semi-nested) PCR for DGGE, in which bacterial primers F968GC and R1401 were used. The resulting products were separated into community fingerprints by DGGE. To assess the reliability of the method, the diversity of Paenibacillus species was evaluated on the basis of DNA extracted directly from the rhizospheres of four different cultivars of maize (Zea mays), i.e. CMS04, CMS11, CMS22 and CMS36, sown in two Brazilian field soils (Cerrado and Várzea). In addition, a clone library was generated from the PCR-generated 16S rDNA fragments, and selected clones were sequenced.The results of the bacterial community analyses showed, at the level of clone libraries, that considerable diversity among Paenibacillus spp. was present. The most dominantly found sequences clustered into 12 groups, each one potentially representing a species complex. Sequences closely affiliated with the P. macerans and P. azotofixans complexes were found in all samples, whereas other sequences were scarcer. Clones affiliated with the latter species complex were most abundant, representing 19% of all clones analysed.The Paenibacillus fingerprints generated via semi-nested PCR followed by DGGE showed a clear distinction between the maize plants grown in Cerrado versus Várzea soils. Thus, soil type, instead of maize cultivar type, was the overriding determinative factor that influenced the community structures of the Paenibacillus communities in the rhizospheres investigated. At a lower level (subcluster), there was a trend for maize cultivars CMS11 and CMS22 on the one hand, and CMS36 and CMS04 on the other hand, to cluster together, indicating that these respective pair of cultivars were similar in their Paenibacillus species composition. This trend was tentatively linked to the growth characteristics of these maize cultivars. These results clearly demonstrated the efficacy of the Paenibacillus-specific PCR-DGGE method in describing Paenibacillus species diversity in rhizosphere soils.     

Genetic profiling of noncultivated bacteria from the rhizospheres of sugar beet (Beta vulgaris) reveal field and annual variability but no effect of a transgenic herbicide resistance

In this field study, we compared the bacterial communities inhabiting the rhizosphere of a transgenic, herbicide-resistant sugar beet (Beta vulgaris) cultivar with those of its nonengineered counterpart, using a genetic profiling technique based on PCR amplifications of partial 16S rRNA gene sequences and single-strand conformation polymorphism (SSCP). As a control for the plasticity of the bacterial community, we also analyzed the influence of herbicides, the field heterogeneity, and the annual variation. DNA was isolated from bacterial cell consortia that were directly collected from root material. PCR was carried out with primers that hybridized to evolutionarily conserved regions flanking variable regions 4 and 5 of the 16S rRNA gene. SSCP patterns of these PCR products were composed of approximately 50 distinguishable bands, as detected by silver staining of the gels after electrophoresis. Patterns of the replicates and the different treatments were highly similar, but digital image and similarity analyses revealed differences that corresponded to the positions of the replicates in the field. In addition, communities collected from sugar beet in two successive growing seasons could be distinguished. In contrast, no effect of the transgenic herbicide resistance was detectable. Sequencing of 24 dominant products of the SSCP profiles indicated the presence of bacteria from different phylogenetic groups, with Proteobacteria and members of the Cytophaga-Flavobacterium-Bacteroides group being most abundant.     

Bacterial diversity in a finished compost and vermicompost: differences revealed by cultivation-independent analyses of PCR-amplified 16S rRNA genes

Bacterial communities are important catalysts in the production of composts. Here, it was analysed whether the diversity of bacteria in finished composts is stable and specific for the production process. Single-strand conformation polymorphism (SSCP) based on polymerase chain reaction amplified partial 16S rRNA genes was used to profile and analyse bacterial communities found in total DNA extracted from finished composts. Different batches of compost samples stored over a period of 12 years and a 1-year-old vermicompost were compared to each other. According to digital image analysis, clear differences could be detected between the profiles from compost and vermicompost. Differences between three different periods of compost storage and between replicate vermicompost windrows were only minor. A total of 41 different 16S rRNA genes were identified from the SSCP profiles by DNA sequencing, with the vast majority related to yet-uncultivated bacteria. Sequences retrieved from compost mainly belonged to the phyla Actinobacteria and Firmicutes. In contrast, vermicompost was dominated by bacteria related to uncultured Chloroflexi, Acidobacteria, Bacteroidetes and Gemmatimonadetes. The differences were underscored with specific gene probes and Southern blot hybridizations. The results confirmed that different substrates and composting processes selected for specific bacterial communities in the finished products. The specificity and consistency of the bacterial communities inhabiting the compost materials suggest that cultivation-independent bacterial community analysis is a potentially useful indicator to characterize the quality of finished composts in regard to production processes and effects of storage conditions.     

Genotypic diversity among rhizospheric bacteria of three legumes assessed by cultivation-dependent and cultivation-independent techniques

The genotypic diversity of rhizospheric bacteria of 3 legumes including Vigna radiata, Arachis hypogaea and Acacia mangium was compared by using cultivation-dependent and cultivation-independent methods. For cultivation-dependent method, Random amplified polymorphic DNA (RAPD) profiles revealed that the bacterial genetic diversity of V. radiata and A. mangium rhizospheres was higher than that of A. hypogaea rhizosphere. For cultivation-independent method, Denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA genes revealed the difference in bacterial community and diversity of rhizospheres collected from 3 legumes. The ribotype richness which indicates species diversity, was highest in V. radiata rhizosphere, followed by A. hypogaea and A. mangium rhizospheres, respectively. Three kinds of media were used to cultivate different target groups of bacteria. The result indicates that the communities of cultivable bacteria in 3 rhizospheres recovered from nutrient agar (NA) medium were mostly different from each other, while Bradyrhizobium selective medium (BJSM) and nitrogen-free medium shaped the communities of cultivable bacteria. Nine isolates grown on BJSM were identified by 16S rRNA gene sequence analysis. These isolates were very closely related (with 96% to 99% identities) to either one of the three groups including Cupriavidus-Ralstonia group, Bacillus group and Bradyrhizobium-Bosea-Afipia group. The rhizospheres were also examined for their enzymatic patterns. Of 19 enzymes tested, 3 rhizospheres were distinguishable by the presence or the absence of leucine acrylamidase and acid phosphatase. The selected cultivable bacteria recovered from NA varied in their abilities to produce indole-acetic acid and ammnonia. The resistance to 10 antibiotics was indistinguishable among bacteria isolated from different rhizospheres.     

Molecular profiling of rhizosphere bacterial communities associated with Prosopis juliflora and Parthenium hysterophorus

Prosopis juliflora and Parthenium hysterophorus are the two arid, exotic weeds of India that are characterized by distinct, profuse growth even in nutritionally poor soils and environmentally stressed conditions. Owing to the exceptional growth nature of these two plants, they are believed to harbor some novel bacterial communities with wide adaptability in their rhizosphere. Hence, in the present study, the bacterial communities associated with the rhizosphere of Prosopis and Parthenium were characterized by clonal 16S rRNA gene sequence analysis. The culturable microbial counts in the rhizosphere of these two plants were higher than bulk soils, possibly influenced by the root exudates of these two plants. The phylogenetic analysis of V1_V2 domains of the 16S rRNA gene indicated a wider range of bacterial communities present in the rhizosphere of these two plants than in bulk soils and the predominant genera included Acidobacteria, Gammaproteobacteria, and Bacteriodetes in the rhizosphere of Prosopis, and Acidobacteria, Betaproteobacteria, and Nitrospirae in the Parthenium rhizosphere. The diversity of bacterial communities was more pronounced in the Parthenium rhizosphere than in the Prosopis rhizosphere. This culture-independent bacterial analysis offered extensive possibilities of unraveling novel microbes in the rhizospheres of Prosopis and Parthenium with genes for diverse functions, which could be exploited for nutrient transformation and stress tolerance in cultivated crops.     

Direct comparison of single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) to characterize a microbial community on the basis of 16S rRNA gene fragments

Characterization of microbial communities using single-strand conformation polymorphism (SSCP) was compared with that using denaturing gradient gel electrophoresis (DGGE). This comparison was based on the V3-4 region (Escherichia coli positions: 341-806) of 16S rRNA gene of bacterial or archaeal communities obtained from a methanogenic bioreactor. Significant differences in the bacterial banding profiles were observed while attempting to detect the diversity of the community and its succession during the reactor operation. The SSCP produced a number of sharp bands and differentiated the bacterial community structures to which the DGGE gave an identical pattern. On the other hand, the SSCP and DGGE provided similar succession patterns for archaeal community.     

Diversity of Peronosporomycete (Oomycete) Communities Associated with the Rhizosphere of Different Plant Species

Peronosporomycete (oomycete) communities inhabiting the rhizospheres of three plant species were characterized and compared to determine whether communities obtained by direct soil DNA extractions (soil communities) differ from those obtained using baiting techniques (bait communities). Using two sets of Peronosporomycete-specific primers, a portion of the 5′ region of the large subunit (28S) rRNA gene was amplified from DNA extracted either directly from rhizosphere soil or from hempseed baits floated for 48 h over rhizosphere soil. Amplicons were cloned, sequenced, and then subjected to phylogenetic and diversity analyses. Both soil and bait communities arising from DNA amplified with a Peronosporomycetidae-biased primer set (Oom1) were dominated by Pythium species. In contrast, communities arising from DNA amplified with a Saprolegniomycetidae-biased primer set (Sap2) were dominated by Aphanomyces species. Neighbor-joining analyses revealed the presence of additional taxa that could not be identified with known Peronosporomycete species represented in GenBank. Sequence diversity and mean sequence divergence ( ) within bait communities were lower than the diversity within soil communities. Furthermore, the composition of Peronosporomycete communities differed among the three fields sampled and between bait and soil communities based on F_st and parsimony tests. The results of our study represent a significant advance in the study of Peronosporomycetes in terrestrial habitats. Our work has shown the utility of culture-independent approaches using 28S rRNA genes to assess the diversity of Peronosporomycete communities in association with plants. It also reveals the presence of potentially new species of Peronosporomycetes in soils and plant rhizospheres.     

Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single-strand-conformation polymorphism.

We describe a new method for studying the structure and diversity of bacterial communities in the natural ecosystem. Our approach is based on single-strand-conformation polymorphism (SSCP) analysis of PCR products of 16S rRNA genes from complex bacterial populations. A pair of eubacterial universal primers for amplification of the variable V3 region were designed from the 16S rRNA sequences of 1,262 bacterial strains. The PCR conditions were optimized by using genomic DNAs from five gram-positive and seven gram-negative strains. The SSCP analysis of the PCR products demonstrated that a bacterial strain generated its characteristic band pattern and that other strains generated other band patterns, so that the relative diversity in bacterial communities could be measured. In addition, this method was sensitive enough to detect a bacterial population that made up less than 1.5% of a bacterial community. The distinctive differences between bacterial populations were observed in an oligotrophic lake and a eutrophic pond in a field study. The method presented here, using combined PCR amplification and SSCP pattern analyses of 16S rRNA genes, provides a useful tool to study bacterial community structures in various ecosystems.     

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。     

A New Approach To Utilize PCR–Single-Strand-Conformation Polymorphism for 16S rRNA Gene-Based Microbial Community Analysis

Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5′ end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR–single-stranded DNA approach for microbial community analysis.     

中国典型冻土区土壤可培养细菌多样性

[目的]对比分析中国典型高纬度冻土区和高海拔冻土区土壤可培养细菌的多样性.[方法]采用NM、TSA 、R2A 3种培养基分离培养不同冻土区土壤可培养细菌,用通用引物扩增分离的细菌16S rRNA基因,根据系统发育分析进行鉴定.[结果]从6个样品中得到冻土土壤可培养细菌的菌落数量为4.70×103 -2.57×105 cfu/g(土壤干重),根据不同的菌落形态分离出144株可培养细菌.纯培养物的16S rRNA基因部分序列分析表明:我国高纬度冻土区土壤样品中的细菌分别属于Firmicutes分支(59.52％)、Gammaproteobacteria 分支(38.10％)、Betaproteobacteria分支(2.38％),其中假单胞菌属(Pseudomonas)、芽胞杆菌属(Bacillus)、类芽胞杆菌属(Paenibacillus)的菌株为该区域的三大优势菌群.我国高海拔冻土区土壤样品中分离细菌属于Gammaproteobacteria分支(89.22％)、Firmicutes分支(8.82％)和Bacteroidetes分支(1.96％)o优势菌群为假单胞菌属( Pseudomonas).[结论]我国高纬度冻土区和高海拔冻土区土壤具有较高的可培养细菌多样性;不同类型冻土区土壤可培养细菌群落组成不同.本文研究结果将为我国冻土区土壤细菌资源研究与利用提供理论依据.     

人工神经原网络处理PCR数据在细菌鉴定中的应用

目的16SrRNA和16S-23SrRNA间区片段是常用细菌分类鉴定靶点,本研究探讨人工神经原网络（ANN）对上述位点PCR扩增产物数据分析在细菌快速鉴定方面的价值。方法2对15SrRNA基因荧光引物和1对16S-23SrRNA区间基因引物用于扩增血液标本中分离出的317株细菌。相关毛细管电泳（CE）限制性片段长度多态性（RFLP）和单链构象多态性（SSCP）数据进行人工神经原网络分析。结果16S-23SrRNA基因的RFLP数据对未知菌鉴定的准确率高于16SrRNA基因的SSCP数据,分别为98．0％和79．6％。结论实验证明了人工神经原网络作为一种模式识别方法对于简化细菌鉴定十分有价值。