Pantoea beijingensis sp. nov., isolated from the fruiting body of Pleurotus eryngii

Four Gram-negative-staining, facultatively anaerobic bacterial isolates were obtained from a fruiting body of the edible mushroom Pleurotus eryngii showing symptoms of soft rot disease in Beijing, China. Partial 16S rRNA gene sequencing, together with partial rpoB sequencing, placed these isolates in the genus Pantoea. Multilocus sequence analysis based on the partial sequences of gyrB, rpoB, infB and atpD revealed Pantoea dispersa and Pantoea gaviniae as their closest phylogenetic relatives and indicated that these isolates constituted a possible novel species. DNA–DNA hybridization studies confirmed the classification of the new isolates as a novel species and phenotypic tests allowed for differentiation from the closest phylogenetic neighbours. The name Pantoea beijingensis sp. nov. [type strain LMG 27579^T = KCTC 32406^T = JZB2120001^T (deposited at Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences)] is proposed.     

Genotypic characteristics of the causal agent of chinaberry gall

Chinaberry (Melia azedarach) is a deciduous tree of moderate size, grown throughout warm countries. The plantation of M. azedarach tree improves the environmental condition of the country. During the years 1390–1391, bacterial gall symptoms were observed on chinaberry. The isolation of the causal agent bacteria was done on nutrient agar medium. Approximately 4 weeks after inoculation, galls developed on the chinaberries inoculated with different isolates. The specific primer was designed based on 16S rRNA gene and all isolates amplified 429 bp fragment. To sequence 16S rRNA gene of Shiraz isolates, the universal primer PS16Sf-FAM/PS23Sr was used to amplify 16S rRNA gene. The Iranian isolates of Pseudomonas meliae showed 99.7% similarity to P. meliae (type strain) in the Gene Bank. Based on the rep-PCR results and sequence of 16S rRNA gene, Pseudomonas syringae and Pseudomonas fluorescens were placed in the different groups.     

Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses

Background

Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported.

Methods

The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains.

Results

Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed.

Conclusion

Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3–100%. However, the inter-species similarities were relatively low, ranging from 68.7–97.9%. The housekeeping genes rpoB and gyrB1 were demonstrated to be alternative classification markers to the species level based on intra- and inter-species comparisons, whereas based on phylogenetic tree rpoB proved to be reliable phylogenetic marker for the genus Prevotella.     

Frederiksenia canicola gen. nov., sp. nov. isolated from dogs and human dog-bite wounds

Polyphasic analysis was done on 24 strains of Bisgaard taxon 16 from five European countries and mainly isolated from dogs and human dog-bite wounds. The isolates represented a phenotypically and genetically homogenous group within the family Pasteurellaceae. Their phenotypic profile was similar to members of the genus Pasteurella. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry clearly identified taxon 16 and separated it from all other genera of Pasteurellaceae showing a characteristic peak combination. Taxon 16 can be further separated and identified by a RecN protein signature sequence detectable by a specific PCR. In all phylogenetic analyses based on 16S rRNA, rpoB, infB and recN genes, taxon 16 formed a monophyletic branch with intraspecies sequence similarity of at least 99.1, 90.8, 96.8 and 97.2 %, respectively. Taxon 16 showed closest genetic relationship with Bibersteinia trehalosi as to the 16S rRNA gene (95.9 %), the rpoB (89.8 %) and the recN (74.4 %), and with Actinobacillus lignieresii for infB (84.9 %). Predicted genome similarity values based on the recN gene sequences between taxon 16 isolates and the type strains of known genera of Pasteurellaceae were below the genus level. Major whole cell fatty acids for the strain HPA 21^T are C_14:0, C_16:0, C_18:0 and C_16:1 ω7c/C_15:0 iso 2OH. Major respiratory quinones are menaquinone-8, ubiquinone-8 and demethylmenaquinone-8. We propose to classify these organisms as a novel genus and species within the family of Pasteurellaceae named Frederiksenia canicola gen. nov., sp. nov. The type strain is HPA 21^T (= CCUG 62410^T = DSM 25797^T).     

Corynebacterium heidelbergense sp. nov., isolated from the preen glands of Egyptian geese (Alopochen aegyptiacus)

Two strains (pedersoli^T and girotti) of a new species of bacteria were isolated from the preen glands of wild Egyptian geese (Alopochen aegyptiacus) from the river Neckar in southern Germany in two subsequent years. The strains were lipophilic, fastidious, Gram-positive rods and belonged to the genus Corynebacterium. Phylogenetically, the isolates were most closely related to Corynebacterium falsenii DSM 44353^T which has been found to be associated with birds before. 16S rRNA gene sequence similarity to all known Corynebacterium spp. was significantly <97%. Corresponding values of rpoB showed low levels of similarity <87% and ANIb was <73%. G + C content of the genomic DNA was 65.0 mol% for the type strain of the goose isolates, as opposed to 63.2 mol% in Corynebacterium falsenii. MALDI-TOF MS analysis of the whole-cell proteins revealed patterns clearly different from the related species, as did biochemical tests, and polar lipid profiles. We therefore conclude that the avian isolates constitute strains of a new species, for which the name Corynebacterium heidelbergense sp. nov. is proposed. The type strain is pedersoli^T (=DSM 104638^T = LMG 30044^T).     

Genetic Diversity of Rhizobia Nodulating Arachis hypogaea L. in Central Argentinean Soils

The diversity of thirty-nine isolates from peanut plants growing at fourteen different sites in the Argentinean province of Córdoba was examined by rep-PCR, RFLP of PCR amplified 16S rRNA gene and complete sequencing of ribosomal genes. The genomic analysis of the peanut isolates indicated that each group encompasses heterogeneity among their members, having distinct rep fingerprints and 16S rRNA alleles. Complete sequencing of 16S rRNA demonstrated that native peanut rhizobia from Córdoba soils representative of the slow and fast growers are phylogenetically related to Bradyrhizobium japonicum and Bradyrhizobium sp. and Rhizobium giardinii and R. tropici species, respectively. The nodC gene sequence analysis showed phylogenetic similarity between fast grower peanut symbionts and Rhizobium tropici.     

Acinetobacter apis sp. nov., isolated from the intestinal tract of a honey bee,Apis mellifera

A novel Gram-negative, obligate aerobic, non-motile, and both coccobacillus- and bacillus-shaped bacterium, designated strain HYN18^T, was isolated from the intestinal tract of a honey bee (Apis mellifera). The isolate was oxidasenegative and catalase-positive. Strain HYN18^T showed optimum growth at 25°C, pH 6–7, and in the presence of 1% (w/v) NaCl in trypticase soy broth medium. The isolate was negative for hydrolyses of starch, casein, gelatin and urea, indole production from tryptone and hemolysis on sheep blood agar. A phylogenetic analysis based on the 16S rRNA gene and rpoB gene sequence showed that strain HYN18^T was most closely related to Acinetobacter nectaris SAP 763.2^T and A. boissieri SAP 284.1^T with 98.3% and 98.1% similarity (16S rRNA gene), respectively, and 84.4% similarity with Acinetobacter nectaris SAP 763.2^T (rpoB gene). The major cellular fatty acids were summed features 3 (comprising C_16:1ω7c /C_16:1ω6c ), C_12:0 and C_16:0. The main isoprenoid quinone was ubiquinone-9 (Q-9). The polar lipids of strain HYN18^T were phosphatidylethanolamine, three unidentified lipids, an unidentified phospholipid and an unidentified glycolipid. The DNA G+C content was 40.6 mol%. DNADNA hybridization experiments indicated less than 33 ± 10% relatedness to the closest phylogenetic species, Acinetobacter nectaris SAP 763.2^T. Thus, the phenotypic, phylogenetic and genotypic analyses indicate that strain HYN18^T is a novel species within the genus Acinetobacter, for which the name Acinetobacter apis is proposed. The type strain is HYN18^T (=KACC 16906^T =JCM 18575^T).     

Detection of <Emphasis Type="Italic">Lactobacillus</Emphasis> species using a gene fragment of the RNA polymerase beta subunit <Emphasis Type="Italic">rpoB</Emphasis>

In this paper, we report on the comparative analysis of nucleotide sequences of the rpoB gene to assess its potential for use as a genetic marker for identification of Lactibacillus spp. recovered from acid milk products in different regions of Kazakhstan. The discriminatory power of the rpoB gene sequence was shown to be significantly higher for identification of closely related species of the Lactobacillus genus as compared to that of the 16S rRNA gene. The phylogeny based on the rpoB gene proved identical to that of the 16S rRNA gene and could be used as a supplement phylogenetic marker.     

Rhizobium smilacinae sp. nov., an endophytic bacterium isolated from the leaf of Smilacina japonica

During a study of endophytic bacteria from traditional Chinese medicinal plants, a bacterial strain, designated PTYR-5^T, was isolated from the leaf of Smilacina japonica A. Gray collected from Taibai Mountain in Shaanxi Province, north-west China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PTYR-5^T is a member of the genus Rhizobium, exhibiting the highest sequence similarities to R. cellulosilyticum LMG 23642^T (97.2 %), R. huautlense LMG 18254^T (97.2 %) and R. alkalisoli CCBAU 01393^T (97.1 %). The levels of 16S rRNA gene sequence similarity with respect to other Rhizobium species with validly published names were less than 97.0 %. Phylogenies of the housekeeping genes atpD, recA and glnII confirmed its distinct position, showing low similarity with respect to those of recognized Rhizobium species (no more than 94.1?, 90.0 and 88.0 ?    % similarity, respectively). The DNA–DNA relatedness values of strain PTYR-5^T with R. cellulosilyticum LMG 23642^T, R. huautlense LMG 18254^T and R. alkalisoli CCBAU 01393^T were 33.6, 21.4 and 29.5 %, respectively. Based on phenotypic, phylogenetic and genotypic data, strain PTYR-5^T is considered to represent a novel species of the genus Rhizobium, for which the name Rhizobium smilacinae sp. nov. is proposed. The type strain is PTYR-5^T (=CCTCC AB 2013016^T=KCTC 32300^T=LMG 27604^T).     

Phytoremediation of Light Crude Oil by Maize (Zea mays L.) Bio-Augmented with Plant Growth Promoting Bacteria

The aim of this study was to evaluate the converged effect of maize and plant growth promoting bacteria on degradation of petroleum hydrocarbons under axenic conditions. Artificially spiked sand with 10 g kg^?1 light crude oil was planted with maize alone and in combination with eight bacterial isolates having plant growth promotion and bioremediation potential to observe the dissipation of petroleum hydrocarbons. Results showed remarkable suppression of maize growth and biomass production due to phytotoxicity of the crude oil contamination. However, bio-augmentation of plants with bacteria having ACC-deaminase activity significantly compensated the reduction in plant growth compared to uninoculated plants. The results revealed that plants bio-augmented with PM32Y exhibited significant increase in root length (75%), plant height (74%), and biomass (67%) as compared to uninoculated plants after 60 days of planting. The same bacterium in convergence with maize caused 43% degradation of petroleum hydrocarbons as compared to the unplanted and uninoculated control. Amplification, sequencing and phylogenetic analysis of 16S rRNA gene sequence identified PM32Y bacterium as Bacillus subtilis strain. It is concluded that bio-augmentation of plants with plant growth promoting bacteria having bioremediation potential and ACC-deaminase activity can successfully be used in phytoremediation of petroleum hydrocarbons.     

Thalassobius aquaeponti sp. nov., an alphaproteobacterium isolated from seawater

A Gram-stain-negative, aerobic, non-motile and rod-shaped or ovoid bacterial strain, GJSW-22^T, which was isolated from seawater at Geoje island in South Korea, was characterized taxonomically. Strain GJSW-22^T was observed to grow optimally at 30 °C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain GJSW-22^T grouped with the type strains of Thalassobius species, forming a stable cluster with the type strain of Thalassobius aestuarii (bootstrap value of 83.2 %). Strain GJSW-22^T exhibited the highest 16S rRNA gene sequence similarity value (98.0 %) to the type strain of T. aestuarii. It exhibited 16S rRNA gene sequence similarity values of 95.6–96.1 % to the type strains of the other Thalassobius species. Strain GJSW-22^T was found to contain Q-10 as the predominant ubiquinone and C_18:1 ω7c and 11-methyl C_18:1 ω7c as the major fatty acids. The major polar lipids of strain GJSW-22^T were identified as phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid. The DNA G + C content of strain GJSW-22^T is 60.3 mol % and its mean DNA–DNA relatedness value with the type strain of T. aestuarii was 23 %. Differential phenotypic properties, together with the phylogenetic and chemotaxonomic data, confirmed that strain GJSW-22^T is distinct from other Thalassobius species. On the basis of the data presented, strain GJSW-22^T is considered to represent a novel species of the genus Thalassobius, for which the name Thalassobius aquaeponti sp. nov. is proposed. The type strain is GJSW-22^T (=KCTC 42115^T = NBRC 110378^T).     

Application of rpoB sequence similarity analysis, REP-PCR and BOX-PCR for the differentiation of species within the genus Geobacillus

Aim:  To investigate the applicability of rpoB gene, which encodes the β subunit of RNA polymerase, to be used as an alternative to 16S rRNA for sequence similarity analysis in the thermophilic genus Geobacillus. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP‐ and BOX‐polymerase chain reaction) were also used. Methods and Results:  rpoB DNA (458 bp) were amplified from 21 Geobacillus‐ and Bacillus type strains, producing different BOX‐ and REP‐PCR profiles, in addition to 11 thermophilic isolates of Geobacillus and Bacillus species from a Santorini volcano habitat. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The results demonstrated between 90–100% (16S rRNA) and 74–100% (rpoB) similarity among examined bacteria. Conclusion:  BOX‐ and REP‐PCR can be applied for molecular typing within Geobacillus genus. rpoB sequence similarity analysis permits a more accurate discrimination of the species within the Geobacillus genus than the more commonly used 16S rRNA. Significance and Impact of the Study:  The obtained results suggested that rpoB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Geobacillus genus.     

Broad Diversity and Newly Cultured Bacterial Isolates from Enrichment of Pig Feces on Complex Polysaccharides

One of the fascinating functions of mammalian intestinal microbiota is fermentation of plant cell wall components. Eight-week continuous culture enrichments of pig feces with cellulose and xylan/pectin were used to isolate bacteria from this community. A total of 575 bacterial isolates were classified phylogenetically using 16S rRNA gene sequencing. Six phyla were represented in the bacterial isolates: Firmicutes (242), Bacteroidetes (185), Proteobacteria (65), Fusobacteria (55), Actinobacteria (23), and Synergistetes (5). The majority of the bacterial isolates had ≥97 % similarity to cultured bacteria with sequences in the RDP, but 179 isolates represent new species and/or genera. Within the Firmicutes isolates, most were classified in the families of Lachnospiraceae, Enterococcaceae, Staphylococcaceae, and Clostridiaceae I. The majority of the Bacteroidetes were most closely related to Bacteroides thetaiotaomicron, Bacteroides ovatus, and B. xylanisolvens. Many of the Firmicutes and Bacteroidetes isolates were identified as species that possess enzymes that ferment plant cell wall components, and the rest likely support these bacteria. The microbial communities that arose in these enrichment cultures had broad bacterial diversity. With over 30 % of the isolates not represented in culture, there are new opportunities to study genomic and metabolic capacities of these members of the complex intestinal microbiota.     

Diverse cellulolytic bacteria isolated from the high humus, alkaline-saline chinampa soils

Aiming at learning the functional bacterial community in the high humus content, saline-alkaline soils of chinampas, the cellulolytic bacteria were quantified and 100 bacterial isolates were isolated and characterized in the present study. Analysis of 16S-23S IGS (intergenic spacer) RFLP (restriction fragment length polymorphism) grouped the isolates into 48 IGS types and phylogenetic analysis of 16S rRNA genes identified them into 42 phylospecies within 29 genera and higher taxa belonging to the phyla Actinobacteria, Firmicutes and Proteobacteria, dominated by the genera Arthrobacter, Streptomyces, Bacillus, Pseudomonas, Pseudoxanthomonas and Stenotrophomonas. Among these bacteria, 63 isolates represent 26 novel putative species or higher taxa, while 37 were members of 17 defined species according to the phylogenetic relationships of 16S rRNA gene. Except for the novel species, the cellulolytic activity was not reported previously in 9 of the 17 species. They degraded cellulose in medium at pH?4.5–10.0 or supplied with NaCl up to 9 %. In addition, 84.8 and 71.7 % of them degraded xylan and Avicel, respectively. These results greatly improved the knowledge about the diversity of cellulolytic bacteria and demonstrated that the chinampa soils contain diverse and novel cellulolytic bacteria functioning at a wide range of pH and salinity levels, which might be a valuable biotechnological resource for biotransformation of cellulose.     

Nitratireductor shengliensis sp. nov., Isolated from an Oil-Polluted Saline Soil

Two Gram-negative, non-motile, short-rod-shaped bacterial isolates, designated 110399^T and 110248, were isolated from an oil-polluted saline soil in Shengli Oilfield, Eastern China. The two strains shared 99.9 % 16S rRNA gene sequence similarity with the DNA–DNA relatedness value being 80.0 %. They were both capable to grow at 20–40 °C, pH 7–9, and 1–9 % (w/v) NaCl with the optimum growth happened at 30 °C, pH 8, and 2–6 % (w/v) NaCl. The phylogenetic analysis based on 16S rRNA gene sequences revealed that the two strains were members of Nitratireductor and most closely related to Nitratireductor pacificus pht-3B^T and N. basaltis J3^T with the 16S rRNA gene sequence similarities being 97.1 and 97.0 %. The DNA–DNA relatedness between the novel strains and two type strains were below 27 ± 7 %. The strains 110399^T and 110248 also differed from N. pacificus and N. basaltis in nitrate reduction, salt tolerance, enzyme activities, and utilization of carbon sources. The major cellular fatty acids of strain 110399^T were C_19:0ω8c cyclo (10.5 %) and Summed Feature 8 (C_18:1ω7c and/or C_18:1ω6c, 41.5 %) which are typical in the genus Nitratireductor. The predominant ubiquinone was Q-10. The genome DNA G+C content of strain 110399^T and 110248 was 61.1 and 61.7 mol%. On the basis of genetic, phenotypic, and chemotaxonomic analyses, strains 110399^T and 110248 represent a novel species within the genus Nitratireductor, for which the name Nitratireductor shengliensis sp. nov. is proposed. The type strain is 110399^T (=CGMCC 1.12519^T = LMG 27405^T).     

Vibrio madracius sp. nov. Isolated from Madracis decactis (Scleractinia) in St Peter & St Paul Archipelago,Mid-Atlantic Ridge,Brazil

Three novel isolates (A-354^T, A-328, and A-384) were retrieved from apparently healthy scleractinian Madracis decactis in the remote St Peter & St Paul Archipelago, Mid-Atlantic Ridge, Brazil. The novel isolates formed a distinct lineage based on the phylogenetic reconstruction using the 16S rRNA and pyrH gene sequences. They fell into the Mediterranei clade and their closest phylogenetic neighbour was V. mediterranei species, sharing upto 98.1 % 16S rRNA gene sequence similarity. Genomic analysis including in silico DDH, MLSA, AAI and genomic signature distinguished A-354^T from V. mediterranei LMG 19703 (=AK1) with values of 33.3, 94.2, 92 %, and 11.3, respectively. Phenotypically, the novel isolates can be differentiated from V. mediterranei based on the four following features. They do not grow at 8 % NaCl; use d-gluconic acid but not l-galactonic acid lactone as carbon source; and do not have the fatty acid C_18:0. Differentiation from both the other Mediterranei clade species (V. maritimus and V. variabilis) is supported by fifteen features. The novel species show lysine decarboxylase and tryptophan deaminase, but not gelatinase and arginine dihydrolase activity; produce acetoin; use α-d-lactose, N-acetyl-d-galactosamine, myo-Inositol, d-gluconic acid, and β-hydroxy-d,l-butyric acid; and present the fatty acids C_14:0 iso, C_15:0 anteiso, C_16:0 iso, C_17:0 anteiso, and C_17:1x8c . Whole-cell protein profiles, based on MALDI-TOF, showed that the isolates are not clonal and also distinguished them from the closes phylogenetic neighbors. The name Vibrio madracius sp. nov. is proposed to encompass these novel isolates. The G+C content of the type strain A-354^T (=LMG 28124^T=CBAS 482^T) is 44.5 mol%.     

Streptosporangium subfuscum sp. nov., isolated from the rhizosphere of marigold (Tagetes erecta L.)

A Gram-stain positive, filamentous bacterial strain, designated strain NEAU-TWSJ13^T, was isolated from the rhizosphere of a marigold (Tagetes erecta L.) plant collected in Heilongjiang Province, northeast China, and characterized using a polyphasic approach. The strain was observed to form abundant aerial hyphae differentiated into spherical sporangia. 16S rRNA gene sequence similarity studies showed that strain NEAU-TWSJ13^T belongs to the genus Streptosporangium, being most closely related to Streptosporangium fragile DSM 43847^T (98.6 %). Phylogenetic analysis of the 16S rRNA gene sequence indicated that it formed a phyletic line with S. fragile DSM 43847^T, Streptosporangium jomthongense NBRC 110047^T (98.4 % 16S rRNA gene similarity) and Streptosporangium violaceochromogenes DSM 43849^T (97.6 % 16S rRNA gene similarity). A combination of DNA–DNA hybridization results and some phenotypic characteristics indicated that strain NEAU-TWSJ13^T can be distinguished from S. fragile DSM 43847^T and S. jomthongense NBRC 110047^T. Moreover, strain NEAU-TWSJ13^T can also be differentiated from S. violaceochromogenes DSM 43849^T and other Streptosporangium species showing high 16S rRNA gene sequence similarity (>98.0 %) by morphological and physiological characteristics. Therefore, it is proposed that strain NEAU-TWSJ13^T represents a novel species of the genus Streptosporangium, for which the name Streptosporangium subfuscum sp. nov. is proposed. The type strain is NEAU-TWSJ13^T ( = CGMCC 4.7146^T = DSM = 46724^T).     

Identification of the rhizobial symbiont of Astragalus glombiformis in Eastern Morocco as Mesorhizobium camelthorni

Astragalus gombiformis is a desert symbiotic nitrogen-fixing legume of great nutritional value as fodder for camels and goats. However, there are no data published on the rhizobial bacteria that nodulate this wild legume in northern Africa. Thirty-four rhizobial bacteria were isolated from root nodules of A. gombifomis grown in sandy soils of the South-Eastern of Morocco. Twenty-five isolates were able to renodulate their original host and possessed a nodC gene copy. The phenotypic and genotypic characterizations carried out illustrated the diversity of the isolates. Phenotypic analysis showed that isolates used a great number of carbohydrates as sole carbon source. However, although they were isolated from arid sandy soils, the isolates do not tolerate drought stress applied in vitro. The phenotypic diversity corresponded mainly to the diversity in the use of some carbohydrates. The genetic analysis as assessed by repetitive extragenic palindromic (REP)-polymerase chain reaction (PCR) showed that the isolates clustered into 3 groups at a similarity coefficient of 81 %. The nearly-complete 16S rRNA gene sequence from a representative strain of each PCR-group showed they were closely related to members of the genus Mesorhizobium of the family Phyllobactericeae within the Alphaproteobacteria. Sequencing of the housekeeping genes atpD, glnII and recA, and their concatenated phylogenetic analysis, showed they are closely related to Mesorhizobium camelthorni. Sequencing of the symbiotic nodC gene from each strain revealed they had 83.53 % identity with the nodC sequence of the type strain M. camelthorni CCNWXJ 40-4^T.     

Molecular serotyping of Salmonella enterica by complete rpoB gene sequencing

Serotyping has been the gold standard for identifying Salmonella, but it requires large amounts of standard antisera. Multilocus sequence typing (MLST) has been applied to identify Salmonella serovars, but the recombination of 4–7 housekeeping genes and multiple analytic steps diminish its applicability. In the present study, we determined the complete sequences of the RNA polymerase beta subunit gene (rpoB) and 7 housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA) for 76 strains of 33 Salmonella enterica serovars and conducted phylogenetic analyses together with the corresponding gene sequences of 24 reference strains registered in the GenBank database. Based on the phylogenetic analyses, 100 strains from 40 serovars and 91 strains from 37 serovars were classified into 60 rpoB (RST) and 49 multilocus sequence types (ST), respectively. The nucleotide similarities were 98.8–100% and 96.9–100% for the complete rpoB gene and the seven concatenated housekeeping genes, respectively. The strains of 35 and 30 serovars formed serovar-specific branches or clusters in the rpoB and housekeeping gene phylogenetic trees, respectively. Therefore, complete rpoB gene sequencing and phylogenetic analysis may be a useful method for identifying Salmonella serovars that is a simpler, more cost-effective, and less time-consuming alternative or complementary method to MLST and conventional serotyping.