Diversity of nifH gene pools in the rhizosphere of two cultivars of sorghum (Sorghum bicolor) treated with contrasting levels of nitrogen fertilizer.

The diversity of nitrogen-fixing bacteria was assessed in the rhizospheres of two cultivars of sorghum (IS 5322-C and IPA 1011) sown in Cerrado soil amended with two levels of nitrogen fertilizer (12 and 120 kg ha(-1)). The nifH gene was amplified directly from DNA extracted from the rhizospheres, and the PCR products cloned and sequenced. Four clone libraries were generated from the nifH fragments and 245 sequences were obtained. Most of the clones (57%) were closely related to nifH genes of uncultured bacteria. NifH clones affiliated with Azohydromonas spp., Ideonella sp., Rhizobium etli and Bradyrhizobium sp. were found in all libraries. Sequences affiliated with Delftia tsuruhatensis were found in the rhizosphere of both cultivars sown with high levels of nitrogen, while clones affiliated with Methylocystis sp. were detected only in plants sown under low levels of nitrogen. Moreover, clones affiliated with Paenibacillus durus could be found in libraries from the cultivar IS 5322-C sown either in high or low amounts of fertilizer. This study showed that the amount of nitrogen used for fertilization is the overriding determinative factor that influenced the nitrogen-fixing community structures in sorghum rhizospheres cultivated in Cerrado soil.     

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.     

Molecular detection of nifH gene-containing Paenibacillus in the rhizosphere of sorghum (Sorghum bicolor) sown in Cerrado soil

Aims:  To develop a polymerase chain reaction (PCR)-based approach for the detection of nifH gene-containing Paenibacillus in environmental samples.
Methods and Results:  The primers, nifHPAENf and nifHPAENr, were designed and tested with DNA from: (i) strains of different nitrogen-fixing Paenibacillus species, (ii) strains of other nitrogen-fixing genera and (iii) rhizosphere of sorghum sown in Cerrado soil amended with either 12 or 120 kg ha⁻¹ of nitrogen fertilizer. All nitrogen-fixing Paenibacillus strains tested and the DNA samples from rhizosphere soil were amplified when these primers were used, generating a 280 bp fragment. When the PCR products obtained from both sorghum rhizospheres were cloned and sequenced, the majority of the clones analysed could be identified as Paenibacillus durus . Moreover, a greater diversity in the nifH sequences could be observed in the rhizosphere treated with a high amount of nitrogen fertilizer.
Conclusions:  Nitrogen fertilization slightly influenced the structure of the nifH gene-containing Paenibacillus community in sorghum rhizospheres cultivated in Cerrado soil.
Significance and Impact of the Study:  The PCR detection method developed is adequate to assess the presence of nifH gene-containing Paenibacillus in the environment and can be used in future to determine the ecological role of this group of micro-organisms for the nitrogen input to the plants.     

Assessment of the diversity of Paenibacillus species in environmental samples by a novel rpoB-based PCR-DGGE method

A specific PCR system based on the gene encoding the RNA polymerase beta subunit, rpoB, was developed for amplification and denaturing gradient gel electrophoresis (DGGE) fingerprinting of Paenibacillus communities in environmental samples. This gene has been previously proven to be a powerful identification tool for the discrimination of species within the genus Paenibacillus and could avoid the limitations of 16S rRNA-based phylogenetic analysis. Initially, the PCR system based on universal rpoB primers were used to amplify DNAs of different Paenibacillus species. A new reverse primer (rpoBPAEN) was further designed based on an insertion of six nucleotides in the Paenibacillus sequences analyzed. This semi-nested PCR system was evaluated for specificity using DNAs isolated from 27 Paenibacillus species belonging to different 16S rRNA-based phylogenetic groups and seven non-Paenibacillus species. The non-Paenibacillus species were not amplified using this PCR approach and one group of Paenibacillus species consisting of strains without the six-base insert also were not amplified; these latter strains were found to be distinct based on 16S rRNA gene phylogeny. In addition, a clone library was generated from the rpoB fragments amplified from two Brazilian soil types (Cerrado and Forest) and all 62 clones sequenced were closely related to one of the 22 sequences from Paenibacillus previously obtained in this study. To assess the diversity of Paenibacillus species in Cerrado and Forest soils and in the rhizosphere of different cultivars of maize, a PCR-DGGE system was used. The Paenibacillus DGGE fingerprints showed a clear distinction between communities of Paenibacillus in Forest and Cerrado soils and rhizosphere samples clustered along Cerrado soil. Profiles of cultivars CMS22 and CMS36 clustered together, with only 53% of similarity to CMS11 and CMS04. The results presented here demonstrate the potential use of the rpoB-based Paenibacillus-specific PCR-DGGE method for studying the diversity of Paenibacillus populations in natural environments.     

Use of rpoB and 16S rRNA genes to analyse bacterial diversity of a tropical soil using PCR and DGGE

AIM: To evaluate the rpoB gene as a biomarker for PCR-DGGE microbial analyses using soil DNA from the Cerrado, Brazil. METHODS: DNA extraction from soil was followed by Polymerase Chain Reaction (PCR) amplification of rpoB and 16S rRNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare gene/community profiles. RESULTS: The rpoB DGGE profiles comprised fewer bands than the 16S rDNA profiles and were easier to delineate and therefore to analyse. Comparison of the community profiles revealed that the methods were complementary. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The gene for the beta subunit of the RNA polymerase, rpoB, is a single copy gene unlike 16S rDNA. Multiple copies of 16S rRNA genes in bacterial genomes complicate diversity assessments made from DGGE profiles. Using the rpoB gene offers a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE.     

应用rpoB和16S rDNA基因的变性梯度凝胶电泳技术对山羊瘤胃细菌多样性的研究

采用免培养的rpoB和16S rDNA基因的变性梯度凝胶电泳技术(DGGE)对3种山羊(波尔山羊,内蒙古绒山羊,四川南江黄羊)瘤胃细菌优势菌群结构进行了比较分析。研究结果显示rpoBDGGE图谱中条带数目少于16S rDNA图谱,并且条带分离效果明显,更有利于分析瘤胃细菌群落组成。从两种DGGE图谱中均可以发现3种山羊瘤胃细菌具有一定的相似性,种内个体间相似性明显高于种间相似性,这说明寄主品种是影响瘤胃细菌种群构成的一个重要因素。同时进行了部分优势细菌16S rDNA基因V6-V8区序列的系统发育分析。基因序列分析表明,DGGE图谱中优势条带的16S rDNA基因序列中有4条克隆的序列与基因库最相似菌的相似性大于97%,余下的克隆序列相似性在89%～96%之间,其中13条序列的与之相似性最高的序列均来自于未被鉴定的瘤胃细菌。     

Use of rpoB gene analysis for identification of nitrogen-fixing Paenibacillus species as an alternative to the 16S rRNA gene

AIM: To avoid the limitations of 16S rRNA-based phylogenetic analysis for Paenibacillus species, the usefulness of the RNA polymerase beta-subunit encoding gene (rpoB) was investigated as an alternative to the 16S rRNA gene for taxonomic studies. METHODS AND RESULTS: Partial rpoB sequences were generated for the type strains of eight nitrogen-fixing Paenibacillus species. The presence of only one copy of rpoB in the genome of P. graminis strain RSA19(T) was demonstrated by denaturing gradient gel electrophoresis and hybridization assays. A comparative analysis of the sequences of the 16S rRNA and rpoB genes was performed and the eight species showed between 91.6-99.1% (16S rRNA) and 77.9-97.3% (rpoB) similarity, allowing a more accurate discrimination between the different species using the rpoB gene. Finally, 24 isolates from the rhizosphere of different cultivars of maize previously identified as Paenibacillus spp. were assigned correctly to one of the nitrogen-fixing species. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study indicate that rpoB is a powerful identification tool, which can be used for the correct discrimination of the nitrogen-fixing species of agricultural and industrial importance within the genus Paenibacillus.     

Evaluation of the diversity of Paenibacillus polymyxa strains by using the DNA of bacteriophage IPy1 as a probe in hybridization experiments

AIMS: To evaluate the genetic diversity within the species Paenibacillus polymyxa. METHODS: Southern hybridization was performed on 102 strains of P. polymyxa using DNA from the phage IPy1 as a probe. Results: All 102 strains hybridized to phage IPy1 DNA. Data from different hybridization patterns obtained were used to construct a dendrogram in which 53 genotypic groups were split into two main clusters. One cluster contained strains from the rhizospheres of sorghum and maize planted in Cerrado soil, Brazil, and the majority of strains received from two culture collections. The other cluster contained strains isolated from different Brazilian soils and rhizospheres and strains deposited in a third culture collection. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach used in this study appears to be a new and a very useful tool to study the diversity within this species.     

Improvements for comparative analysis of changes in diversity of microbial communities using internal standards in PCR-DGGE

The use of internal standards both during DNA extraction and PCR-DGGE procedure gives the opportunity to analyse the relative abundance of individual species back to the original sample, thereby facilitating relative comparative analysis of diversity. Internal standards were used throughout the DNA extraction and PCR-DGGE to compensate for experimental variability. Such variability causes decreased reproducibility among replicate samples as well as compromise comparisons between samples, since experimental errors cannot be differentiated from actual changes in the community abundance and structure. The use of internal standards during DNA extraction and PCR-DGGE is suitable for ecological and ecotoxicological experiments with microbial communities, where relative changes in the community abundance and structure are studied. We have developed a protocol Internal Standards in Molecular Analysis of Diversity (ISMAD) that is simple to use, inexpensive, rapid to perform and it does not require additional samples to be processed. The internal standard for DNA extraction (ExtrIS) is a fluorescent 510-basepair PCR product which is added to the samples prior to DNA extraction, recovered together with the extracted DNA from the samples and analysed with fluorescence spectrophotometry. The use of ExtrIS during isolation of sample DNA significantly reduced variation among replicate samples. The PCR internal standard (PCR(IS)) originates from the Drosophila melanogaster genome and is a 140-basepair long PCR product, which is amplified by non-competitive primers in the same PCR reaction tubes as the target DNA and analysed together with the target PCR product on the same DGGE gel. The use of PCR(IS) during PCR significantly reduced variation among replicate samples both when assessing total PCR product and when comparing bands representing species on a DGGE gel. The entire ISMAD protocol was shown to accurately describe changes in relative abundance in an environmental sample using PCR-DGGE. It should, however, be mentioned that despite the use of ISMAD some inherent biases still exist in DNA extraction and PCR-DGGE and these should be taken into consideration when interpreting the diversity in a sample based on a DGGE gel.     

硫素对不同大豆品种成熟期AM真菌群落多样性的影响

研究硫素对不同大豆品种成熟期丛枝菌根（arbuscular mycorrhizal,AM）真菌群落多样性的影响,探索有利于提高3个特定大豆品种根围土壤和根系AM真菌多样性的最佳施硫量,为提高大豆产量和改善大豆品质提供理论依据。试验采用盆栽,选用黑农44（HN44）、黑农48（HN48）、黑农37（HN37）3个大豆品种作为试验材料,设4个硫素处理S1（对照）,S2（0.02g/kg）,S3（0.04g/kg）和S4（0.06g/kg）。采用PCR-DGGE技术分析3个大豆品种根围土壤和根系中AM真菌群落多样性。结果表明：在S2处理下HN37和HN44根围土壤和根系AM真菌多样性最高,而在S3处理下HN48根围土壤和根系AM真菌多样性最高;DGGE图谱中各样品优势种群变化显著,球囊霉属Glomus和柄囊霉属Funneliformis真菌为3个大豆品种根围土壤和根系中AM真菌的优势菌群。由此可见,硫素对不同大豆品种根围土壤和根系AM真菌群落多样性有显著影响,适量施硫能够提高大豆根围土壤和根系中AM真菌的多样性,不施或过高施硫反而抑制AM真菌的多样性。     

Medium-term fertilization of grassland plant communities masks plant species-linked effects on soil microbial community structure

According to the singular hypothesis of plant diversity, different plant species are expected to make unique contributions to ecosystem functioning. Hence, individual species would support distinct microbial communities. It was hypothesized that microbial community dynamics in the respective rhizospheres of, two floristically divergent species, Agrostis capillaris and Prunella vulgaris that were dominant in a temperate, upland grassland in northern Greece, would support distinct microbial communities, in agreement to the singular hypothesis. Phospholipid lipid fatty acid (PLFA) profiles of the rhizosphere soil microbial community were obtained from the grassland which had been subjected to factorial nitrogen (N) and phosphorus (P) fertilization over five plant growth seasons. The soil cores analyzed were centered on stands of the two co-occurring target plant species, sampled from five blocks in all four factorial N and P fertilization combinations. Distinct PLFA clustering patterns following principle component analysis of PLFA concentrations revealed that, in the absence of P fertilization, soils under the two plant species supported divergent microbial communities. In the P fertilized plots, however, no such distinction could be observed. Results reveal that nutrient fertilization may mask the ability of plant species to shape their own rhizosphere microbial community.     

Sorghum lysine-rich cultivar verification by SDS-PAGE and Southern blot

The aim of this study was to assess the genetic variability among lysine-rich cultivars of sorghum (IS 21702; CVS B65, G 1058, G205 and CVS 549) and to compare with low lysine cultivar White Martin and a chemically induced high lysine mutant P721O. The lysine-rich cultivars contain approximately 1.5 to 2 times more lysine when compared to low-lysine cultivar. Sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of kafirins showed the absence of both 25.3 kD and 25.9 kD α-kafirins in lysine rich cultivars IS 21702, G 1058 and CV 365 and only the 25.9 kD protein was not present in G 205 compared with a low-lysine cultivar White Martin and with a chemically induced high-lysine mutant P721O. Southern blot analysis with RsaI enzyme gave significantly different banding pattern indicating the absence of 1.0 kb band in lysine-rich cultivars IS 21702, G 1058 and CVS 365 compared to White Martin indicating genetic variability among these cultivars. The detected variability among kafirins both in SDS-PAGE and Southern blot could be effectively used as markers in selection of lysine-rich cultivars for further use in breeding programme.     

Actinobacterial community and diversity in rhizosphere soils of Aquilaria crassna Pierre ex Lec assessed by RT-PCR and PCR-DGGE

The actinobacterial community in rhizospheres of eaglewood (Aquilaria crassna Pierre ex Lec) was analyzed using culture-independent methods of RT-PCR and PCR DGGE of 16S rRNA gene. We conducted the experiments to investigate the difference in diversity and community structure of actinobacteria with respect to sampling sites and seasons and to determine effect of plant species on selection of rhizosphere community from different sampling sites. Total genomic DNA and RNA were extracted from rhizosphere soils collected from two plantations in Phetchabun province and one plantation in each Nakhonnayok province, Rayong province and Chiang Mai province of Thailand during dry and rainy seasons. The UPGMA dendrogram generated from DGGE fingerprints showed that the actinobacterial community was separated corresponding to sampling sites, suggesting sampling sites effect. The shift in community and diversity between two seasons was detected in all sampling sites. RNA-based analyses showed that several actinobacterial groups appeared to be ubiquitous but different in metabolic activity in different environments. Species diversity (S) and simple indexes (I) indicate the increase in species diversity of actinobacteria from all sampling sites in rainy season. Cloning and sequencing of 16S rRNA gene fragments obtained from DGGE bands revealed that 14 of 40 dominant species of actinobacteria in the rhizospheres of this plant belonged to uncultured actinobacteria. Besides the uncultured actinobacteria, Nocardioides sp., Streptomyces sp., Mycobacterium sp., Rhodococcus sp. and Actinoplanes sp. were indentified and frequently found more than other genera.     

Effects of T4 Lysozyme Release from Transgenic Potato Roots on Bacterial Rhizosphere Communities Are Negligible Relative to Natural Factors

Rhizosphere bacterial communities of two transgenic potato lines which produce T4 lysozyme for protection against bacterial infections were analyzed in comparison to communities of wild-type plants and transgenic controls not harboring the lysozyme gene. Rhizosphere samples were taken from young, flowering, and senescent plants at two field sites in three consecutive years. The communities were characterized in a polyphasic approach. Cultivation-dependent methods included heterotrophic plate counts, determination of species composition and diversity based on fatty acid analysis of isolates, and community level catabolic profiling. Cultivation-independent analyses were based on denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from rhizosphere DNA using primers specific for Bacteria, Actinomycetales, or α- or β-Proteobacteria. Several bands of the DGGE patterns were further characterized by sequence analysis. All methods revealed that environmental factors related to season, field site, or year but not to the T4 lysozyme expression of the transgenic plants influenced the rhizosphere communities. For one of the T4 lysozyme-producing cultivars, no deviation in the rhizosphere communities compared to the control lines was observed. For the other, differences were detected at some of the samplings between the rhizosphere community structure and those of one or all other cultivars which were not attributable to T4 lysozyme production but most likely to differences observed in the growth characteristics of this cultivar.     

Identification of nitrogen-fixing Paenibacillus from different plant rhizospheres and a novel nifH gene detected in the P. stellifer

A total of 534 isolates were selectively obtained from different plant rhizospheres based on their growth on nitrogen-free medium and their resistance to 80 degrees C for 15 min. Of the 534 isolates, 23 isolates had nifH gene and exhibited nitrogenase activities. Based on 16S rDNA sequence, G + C content assay and DNA-DNA hybridization, by the 23 isolates, which were divided into four monophyletic clusters, all belonged to the Paenibacillus genus. NifH gene deduced amino acid alignment analysis revealed that cluster I, including 15 isolates, showed the highest NifH identity with Paenibacillus genus; while cluster II identified as P stellifer by DNA-DNA hybridization was consistent with four uncultured bacterial clones. This study suggested that the nitrogen-fixing Paenibacillus were distributed in various ecosystems and prevalent in different plant rhizospheres. It was the first demonstration that nitrogen fixation existed in P. jamilae and P. stellifer. In eight isolates identified as P. stellfer species, a novel nifH gene was detected in Paenibacillus.     

Genotypic diversity effects on biomass production in native perennial bioenergy cropping systems

The perennial grass species that are being developed as biomass feedstock crops harbor extensive genotypic diversity, but the effects of this diversity on biomass production are not well understood. We investigated the effects of genotypic diversity in switchgrass (Panicum virgatum) and big bluestem (Andropogon gerardii) on perennial biomass cropping systems in two experiments conducted over 2008–2014 at a 5.4‐ha fertile field site in northeastern Illinois, USA. We varied levels of switchgrass and big bluestem genotypic diversity using various local and nonlocal cultivars – under low or high species diversity, with or without nitrogen inputs – and quantified establishment, biomass yield, and biomass composition. In one experiment (‘agronomic trial’), we compared three switchgrass cultivars in monoculture to a switchgrass cultivar mixture and three different species mixtures, with or without N fertilization. In another experiment (‘diversity gradient’), we varied diversity levels in switchgrass and big bluestem (1, 2, 4, or 6 cultivars per plot), with one or two species per plot. In both experiments, cultivar mixtures produced yields equivalent to or greater than the best cultivars. In the agronomic trial, the three switchgrass mixture showed the highest production overall, though not significantly different than best cultivar monoculture. In the diversity gradient, genotypic mixtures had one‐third higher biomass production than the average monoculture, and none of the monocultures were significantly higher yielding than the average mixture. Year‐to‐year variation in yields was lowest in the three‐cultivar switchgrass mixtures and Cave‐In‐Rock (the southern Illinois cultivar) and also reduced in the mixture of switchgrass and big bluestem relative to the species monocultures. The effects of genotypic diversity on biomass composition were modest relative to the differences among species and genotypes. Our findings suggest that local genotypes can be included in biomass cropping systems without compromising yields and that genotypic mixtures could help provide high, stable yields of high‐quality biomass feedstocks.     

Denaturing gradient gel electrophoresis and barcoded pyrosequencing reveal unprecedented archaeal diversity in mangrove sediment and rhizosphere samples

Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages.     

Auxin production and detection of the gene coding for the Auxin Efflux Carrier (AEC) protein in <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis>

Different species of Paenibacillus are considered to be plant growth-promoting rhizobacteria (PGPR) due to their ability to repress soil borne pathogens, fix atmospheric nitrogen, induce plant resistance to diseases and/or produce plant growth-regulating substances such as auxins. Although it is known that indole-3-acetic acid (IAA) is the primary naturally occurring auxin excreted by Paenibacillus species, its transport mechanisms (auxin efflux carriers) have not yet been characterized. In this study, the auxin production of P. polymyxa and P. graminis, which are prevalent in the rhizospheres of maize and sorghum sown in Brazil, was evaluated. In addition, the gene encoding the Auxin Efflux Carrier (AEC) protein from P. polymyxa DSM36(T) was sequenced and used to determine if various strains of P. polymyxa and P. graminis possessed this gene. Each of the 68 P. polymyxa strains evaluated in this study was able to produce IAA, which was produced at concentrations varying from 1 to 17 microg/ml. However, auxin production was not detected in any of the 13 P. graminis strains tested in this study. Different primers were designed for the PCR amplification of the gene coding for the AEC in P. polymyxa, and the predicted protein of 319 aa was homologous to AEC from Bacillus amyloliquefaciens, B. licheniformis, and B. subtilis. However, no product was observed when these primers were used to amplify the genomic DNA of seven strains of P. graminis, which suggests that this gene is not present in this species. Moreover, none of the P. graminis genomes tested were homologous to the gene coding for AEC, whereas all of the P. polymyxa genomes evaluated were. This is the first study to demonstrate that the AEC protein is present in P. polymyxa genome.     

Variation of bacterial and fungal community structures in the rhizosphere of hybrid and standard rice cultivars and linkage to CO2 flux

A field experiment was conducted with cultivation of hybrid and conventional cultivars in a rice paddy from China. Rhizosphere soil was sampled and CO(2) flux was measured at tillering (S1), grain filling (S2) and ripening (S3) across the growth stages. Microbial community structure, abundance and activity were analyzed using a combination of functional (enzymes) and denaturing gradient gel electrophoresis (DGGE) and real-time PCR molecular approaches. Invertase and urease activities, total microbial biomass carbon, bacterial 16S rRNA and fungal internal transcribed spacer rRNA gene copies were found to be the highest at S2 under both cultivars, being greater under the hybrid cultivar than under the conventional cultivar across the stages. Moreover, the CO(2) flux was 11%, 16% and 25% higher under the hybrid cultivar than under the conventional cultivar at S1, S2 and S3, respectively. Principal component analyses of the PCR-DGGE profile revealed a significant difference between conventional and hybrid cultivars across growth stages. Sequencing DGGE bands of the bacterial 16S rRNA gene showed that a particular bacterial group of Alphaproteobacteria was enhanced and several distinct operational taxonomic units markedly resembled Ascomycota under the hybrid cultivar. These illustrate a significant selection of a particular group of bacteria and fungi of the hybrid cultivar. However, the potential impacts of these cultivar effects in soil C and N cycling deserve further field studies.     

不同施肥模式对雷竹林土壤真菌群落特征的影响

为探明施肥处理对雷竹林土壤真菌群落特征的影响,采用末端限制性片段长度多态性(T-RFLP)和荧光定量PCR技术,分析有机肥(M)、化肥(CF)、化肥配施有机肥(CFM)、化肥配施有机肥加覆盖(CFMM)及不施肥(CK)处理土壤真菌群落结构和数量特征.结果表明:施肥显著影响真菌群落结构与多样性,表层(0～20 cm)土壤中M、CFMM处理与CK,亚表层(20～40 cm)土壤中CF、CFMM处理与CK之间真菌群落结构均存在明显差异;且表层土壤中CF、CFMM处理真菌Shannon指数和均匀度指数显著低于CK.M、CFM处理表层土壤真菌数量显著高于CK.土壤有机质、全氮、铵态氮和速效钾含量显著影响了真菌群落结构的变异;全氮、铵态氮、硝态氮含量与真菌数量均呈显著正相关.表明雷竹林表层和亚表层土壤中真菌群落对施肥处理的响应存在明显差异,表层土壤真菌群落明显受有机质添加的影响,而亚表层则对化肥投入较为敏感;施肥对真菌群落多样性的影响主要集中在表层土壤.