Amplified 16S rDNA Restriction Analysis Research Articles

Antibiotic gene specific characterization and ARDRA analysis of native isolates of Pseudomonas spp. from Jammu, India

The genus Pseudomonas is well known biocontrol agent established for its diverse role and antagonistic properties against phytopathogens due its ability of producing antibiotics. The studies were conducted to characterize Pseudomonas spp. for antibiotic production on the basis of gene specific primer. 150 isolates of Pseudomonas spp. were isolated from different agricultural fields of Jammu on King’s B medium using plate dilution method. Out of the total, thirty isolates were selected. The antibiotic i.e. 2,4-Diacetylphloroglucinol (2,4-DAPG) extracted from the various isolates showed spots having Rf values in the range of 0.7–0.89 which is very close to the Rf value of synthetic phloroglucinol (0.89). 2,4-DAPG positive isolates were tested against Fusarium oxysporum using dual culture assay and they were found to inhibit the mycelial growth of F. oxysporum by 52.76–65.45%. PCR analysis with 2,4-DAPG gene (phlD) specific primer indicated that a DNA fragment of approximately 750 bp in size was obtained in all the strains except PS 3 and 5 which is closely corroborated with the already reported size of 2,4-DAPG gene in literature. This confirms that 28 isolates out of 30 isolates have the genetic ability for the production of 2,4-DAPG antibiotic and thus these isolates can be promoted as potential biocontrol agents. The genetic diversity of anti-fungal producing rhizobacteria Pseudomonas spp. was investigated using amplified 16S rDNA restriction analysis. 16S rDNA was amplified using PCR technique and digestion with restriction endonuclease HaeIII and dendrogram analysis was carried out by the SHAN clustering UPGMA method (NTSYS 2.1). ARDRA analysis revealed the variability of the Pseudomonas spp. based on the restriction sites and the amplified 16S rDNA distinguished into 18 clusters/types including twelve minor groups and six major groups.

Isolation, characterization and exploring biotechnological potential of halophilic archaea from salterns of western India.

Thirteen halophilic archaea were isolated from Kandla and Bhayander salt pans. These isolates were grouped into three different genera Halobacterium, Haloferax and Haloarcula based on morphological and biochemical characterization, polar lipid analysis, Amplified 16S rDNA restriction analysis (ARDRA) and 16S rDNA sequence analysis. Biochemical characterization suggested the ability of isolates to produce protease, amylase and poly-hydroxybutyrate (PHB) indicating their biotechnological potential. The isolates were further screened for the amount of extracellular protease produced. Halobacterium sp. SP1(1) showed significant protease production compared to other isolates. Protease producing ability of the isolate was influenced by several factors such as NaCl concentration, type of protein source, metal ions and surfactants, and presence of amino acid supplements in the production medium. Soybean flour, FeCl3 and dicotylsulfosuccinate were found to increase protease production by 2.36, 1.54 and 1.26 folds, respectively compared to production in basal medium. Effect of organic solvents used in paints (n-decane, n-undecane and n-dodecane) was also investigated on protease production by the isolate. Protease production by Halobacterium sp. SP1(1) was enhanced by 1.2 folds in presence of n-decane compared to control. Furthermore, the ability of isolate to hydrolyse fish protein was investigated using three different edible fishes (Pomfret, Flat fish and Seer fish) as sole protein source. Pomfret was found to be a good protein source for protease production by the isolate. These results revealed that Halobacterium sp. SP1(1) may have potential for paint-based antifouling coating preparations and fish sauce preparation by virtue of its extracellular protease.

Plant genotype and nitrogen fertilization effects on abundance and diversity of diazotrophic bacteria associated with maize (Zea mays L.)

This research has investigated the effect of two genotypes of maize (Zea mays L.) on the number and diversity of diazotrophic bacteria under different N-fertilization rates. Cultivars NK940 and PAU871 were grown in soil:sand with 0, 40, and 80 kg N ha−1 added as NH4NO3 under controlled conditions for 60 days. The number of diazotrophic bacteria of rhizosphere (S), disinfected roots (R), and stems (T) was determined by most probable number (MPN) method. Diazotrophic bacteria were isolated by N-free media, confirmed by amplification of nifH gene fragments, characterized by amplified 16S rDNA restriction analysis (ARDRA) and (GTG)5 fingerprinting, and identified by partial 16S ribosomal DNA (rDNA) sequence analysis. The diversity of the diazotrophic community from S and R was determined by Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis considering the nifH gene. Maize genotype had a marked effect on the number and diversity of endophytic communities, the NK940 community being more abundant and diverse than that of PAU871. Additionally, N-fertilization increased the number and diversity of diazotrophs in endophytic communities but not in rhizosphere samples. One hundred and six diazotrophs were isolated from S, R, and T samples. Pseudomonas and Enterobacter were the dominant and ubiquitous genera isolated and detected by culture-independent method. T-RFLP showed that the N-fixing populations of the rhizosphere of both cultivars were more diverse than those of inside roots. Principal component analysis (PCA) separated the samples by cultivar and demonstrated a more marked effect of N-fertilization on the NK940 diazotrophic community than on PAU871. These results support the hypothesis that plant genotype and fertilization conditions should be taken into account when searching for N-fixer inocula.

Genotypic diversity in native rhizobial population nodulating Vicia faba in arid and semi-arid regions of Haryana state (India)

A Rhizobium–legume interaction stands out from other plant–microbe interactions as one in which a true developmental mutualism occurs. To study the genotypic diversity in native population of rhizobia-nodulating Vicia faba plants, we retrieved 64 rhizobial isolates from root nodules of faba bean grown in pots holding soils collected from arid and semi-arid regions of the state of Haryana, India. The amplification of nodC in all the isolates authenticated these as rhizobia. The nitrogen-fixing potential of the isolates was tested by the amplification of the nifH gene. Only 50 isolates out of 64 showed nifH gene amplification. The characterization of the isolates by amplified 16S rDNA restriction analysis (ARDRA) categorized these into 36 16S rDNA genotypes using a combination of MspI and HaeIII restriction enzymes. Majority of the isolates resolved into separate genotypes, indicating a wide diversity among them, which seemed to arise from their geographical origin and soil characteristics. These findings may be immensely useful in agriculture towards developing rhizobial inoculants specific for faba beans under arid and semi-arid conditions.

Ferric iron amendment increases Fe(III)-reducing microbial diversity and carbon oxidation in on-site wastewater systems

Onsite wastewater systems, or septic tanks, serve approximately 25% of the United States population; they are therefore a critical component of the total carbon balance for natural water bodies. Septic tanks operate under strictly anaerobic conditions, and fermentation is the dominant process driving carbon transformation. Nitrate, Fe(III), and sulfate reduction may be operating to a limited extent in any given septic tank. Electron acceptor amendments will increase carbon oxidation, but nitrate is toxic and sulfate generates corrosive sulfides, which may damage septic system infrastructure. Fe(III) reducing microorganisms transform all major classes of organic carbon that are dominant in septic wastewater: low molecular weight organic acids, carbohydrate monomers and polymers, and lipids. Fe(III) is not toxic, and the reduction product Fe(II) is minimally disruptive if the starting Fe(III) is added at 50–150mgL−1. We used 14C radiolabeled acetate, lactate, propionate, butyrate, glucose, starch, and oleic acid to demonstrate that short and long-term carbon oxidation is increased when different forms of Fe(III) are amended to septic wastewater. The rates of carbon mineralization to 14CO2 increased 2–5times (relative to unamended systems) in the presence of Fe(III). The extent of mineralization reached 90% for some carbon compounds when Fe(III) was present, compared to levels of 50–60% in the absence of Fe(III). 14CH4 was not generated when Fe(III) was added, demonstrating that this strategy can limit methane emissions from septic systems. Amplified 16S rDNA restriction analysis indicated that unique Fe(III)-reducing microbial communities increased significantly in Fe(III)-amended incubations, with Fe(III)-reducers becoming the dominant microbial community in several incubations. The form of Fe(III) added had a significant impact on the rate and extent of mineralization; ferrihydrite and lepidocrocite were favored as solid phase Fe(III) and chelated Fe(III) (with nitrilotriacetic acid or EDTA) as soluble Fe(III) forms.

Biochemical and molecular characterisation of the bacterial endophytes from native sugarcane varieties of Himalayan region.

Seven endophytic bacterial isolates were finally recovered from native sugarcane varieties at hilly areas namely Berinag, Champawat and Didihat of Uttarakhand state in northern Himalayan region. New isolates and two standard cultures—Azospirillum brasilense and Gluconacetobacter diazotrophicus, were evaluated for their morphological, biochemical and molecular characteristics. Morphologically all were rod shaped, Gram-negative bacteria. Their plant growth promotory properties were also assessed which proved isolates RtBn and StBn as IAA producing. Except isolate StBn, all were phosphate solubilising and except RtBn all produced siderophores. Molecular characterisation of the isolates was performed using amplified 16S r-DNA restriction analysis. Similarity index in unweighted pair group method with arithmetic mean programme clustered the isolates according to their geographical distribution. Native isolates showed insignificant similarity with South American strains used as standards. nifH amplification was observed with all the isolates used in the study which again establish them as potential N-fixers.

Occurrence of Biosurfactant Producing Bacillus spp. in Diverse Habitats.

Diversity among biosurfactant producing Bacillus spp. from diverse habitats was studied among 77 isolates. Cluster analysis based on phenotypic characteristics using unweighted pair-group method with arithmetic averages (UPGMAs) method was performed. Bacillus isolates possessing high surface tension activity and five reference strains were subjected to amplified 16S rDNA restriction analysis (ARDRA). A correlation between the phenotypic and genotypic characterization of Bacillus spp. is explored. Most of the oil reservoir isolates showing high surface activity clustered with B. licheniformis and B. subtilis, the hot water spring isolates clustered in two ingroups, while the petroleum contaminated soil isolates were randomly distributed in all the three ingroups. Present work revealed that diversity exists in distribution of Bacillus spp. from thermal and hydrocarbon containing habitats where majority of organisms belonged to B. licheniformis and B. subtilis group. Isolate B. licheniformis TT42 produced biosurfactant which reduced the surface tension of water from 72 mNm−1 to 28 mNm−1, and 0.05 mNm−1 interfacial tension against crude oil at 80°C. This isolate clustered with B. subtilis and B. licheniformis group on the basis of ARDRA. These findings increase the possibility of exploiting the Bacillus spp. from different habitats and their possible use in oil recovery.

Identification of lactobacilli isolated from food by genotypic methods and MALDI-TOF MS

Lactobacilli are bacteria with important implications in food and feed fermentation. Detailed knowledge of the lactobacilli composition is of high relevance to food and health control, various industrial or biotechnological applications, etc., but accurate identification of the Lactobacillus species is not an easy task. In this study, three methods, i.e. polymerase chain reaction (PCR), amplified 16S rDNA restriction analysis with restrictase MseI (16S-ARDRA), and the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were evaluated for their capabilities to identify Lactobacillus species. After comparison of the three methods on a set of 19 type/reference Lactobacillus strains, 148 strains isolated from dairy and meat products were characterized by PCR and MALDI-TOF MS. The strains were differentiated into nine and ten lactobacilli species by PCR and MALDI-TOF MS, respectively, with nine of these species being congruent. The success rates of species level assignment were 77% for PCR and 93% for MALDI-TOF MS that proved superior in the species identification. However, to differentiate between closely related Lactobacillus species, MALDI-TOF MS needs to be used in combination with genotypic techniques to achieve a more reliable identification.

Molecular and phenotypic characterization of Lactobacillus curvatus isolated from handmade Brazilian salami.

17 Lactobacillus curvatus and one Lactobacillus brevis isolate were obtained from salami. The isolates were previously selected according to the desirable characteristics regarding technological criteria. Identification was based primarily on the biochemical carbohydrate assimilation profile, and later by amplified 16S rDNA restriction analysis (ARDRA). Due to inconsistencies in the results found by the identification methods employed, the 16S rRNA gene was partially sequenced and the result confirmed identity of L. curvatus, as predicted by ARDRA, which shows that this molecular technique is efficient in identifying the species. All isolates of L. curvatus were characterized by the molecular technique of random amplified polymorphic DNA (RAPD). In order to analyze the relationships existing between the isolates, taxonomic dendrograms were obtained based on molecular profile, what was suitable for the typing. The molecular techniques are efficient in the identification of Lactobacillus sp . isolates, which can be complementary or even substitute biochemical techniques. Thus, these molecular methods offer a fast and economic way for the identification and characterization of lactic acid bacterial with a potential use as starter cultures in the elaboration of handmade fermented products. Key words: 16S amplified ribosomal DNA restriction analysis (ARDRA), random amplified polymorphic DNA (RAPD), lactobacilli, microbial genetics.

Inventory of non starter lactic acid bacteria from ripened Parmigiano Reggiano cheese as assessed by a culture dependent multiphasic approach

The objective of this study was to investigate microbial species diversity and strain complexity of the cultivable non starter lactic acid bacteria (NSLAB) occurring in 31 ripened Parmigiano Reggiano (PR) cheeses. Dereplication of 127 lactobacilli isolates by (GTG)5-PCR fingerprinting yielded a total of 51 genotypes. Phylogenetic relatedness of all the genotypes with known Lactobacillus species was determined by a novel combined amplified 16S rDNA restriction analysis (16S-ARDRA), species-specific PCR assays and 16S rRNA gene sequencing. The species Lactobacillus rhamnosus and Lactobacillus paracasei comprise the largest portions of the cultivable NSLAB community in PR cheese, with an inter-individual diversity ranging from one to four dominant genotypes per sample. Lactobacillus casei, Lactobacillus harbinensis and Lactobacillus fermentum species were also detected at low frequency. The data showed differences in cultivable NSLAB population, with an overall decrease in diversity and complexity from early to advanced stages of ripening. Finally the de-replicated collection of genotypes resulting from this work is the bases for further functional screening.

Isolation and screening of phlD + plant growth promoting rhizobacteria antagonistic to Ralstonia solanacearum

Tomato (Lycopersicon esculentum) is important widely grown vegetable in India and its productivity is affected by bacterial wilt disease infection caused by Ralstonia solanacearum. To prevent this disease infection a study was conducted to isolate and screen effective plant growth promoting rhizobacteria (PGPR) antagonistic to R. solanacearum. A total 297 antagonistic bacteria were isolated through dual culture inoculation technique, out of which forty-two antagonistic bacteria were found positive for phlD gene by PCR amplification using two primer sets Phl2a:Phl2b and B2BF:BPR4. The genetic diversity of phlD (+) bacteria was studied by amplified 16S rDNA restriction analysis and demonstrated eleven groups at 65% similarity level. Out of these 42 phlD (+) antagonistic isolates, twenty exhibited significantly fair plant growth promoting activities like phosphate solubilization (0.92-5.33%), 25 produced indole acetic acid (1.63-7.78 μg ml(-1)) and few strains show production of antifungal metabolites (HCN and siderophore). The screening of PGPR (phlD (+)) for suppression of bacterial wilt disease in glass house conditions was showed ten isolated phlD (+) bacteria were able to suppress infection of bacterial wilt disease in tomato plant (var. Arka vikas) in the presence R. solanacearum. The PGPR (phlD (+)) isolates s188, s215 and s288 was observed to be effective plant growth promoter as it shows highest dry weight per plant (3.86, 3.85 and 3.69 g plant(-1) respectively). The complete absence of wilt disease symptoms in tomato crop plants was observed by these treatments compared to negative control. Therefore inoculation of tomato plant with phlD (+) isolate s188 and other similar biocontrol agents may prove to be a positive strategy for checking wilt disease and thus improving plant vigor.

Phylogenetic profiling of culturable bacteria associated with early phase of mushroom composting assessed by amplified rDNA restriction analysis

The edible mushroom Agaricus bisporus is grown commercially on composted manure/straw mixtures. Mushroom composting is a fermentation process in which various groups of microorganisms play important roles at different stages of composting. The present study was conducted to explore the mesophilic bacterial diversity in the early phase of mushroom composting. Morphologically all the isolated bacteria were either Gram-positive rods, cocci or Gram-negative rods. The functional diversity of the bacterial isolates was examined by plate enzyme assays, siderophore production and antagonistic property. Good enzymatic activity for amylase, cellulase, xylanase and protease was reported for different bacterial isolates. Nine bacterial isolates showed siderophore production activity. During antibiosis assay, most of the isolates inhibited growth of Verticillium fungicola and Mycogone perniciosa. However, a consortium of selected bacterial isolates produced good amounts of lytic enzymes (amylase, cellulase, xylanase and protease) in solid state fermentation experiments that might help in enhancing the composting process. Amplified 16S-rDNA restriction analysis (ARDRA) of bacterial isolates indicated that four groups of nine bacterial isolates had 100% similarity in all the restriction profiles. However, other isolates exhibited discriminatory relationships with each other. The present study reveals culturable mesophilic bacterial diversity and community succession in the early phase of mushroom composting process as well as emphasizing the application of a bacterial consortium to enhance the composting process.

Characterization of an anaerobic population digesting a model substrate for maize in the presence of trace metals

The influence of a defined trace metal solution and additionally Ni2+ on anaerobic digestion of biomass was investigated. A novel synthetic model substrate was designed consisting of cellulose, starch and urea as carbon and nitrogen source in a ratio mimicking the basic composition of maize silage. Two independent batch fermentations were carried out over 21d with the synthetic model substrate in the presence of the trace metal solution. Particularly an increase in nickel concentrations (17 and 34μM) enhanced methane formation by up to 20%. This increased activity was also corroborated by fluorescence microscopy measurements based on cofactor F420. The eubacterial and methanogenic population was characterized with the single strand conformational polymorphism analysis and the amplified 16S rDNA restriction analysis of 16S rRNA genes amplified by different primer systems. Nearly the half of the analyzed bacteria were identified as Firmicutes while 70% in this phylum belonged to the class of Clostridiales and 30% to the class of Bacilli. Bacteroides and uncultured bacteria represented each a quarter of the analyzed community. Methanogenic archaea were investigated with ARDRA, too. The hydrogenotrophic Methanoculleus sp. was the dominant genus which is commonly described for maize digestion thus confirming the value of the model substrate.

Effects of intercropping and Rhizobium inoculation on yield and rhizosphere bacterial community of faba bean (Vicia faba L.)

The effects of intercropping with maize and Rhizobium inoculation on the yield of faba bean and rhizosphere bacterial diversity were analyzed by terminal restriction fragment length polymorphism, amplified 16S rDNA restriction analysis (ARDRA), and 16S rDNA sequencing. The results showed that intercropping but not Rhizobium inoculation significantly increased the faba bean yield. Probably the relatively high level of native rhizobia in soil annulled the effect of rhizobia inoculation. ARDRA results showed that intercropping did not affect bacterial diversity whereas Rhizobium inoculation decreased bacterial diversity. The canonical correspondence analysis showed that the composition of bacterial community was changed apparently by intercropping, and there was a positive correlation (P = 0.724) between faba bean yields and intercropping and an apparent correlation (P = 0.648) between intercropping and total N. The available content of K and P had a lower effect on the bacterial community composition than did the total N content, Rhizobium inoculation, and microbial biomass C. Rhizobium inoculation negatively correlated with microbial biomass C (P = −0.827). These results revealed a complex interaction among the intercropped crops, inoculation with rhizobia, and indigenous bacteria and implied that the increase of faba bean production in intercropping might be related to the modification of rhizosphere bacterial community.

Electron shuttle-stimulated RDX mineralization and biological production of 4-nitro-2,4-diazabutanal (NDAB) in RDX-contaminated aquifer material

The potential for extracellular electron shuttles to stimulate RDX biodegradation was investigated with RDX-contaminated aquifer material. Electron shuttling compounds including anthraquinone-2,6-disulfonate (AQDS) and soluble humic substances stimulated RDX mineralization in aquifer sediment. RDX mass-loss was similar in electron shuttle amended and donor-alone treatments; however, the concentrations of nitroso metabolites, in particular TNX, and ring cleavage products (e.g., HCHO, MEDINA, NDAB, and NH(4) (+)) were different in shuttle-amended incubations. Nitroso metabolites accumulated in the absence of electron shuttles (i.e., acetate alone). Most notably, 40-50% of [(14)C]-RDX was mineralized to (14)CO(2) in shuttle-amended incubations. Mineralization in acetate amended or unamended incubations was less than 12% within the same time frame. The primary differences in the presence of electron shuttles were the increased production of NDAB and formaldehyde. NDAB did not further degrade, but formaldehyde was not present at final time points, suggesting that it was the mineralization precursor for Fe(III)-reducing microorganisms. RDX was reduced concurrently with Fe(III) reduction rather than nitrate or sulfate reduction. Amplified 16S rDNA restriction analysis (ARDRA) indicated that unique Fe(III)-reducing microbial communities (β- and γ-proteobacteria) predominated in shuttle-amended incubations. These results demonstrate that indigenous Fe(III)-reducing microorganisms in RDX-contaminated environments utilize extracellular electron shuttles to enhance RDX mineralization. Electron shuttle-mediated RDX mineralization may become an effective in situ option for contaminated environments.

Genetic Diversity of Antifungi-Producing Rhizobacteria of Pseudomonas sp. Isolated from Rhizosphere of Soybean Plant

Antifungi-producing rhizobacteria have been recognized playing an important role in plant disease suppression. In our laboratory, 13 indigenous soybeans' rhizobacteria Pseudomonas sp. that showed strong growth inhibition of root pathogenic fungi, Rhizoctonia solani , Fusarium oxysporum and Sclerotium rolfsii, have been isolated from rhizosphere of soybean plant. For further understanding, the genetic diversity of the antifungi-producing Pseudomonas sp. was investigated using Amplified 16S rDNA Restriction Analysis (ARDRA) and 16S rRNA gene sequences analysis. 16S rDNA were amplified by PCR technique and digested with restriction endonuclease HaeIII, RsaI and AluI. Sequences of 16S rRNA gene were analyzed using the BLAST program for similarity searches on sequence databases. ARDRA based dendrogram analysis was carried out by neighbor-joining of TREECON 1.3b software package. ARDRA indicated the variability of Pseudomonas sp. based on the digestion sites. Dendrogram clustering analysis based on the restriction enzymes profile of the amplified rDNA distinguished Pseudomonas sp. into 7 ribotype groups. The sequences of 16S rRNA gene confirmed that the isolates belonging to Pseudomonas sp. and the phylogenetic tree formed 4 clusters. There was a quite overlap among ARDRA groups and 16S rRNA sequence clusters. This finding suggested that antifungal producing Pseudomonas sp. were present in the rhizosphere of soybean plant and the level of genetic diversity exist within these species. Sequence analysis of the 16S rRNA gene of the Pseudomonas sp. with an identical ARDRA pattern confirmed that members of an ARDRA group were closely related to each other.

Validation of a partialrpoBgene sequence as a tool for phylogenetic identification of aeromonads isolated from environmental sources

A collection of 50 aeromonads isolated from environmental sources were studied, together with all known Aeromonas nomenspecies, by phenotypic, amplified 16S rDNA restriction analysis (16S rDNA RFLP) and by partial sequence alignment of both 16S rDNA and rpoB genes. Although most of the type strain showed a unique phenotypic pattern, a database constructed on type strain phenotype allowed the identification of only 24% of the isolates. Analysis of 16S rDNA RFLP and the rpoB sequence were almost concordant in identifying environmental isolates at the species level, except for strains belonging to Aeromonas caviae spp., which were not differentiated from Aeromonas aquariorum, nor Aeromonas hydrophila susbsp. dhakensis by 16S rDNA RFLP. In addition, rpoB gene analysis clustered separately a group of isolates found in snails within the A. hydrophila species. In contrast to 16S rDNA RFLP and rpoB, the partial 16S rDNA sequence analysis was weak in resolving species identity. Part of these results, phenotypic and phylogenetic data, showed that Aeromonas molluscorum and Aeromonas sharmana are distant from all other Aeromonas species and that the type species of A. hydrophila subsp. anaerogenes is similar to A. caviae and should be considered synonymous.

Robinia pseudoacacia in Poland and Japan is nodulated by Mesorhizobium amorphae strains

Robinia pseudoacacia microsymbionts from plants growing in Poland and Japan were evaluated for phylogeny and taxonomic position by genomic approach. Based on the comparative analyses of atpD (368 bp) and dnaK (573 bp) gene sequences as well as 16S rDNA restriction analysis (RFLP-16S rDNA), R. pseudoacacia microsymbionts were identified as Mesorhizobium strains. In dnaK and atpD gene phylograms R. pseudoacacia nodulators formed robust, monophyletic clusters with Mesorhizobium species with the nucleotide sequence similarity of 91-98% and 90-98%, respectively. The classification of R. pseudoacacia rhizobia to the genus Mesorhizobium was also supported by amplified 16S rDNA restriction analysis. The studied bacteria formed common clusters with Mesorhizobium species, and their DNA patterns were identical or nearly identical to Mesorhizobium genus strains. When DNA-DNA hybridization was performed, the total DNA of the representative R. pseudoacacia rhizobia exhibited 51-75% relatedness to DNA of Mesorhizobium amorphae ICMP15022 strain and below 41% to DNA of other Mesorhizobium species. These results showed that R. pseudoacacia and M. amorphae belong to the same genomospecies. The G+C content of DNA of R. pseudoacacia two microsymbionts was 59.7 and 60.6 mol% compared to 61-64 mol% across M. amorphae strains.

Bacterial community structure and diversity during establishment of an anaerobic bioreactor to treat swine wastewater

An upflow anaerobic sludge blanket (UASB) reactor was successfully established to treat swine wastewater. The bacterial structure and biodiversity of activated sludge in the anaerobic bioreactor were analyzed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and amplified 16S rDNA restriction analysis (ARDRA). The UASB reactor was acclimatized with swine wastewater for a period of 36 days and exhibited an increase in daily COD removal rate up to 90% and methane production up to 9.5 L/day for an influent COD of 3,500 mg/L at the end of the start-up period. The reactor was then run continuously with an influent COD of 3,000-6,000 mg/L in the following two months of steady operation, reaching COD removal rate of 90-95% and methane production of 9.5-13.2 L/day respectively. The results of microbial community analysis showed a diversified and abundant bacterial structure and biodiversity during the start-up period, which then changed insignificantly in the steady operation period. The change patterns of the bacterial population function were similar to those of the bioreactor performance, indicating a close relationship between bacterial community structure and treatment efficiency.

Stress tolerance, genetic analysis and symbiotic properties of root-nodulating bacteria isolated from Mediterranean leguminous shrubs in Central Spain

Nine root-nodulating bacterial isolates were obtained from the leguminous shrubs Spartium junceum, Adenocarpus hispanicus, Cytisus purgans, Cytisus laburnuum, Retama sphaerocarpa and Colutea arborescens in areas of Central Spain. A poliphasic approach analyzing phenotypic, symbiotic and genetic properties was used to study their diversity and characterize them in relation to Mediterranean conditions. Stress tolerance assays revealed marked variations in salinity, extreme pH and cadmium tolerance compared with reference strains, with the majority showing salinity, alkalinity and Cd tolerance and three of them growing at acid pH. Variation within the 16S rRNA gene was examined by amplified 16S rDNA restriction analysis (ARDRA) and direct sequencing to show genetic diversity. Phylogeny confirmed the close relationship of four isolates with Bradyrhizobium canariense, three with Phylobacterium myrsinacearum, one with Rhizobium rhizogenes and another with Mesorhizobium huakuii. The cross inoculation tests revealed wide spectra of nodulation. This is the first report of P. myrsinacearum being able to nodulate these leguminous shrubs, and also the first time reported the association between B.canariense, R. rhizogenes and M. huakuii and C. laburnuum, C. purgans and C. arborescens, respectively. These results suggested that native rhizobia could be suitable candidates as biofertilizers and/or inoculants of leguminous shrubs with restoration or revegetation purposes in Mediterranean areas.