Identification of bacteria and fungi inhabiting fruiting bodies of Burgundy truffle (Tuber aestivum Vittad.).

Tuber species may be regarded as complex microhabitats hosting diverse microorganisms inside their fruiting bodies. Here, we investigated the structure of microbial communities inhabiting the gleba of wild growing (in stands) T. aestivum, using Illumina sequencing and culture-based methods. The two methods used in combination allowed to extract more information on complex microbiota of Tuber aestivum gleba. Analysis of the V3-V4 region of 16S rDNA identified nine phyla of bacteria present in the gleba of T. aestivum ascomata, mostly Proteobacteria from the family Bradyrhizobiaceae. Our results ideally match the earlier data for other Tuber species where the family Bradyrhizobiaceae was the most represented. The ITS1 region of fungal rDNA represented six alien fungal species belonging to three phyla. To complement the metagenomic analysis, cultivable fungi and bacteria were obtained from the gleba of the same T. aestivum fruiting bodies. The identified fungi mostly belong to the phylum Basidiomycota and same to Ascomycota. Analysis of cultivable bacteria revealed that all the specimens were colonized by different strains of Bacillus. Fungal community inhabiting T. aestivum fruiting bodies was never shown before.

Bosea caraganae sp. nov. a new species of slow-growing bacteria isolated from root nodules of the relict species Caragana jubata (Pall.) Poir. originating from Mongolia.

Two Gram-stain-negative strains, RCAM04680T and RCAM04685, were isolated from root nodules of the relict legume Caragana jubata (Pall.) Poir. originating from the south-western shore of Lake Khuvsgul (Mongolia). The 16S rRNA gene (rrs) sequencing data showed that these novel isolates belong to the genus Bosea and are phylogenetically closest to the type strains Bosea lathyri LMG 26379T, Bosea vaviloviae LMG 28367T, Bosea massiliensis LMG 26221T and Bosea lupini LMG 26383T (the rrs-similarity levels were 98.7-98.8 %). The recA gene of strain RCAM04680T showed the highest sequence similarity to the type strain B. lupini LMG 26383T (95.4 %), while its atpD gene was closest to that of B. lathyri LMG 26379T (94.4 %). The ITS, dnaK and gyrB sequences of this isolate were most similar to the B. vaviloviae LMG 28367T (86.8 % for ITS, 90.4 % for the other genes). The most abundant fatty acid was C18 : 1ω7c (40.8 %). The whole genomes of strains RCAM04680T and RCAM04685 were identical (100 % average nucleotide identity). The highest average nucleotide identity value (82.8 %) was found between the genome of strain RCAM04680T and B. vaviloviae LMG 28367T. The common nodABC genes required for legume nodulation were absent in both strains; however, some other symbiotic nol, nod, nif and fix genes were detected. Based on the genetic study, as well as analyses of the whole-cell fatty acid compositions and phenotypic properties, a new species, Boseacaraganae sp. nov. (type strain RCAM04680T (=LMG 31125T), is proposed.

Bosea psychrotolerans sp. nov., a psychrotrophic alphaproteobacterium isolated from Lake Michigan water.

Three strains of a Gram-stain negative bacterium were isolated from Lake Michigan water. 16S rRNA gene sequence analysis revealed that strain 1131 had sequence similarities to Bosea vaviloviae LMG 28367T, Bosea lathyri LMG 26379T, Bosea lupini LMG 26383T, Bosea eneae CCUG 43111T, Bosea vestrisii CCUG 43114T and Boseamassiliensis CCUG 43117T of 99.8, 99.1, 98.4, 98.4, 98.4 and 98.2 %, respectively. The average nucleotide identity value between strain 1131T and Bosea vaviloviae Vaf-18T was 93.4 % and the DNA relatedness was 38 %. The primary cellular fatty acids of strain 1131T were C16 : 1ω7c and C18 : 1ω7c. The primary polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The major compound in the quinone system was ubiquinone Q-10 and in the polyamine pattern sym-homospermidine was predominant. Additional phenotypic characteristics included growth at 5-35 °C, pH values of pH 5.5-8.0, a salt tolerance range of 0.0-1.2 % (w/v), and production of an unknown water soluble brown pigment. After phenotypic, chemotaxonomic and genomic analyses, this isolate was identified as a novel species for which the name Bosea psychrotolerans is proposed. The type strain is 1131T (NRRL B-65405=LMG 30034).

The name Bradyrhizobiaceae Garrity et al. 2006 contains Nitrobacter Winogradsky 1892 (Approved Lists 1980), the nomenclatural type of the family Nitrobacteraceae Buchanan 1917 (Approved Lists 1980), is illegitimate and proposals to alter the wording of Rule 54 of the International Code of Nomenclature of Prokaryotes to clarify the fact that the family name Bradyrhizobiaceae Garrity et al. 2006 is replaced by the family name Nitrobacteraceae Buchanan 1917 (Approved Lists 1980) the only correct name.

The new name at the rank of family Bradyrhizobiaceae Garrity et al. 2006 was created to include the genera Afipia Brenner et al. 1992, Agromonas Ohta and Hattori 1985, Blastobacter Zavarzin 1961 (Approved Lists 1980), Bosea Das et al. 1996, Bradyrhizobium Jordan 1982 (the nomenclatural type), Nitrobacter Winogradsky 1892 (Approved Lists 1980), Oligotropha Meyer et al. 1994, Rhodoblastus Imhoff 2001 and Rhodopseudomonas Czurda and Maresch 1937 (Approved Lists 1980). However, Nitrobacter Winogradsky 1892 (Approved Lists 1980) is the nomenclatural type of Nitrobacteraceae Buchanan 1917 (Approved Lists 1980) a name at the rank of family that was validly published prior to the valid publication of Bradyrhizobiaceae Garrity et al. 2006 and has priority. In addition Rule 51b (1) of the International Code of Nomenclature of Prokaryotes rules that under these circumstances Bradyrhizobiaceae Garrity et al. 2006 is an illegitimate name. Illegitimate names may not be used (Rule 51a) and illegitimate names are also not taken into consideration when determining priority (Rule 23a). Nitrobacteraceae Buchanan 1917 (Approved Lists 1980) is the only correct name (Rule 23a). Despite these facts the name Bradyrhizobiaceae Garrity et al. 2006 continues to be used, perhaps because the fact that it is an illegitimate name and the consequences of that status are not fully understood. A revision of Rule 54 would also appear to be appropriate in order to further emphasise the fact that the family name Bradyrhizobiaceae Garrity et al. 2006 must be replaced by the family name Nitrobacteraceae Buchanan 1917 (Approved Lists 1980), which is the oldest legitimate name and is the only correct name that may be used for the taxon that includes Bradyrhizobium Jordan 1982 and Nitrobacter Winogradsky 1892 (Approved Lists 1980).

Diversity of endemic rhizobia on Christmas Island: Implications for agriculture following phosphate mining.

Given that phosphate supplies may diminish and become uneconomic to mine after 2020, there is a compelling need to develop alternative industries to support the population on Christmas Island. Former mine sites could be turned into productive agricultural land, however, large-scale commercial agriculture has never been attempted, and, given the uniqueness of the island, the diversity of rhizobia prior to introducing legumes needed evaluation. Therefore, 84 rhizobia isolates were obtained from nine different hosts, both crop and introduced legumes, located at seven sites across the island. Based on 16S rRNA and recA gene sequence analysis, the isolates grouped into 13 clades clustering within the genus Bradyrhizobium, Ensifer, Cupriavidus and Rhizobium. According to the sequences of their symbiosis genes nodC and nifH, the isolates were classified into 12 and 11 clades, respectively, and clustered closest to tropical or crop legume isolates. Moreover, the symbiosis gene phylogeny and Multi Locus Sequence Analysis gene phylogeny suggested vertical transmission in the Alpha-rhizobia but horizontal transmission within the Beta-rhizobia. Furthermore, this study provides evidence of a large diversity of endemic rhizobia associated with both crop and introduced legumes, and highlights the necessity of inoculation for common bean, chickpea and soybean on the Island.

Diversity and functions of bacterial community in drinking water biofilms revealed by high-throughput sequencing.

The development of biofilms in drinking water (DW) systems may cause various problems to water quality. To investigate the community structure of biofilms on different pipe materials and the global/specific metabolic functions of DW biofilms, PCR-based 454 pyrosequencing data for 16S rRNA genes and Illumina metagenomic data were generated and analysed. Considerable differences in bacterial diversity and taxonomic structure were identified between biofilms formed on stainless steel and biofilms formed on plastics, indicating that the metallic materials facilitate the formation of higher diversity biofilms. Moreover, variations in several dominant genera were observed during biofilm formation. Based on PCA analysis, the global functions in the DW biofilms were similar to other DW metagenomes. Beyond the global functions, the occurrences and abundances of specific protective genes involved in the glutathione metabolism, the SoxRS system, the OxyR system, RpoS regulated genes, and the production/degradation of extracellular polymeric substances were also evaluated. A near-complete and low-contamination draft genome was constructed from the metagenome of the DW biofilm, based on the coverage and tetranucleotide frequencies, and identified as a Bradyrhizobiaceae-like bacterium according to a phylogenetic analysis. Our findings provide new insight into DW biofilms, especially in terms of their metabolic functions.

Biosorption of Cr(VI) ions from aqueous solutions by a newly isolated Bosea sp. strain Zer-1 from soil samples of a refuse processing plant.

The adsorption behavior of Cr(VI) ions from aqueous solution by a chromium-tolerant strain was studied through batch experiments. An isolate designated Zer-1 was identified as a species of Bosea on the basis of 16S rRNA results. It showed a maximum resistance to 550 mg·L(-1) Cr(VI). The effects of 3 important operating parameters, initial solution pH, initial Cr(VI) concentration, and biomass dose, were investigated by central composite design. On the basis of response surface methodology results, maximal removal efficiency of Cr(VI) was achieved under the following conditions: pH, 2.0; initial concentration of metal ions, 55 mg·L(-1); and biomass dose, 2.0 g·L(-1). Under the optimal conditions, the maximum removal efficiency of Cr(VI) ions was found to be nearly 98%. The experimental data exhibited a better fit with the Langmuir model than the Freundlich model. The biosorption mechanisms were investigated with pseudo-first-order, pseudo-second-order, and intraparticle diffusion kinetics models. These results revealed that biosorption of Cr(VI) onto bacterial biomass could be an alternative method for the removal of metal ions from aqueous solution.

Extra-slow-growing Tardiphaga strains isolated from nodules of Vavilovia formosa (Stev.) Fed.

Eleven extra-slow-growing strains were isolated from nodules of the relict legume Vavilovia formosa growing in North Ossetia (Caucasus) and Armenia. All isolates formed a single rrs cluster together with the type strain Tardiphaga robiniae LMG 26467(T), while the sequencing of the 16S-23S rDNA intergenic region (ITS) and housekeeping genes glnII, atpD, dnaK, gyrB, recA and rpoB divided them into three groups. North Ossetian isolates (in contrast to the Armenian ones) were clustered separately from the type strain LMG 26467(T). However, all isolates were classified as T. robiniae because the DNA-DNA relatedness between them and the type strain LMG 26467(T) was 69.6% minimum. Two symbiosis-related genes (nodM and nodT) were amplified in all isolated Tardiphaga strains. It was shown that the nodM gene phylogeny is similar to that of ITS and housekeeping genes. The presence of the other symbiosis-related genes in described Tardiphaga strains, which is recently described genus of rhizobia, as well as their ability to form nodules on any plants are under investigation.

Metagenome sequence of Elaphomyces granulatus from sporocarp tissue reveals Ascomycota ectomycorrhizal fingerprints of genome expansion and a Proteobacteria-rich microbiome.

Many obligate symbiotic fungi are difficult to maintain in culture, and there is a growing need for alternative approaches to obtaining tissue and subsequent genomic assemblies from such species. In this study, the genome of Elaphomyces granulatus was sequenced from sporocarp tissue. The genome assembly remains on many contigs, but gene space is estimated to be mostly complete. Phylogenetic analyses revealed that the Elaphomyces lineage is most closely related to Talaromyces and Trichocomaceae s.s. The genome of E. granulatus is reduced in carbohydrate-active enzymes, despite a large expansion in genome size, both of which are consistent with what is seen in Tuber melanosporum, the other sequenced ectomycorrhizal ascomycete. A large number of transposable elements are predicted in the E. granulatus genome, especially Gypsy-like long terminal repeats, and there has also been an expansion in helicases. The metagenome is a complex community dominated by bacteria in Bradyrhizobiaceae, and there is evidence to suggest that the community may be reduced in functional capacity as estimated by KEGG pathways. Through the sequencing of sporocarp tissue, this study has provided insights into Elaphomyces phylogenetics, genomics, metagenomics and the evolution of the ectomycorrhizal association.

Bosea vaviloviae sp. nov., a new species of slow-growing rhizobia isolated from nodules of the relict species Vavilovia formosa (Stev.) Fed.

The Gram-negative, rod-shaped slow-growing strains Vaf-17, Vaf-18(T) and Vaf-43 were isolated from the nodules of Vavilovia formosa plants growing in the hard-to-reach mountainous region of the North Ossetian State Natural Reserve (north Caucasus, Russian Federation). The sequencing of 16S rDNA (rrs), ITS region and five housekeeping genes (atpD, dnaK, recA, gyrB and rpoB) showed that the isolated strains were most closely related to the species Bosea lathyri (class Alphaproteobacteria, family Bradyrhizobiaceae) which was described for isolates from root nodules of Lathyrus latifolius. However the sequence similarity between the isolated strains and the type strain B. lathyri LMG 26379(T) for the ITS region was 90 % and for the housekeeping genes it was ranged from 92 to 95 %. All phylogenetic trees, except for the rrs-dendrogram showed that the isolates from V. formosa formed well-separated clusters within the Bosea group. Differences in phenotypic properties of the B. lathyri type strain and the isolates from V. formosa were studied using the microassay system GENIII MicroPlate BioLog. Whole-cell fatty acid analysis showed that the strains Vaf-17, Vaf-18(T) and Vaf-43 had notable amounts of C16:0 (4.8-6.0 %), C16:0 3-OH (6.4-6.6 %), C16:1 ω5c (8.8-9.0 %), C17:0 cyclo (13.5-13.9 %), C18:1 ω7c (43.4-45.4 %), C19:0 cyclo ω8c (10.5-12.6 %) and Summed Feature (SF) 3 (6.4-8.0 %). The DNA-DNA relatedness between the strains Vaf-18(T) and B. lathyri LMG 26379(T) was 24.0 %. On the basis of genotypic and phenotypic analysis a new species Bosea vaviloviae sp. nov. (type strain RCAM 02129(T) = LMG 28367(T) = Vaf-18(T)) is proposed.

Genetic diversity of rhizobia isolated from nodules of the relic species Vavilovia formosa (Stev.) Fed.

Sixteen bacterial strains were isolated from root nodules of Vavilovia formosa plants originated from the North Ossetian State Natural Reserve (Caucasus, Russia). Phylogenetic analysis of these strains was performed using partial 16S rRNA gene and internally transcribed spacer (ITS) sequences. The results showed that the isolates belong to three families of root nodule bacteria. Twelve of them were related to the genus Rhizobium (family Rhizobiaceae) but four strains can be most probably identified as Phyllobacterium-related (family Phyllobacteriaceae), Bosea- and Rhodopseudomonas-related (family Bradyrhizobiaceae). Amplified fragment length polymorphism clustering was congruent with ITS phylogeny but displayed more variability for Rhizobium isolates, which formed a single group at the level of 30 % similarity. We expect that the isolates obtained can belong to new taxa at genus, species or subspecies levels. The results of PCR amplification of the nodulation genes nodC and nodX showed their presence in all Rhizobium isolates and one Rhodopseudomonas-related isolate. The nodC gene sequences of V. formosa isolates were closely related to those of the species Rhizobium leguminosarum bv. viciae but formed separate clusters and did not intermingle with any reference strains. The presence of the nodX gene, which is necessary for nodulation of Afghan peas (Pisum sativum L.) originated from the Middle East, allows the speculation that these wild-type pea cultivars may be the closest existing relatives of V. formosa. Thus, the studies of genetic diversity and symbiotic genes of V. formosa microsymbionts provide the primary information about their phylogeny and contribute to the conservation of this relict leguminous species.

[Identification of a new heterotrophic nitrobacterium strain Colloides sp. JZ1-1 and its nitrifying capability].

A heterotrophic nitrobacterium strain JZ1-1 with higher nitrifying capability was isolated and mutagenized from an acclimated activated sludge. The JZ1-1 was identified as Colloides sp., according to its morphological and physiological features. The factors affecting the nitrifying capability of JZ1-1 were investigated, including medium carbon source, C/N ratio, pH value, dissolved oxygen, temperature, and ammonium nitrogen concentration. The optimal carbon source was sodium citrate, and the nitrification was favored when the C/N ratio was from 10 to 14, temperature was 30 degrees C, and pH value was 6-9 when cultured at a rotating speed of more than 150 r x min(-1). JZ1-1 could degrade ammonium nitrogen effectively when the initial concentration of ammonium nitrogen was from 100 mg x L(-1) to 500 mg x L(-1). JZ1-1 was stable after 5 generations of subculture.

[Community structure and spatial distribution of anaerobic ammonium oxidation bacteria in the sediments of Chongming eastern tidal flat in summer].

The objectives of this study were to identify whether there were Anaerobic Ammonium Oxidation (ANAMMOX) bacteria in the surface sediments of Chongming eastern tidal flat in the Yangtze estuary and the feature of their community structure and spatial distribution. Based on the total DNA extracted from the surface sediments of Chongming eastern tidal flat, ANAMMOX-specific 16S rDNA fragments were amplified. PCR products were cloned and sequenced, and an ANAMMOX-specific 16S rDNA gene library was established. Phylogenetic tree was constructed using MEGA5 after the sequences were checked in the GenBank database. Phylogenetic analysis indicated that the clone sequences CM-L-7 and CM-L-18 had 98% identities with ANAMMOX bacteria Candidatus 'Scalindua sp.'. CM-L-13 had 94% identities with Candidatus 'Scalindua wagneri'. CM-M-6 had 94% identities with Candidatus 'Kuenenia sp.'. CM-M-22 had 95% identities with Anaerobic ammonium-oxidizing planctomycete JMK-1. CM-H-15 had 94% identities with Candidatus 'Kuenenia stuttgartiensis'. The results indicated that there were ANAMMOX bacteria in the surface sediments of Chongming eastern tidal flat, but the ANAMMOX species were diverse in different tidal flats: Candidatus 'Scalindua' was the predominant group in the low tidal flat, while Candidatus 'Kuenenia' was the major population in the high tidal flat and the middle tidal flat. In comparison with the high and low tidal flats, the community structure of ANAMMOX bacteria in the middle tidal flat was the most complicated. A portion of the sequences related to uncultivated bacteria outside the known ANAMMOX cluster, probably indicated that there were potential ANAMMOX bacteria in the sediments of Chongming eastern tidal flat.

Tardiphaga robiniae gen. nov., sp. nov., a new genus in the family Bradyrhizobiaceae isolated from Robinia pseudoacacia in Flanders (Belgium).

Gram-negative, rod-shaped bacteria were isolated from Robinia pseudoacacia root nodules. On the basis of the 16S rRNA gene phylogeny, they are closely related to Bradyrhizobium, Rhodopseudomonas and Nitrobacter species (97% sequence similarity), belonging to the class Alphaproteobacteria and family Bradyrhizobiaceae. The results of physiological and biochemical tests together with sequence analysis of housekeeping genes (atpD, dnaK, gyrB, recA and rpoB) allowed differentiation of this group from other validly published Bradyrhizobiaceae genera. NodA, nodC and nifH genes could not be amplified. On the basis of genotypic and phenotypic data, these organisms represent a novel genus and species for which the name Tardiphaga robiniae gen. nov., sp. nov. (LMG 26467(T)=CCUG 61473(T)), is proposed.

Phylogeny and photoheterotrophy in the acidophilic phototrophic purple bacterium Rhodoblastus acidophilus.

Norbert Pfennig isolated the first acidophilic purple bacterium over 40 years ago and named the organism Rhodopseudomonas acidophila (now Rhodoblastusacidophilus). Since the original work of Pfennig, no systematic study has been conducted on the phylogeny and carbon nutrition of a collection of strains of Rbl. acidophilus. We have isolated six new strains of Rbl. acidophilus from a Canadian peat bog. These strains, three of the original Pfennig strains and two additional putative R. acidophilus strains isolated several years ago in this laboratory,were characterized as to their pigments, phylogeny, and carbon sources supporting photoheterotrophic growth. Phototrophic cultures were either purple or orange in color,and the color of a particular strain was linked to phylogeny. As for the Pfennig strains of Rbl. acidophilus, all new strains grew photoheterotrophically at pH 5 on a variety of organic and fatty acids. However, in addition to methanol and ethanol, the new strains as well as the Pfennig strains grew on several other primary alcohols, results not reported in the original species description. Our work shows that some phylogenetic and physiological diversity exists within the species Rbl. acidophilus and supports the observation that few species of acidophilic purple bacteria appear to exist in nature.

Multilocus sequence analysis of Bosea species and description of Bosea lupini sp. nov., Bosea lathyri sp. nov. and Bosea robiniae sp. nov., isolated from legumes.

Gram-negative, rod-shaped bacteria were isolated from root nodules of Lupinus polyphyllus, Lathyrus latifolius and Robinia pseudoacacia. Based on the 16S rRNA gene phylogeny, they were closely related to Bosea species (100-97 % similarity), belonging to the class Alphaproteobacteria, family Bradyrhizobiaceae. The closest relatives of LMG 26383(T), LMG 26379(T) and LMG 26381(T) were respectively the type strains of Bosea thiooxidans (99.6 %), B. eneae (98.3 %) and B. minatitlanensis (99.0 %). Chemotaxonomic data, including major fatty acid profiles, supported the assignment of our strains to the genus Bosea. Analysis of the concatenated sequences of five housekeeping genes (atpD, dnaK, gyrB, recA and rpoB) and the results of DNA-DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of our strains from each other and from the five Bosea species with validly published names. No nodA or nodC genes could be amplified, while nifH PCR gave non-specific products. On the basis of genotypic and phenotypic data, three novel species, Bosea lupini sp. nov. (type strain LMG 26383(T)  = CCUG 61248(T)  = R-45681(T)), Bosea lathyri sp. nov. (type strain LMG 26379(T)  = CCUG 61247(T)  = R-46060(T)) and Bosea robiniae sp. nov. (type strain LMG 26381(T)  = CCUG 61249(T)  = R-46070(T)), are proposed.

Rhizobium rosettiformans sp. nov., isolated from a hexachlorocyclohexane dump site, and reclassification of Blastobacter aggregatus Hirsch and Muller 1986 as Rhizobium aggregatum comb. nov.

A Gram-negative, rod-shaped, motile, aerobic bacterial strain, W3(T), was isolated from hexachlorocyclohexane (HCH)-contaminated groundwater from Lucknow, India, and its taxonomic position was determined using a polyphasic approach. Strain W3(T) shared highest 16S rRNA gene sequence similarity of 97.8 % with Rhizobium selenitireducens B1(T), followed by Rhizobium daejeonense L61(T) (97.7 %), Rhizobium radiobacter ATCC 19358(T) (97.5 %) and Blastobacter aggregatus IFAM 1003(T) (97.2 %). Strain W3(T) formed a monophyletic clade with Blastobacter aggregatus IFAM 1003(T) ( = DSM 1111(T)) in the cluster of species of the genus Rhizobium. Phylogenetic analyses of strain W3(T) using atpD and recA gene sequences confirmed the phylogenetic arrangements obtained by using 16S rRNA gene sequences. Hence, the taxonomic characterization of B. aggregatus DSM 1111(T) was also undertaken. Strains W3(T) and B. aggregatus DSM 1111(T) contained summed feature 8 (18 : 1ω7c and/or 18 : 1ω6c; 65.4 and 70.8 %, respectively) as the major fatty acid, characteristic of the genus Rhizobium. DNA-DNA relatedness of strain W3(T) with Rhizobium selenitireducens LMG 24075(T), Rhizobium daejeonense DSM 17795(T), Rhizobium radiobacter DSM 30147(T) and B. aggregatus DSM 1111(T) was 42, 34, 30 and 34 %, respectively. The DNA G+C contents of strain W3(T) and B. aggregatus DSM 1111(T) were 62.3 and 62.7 mol%, respectively. A nifH gene encoding dinitrogenase reductase was detected in strain W3(T) but not in B. aggregatus DSM 1111(T). Based on the results obtained by phylogenetic and chemotaxonomic analyses, phenotypic characterization and DNA-DNA hybridization, it is concluded that strain W3(T) represents a novel species of the genus Rhizobium, for which the name Rhizobium rosettiformans sp. nov. is proposed (type strain W3(T)  = CCM 7583(T)  = MTCC 9454(T)). It is also proposed that Blastobacter aggregatus Hirsch and Müller 1986 be transferred to the genus Rhizobium as Rhizobium aggregatum comb. nov. (type strain IFAM 1003(T)  = DSM 1111(T)  = ATCC 43293(T)).

Heavy metal tolerant Metalliresistens boonkerdii gen. nov., sp. nov., a new genus in the family Bradyrhizobiaceae isolated from soil in Thailand.

Bacterial strains from inoculated soybean field soil in Thailand were directly isolated using Bradyrhizobium japonicum selective medium (BJSM), on the basis of Zn(2+) and Co(2+) resistance of B. japonicum and B. elkanii. The isolates were classified into symbiotic and non-symbiotic groups by inoculation assays and Southern hybridization of nod and nif genes. In this study, a nearly full-length 16S rRNA gene sequence showed that the non-symbiotic isolates were more closely related to members of Rhodopseudomonas and to a number of uncultured bacterial clones than to members of Bradyrhizobium. Therefore, a polyphasic study was performed to determine the taxonomic positions of four representatives of the non-symbiotic isolates. Multilocus phylogenetic analysis of individual genes and a combination of the 16S rRNA and three housekeeping genes (atpD, recA and glnII) supported the placement of the non-symbiotic isolates in a different genus. The ability of heavy metal resistance in conjunction with phenotypic analyses, including cellular fatty acid content and biochemical characteristics, showed that the non-symbiotic isolates were differentiated from the other related genera in the family Bradyrhizobiaceae. Therefore, the non-symbiotic isolates represented a novel genus and species, for which the name Metalliresistens boonkerdii gen. nov., sp. nov. is proposed. The type strain is NS23 (= NBRC 106595(T)=BCC 40155(T)).

Diversity analyses of Aeschynomene symbionts in Tropical Africa and Central America reveal that nod-independent stem nodulation is not restricted to photosynthetic bradyrhizobia.

Tropical aquatic legumes of the genus Aeschynomene are unique in that they can be stem-nodulated by photosynthetic bradyrhizobia. Moreover, a recent study demonstrated that two Aeschynomene indica symbionts lack canonical nod genes, thereby raising questions about the distribution of such atypical symbioses among rhizobial-legume interactions. Population structure and genomic diversity were compared among stem-nodulating bradyrhizobia isolated from various Aeschynomene species of Central America and Tropical Africa. Phylogenetic analyses based on the recA gene and whole-genome amplified fragment length polymorphism (AFLP) fingerprints on 110 bacterial strains highlighted that all the photosynthetic strains form a separate cluster among bradyrhizobia, with no obvious structuring according to their geographical or plant origins. Nod-independent symbiosis was present in all sampling areas and seemed to be linked to Aeschynomene host species. However, it was not strictly dependent on photosynthetic ability, as exemplified by a newly identified cluster of strains that lacked canonical nod genes and efficiently stem-nodulated A. indica, but were not photosynthetic. Interestingly, the phenotypic properties of this new cluster of bacteria were reflected by their phylogenetical position, as being intermediate in distance between classical root-nodulatingBradyrhizobium spp. and photosynthetic ones. This result opens new prospects about stem-nodulating bradyrhizobial evolution.

Description of Microvirga aerophila sp. nov. and Microvirga aerilata sp. nov., isolated from air, reclassification of Balneimonas flocculans Takeda et al. 2004 as Microvirga flocculans comb. nov. and emended description of the genus Microvirga.

Two bacterial strains, 5420S-12(T) and 5420S-16(T), isolated from air samples, were characterized using a polyphasic approach. 16S rRNA gene sequence analysis showed that strain 5420S-12(T) was related phylogenetically to Microvirga subterranea FaiI4(T) (97.4 % sequence similarity) and Microvirga guangxiensis 25B(T) (97.1 %) and that strain 5420S-16(T) was closely related to Balneimonas flocculans TFB(T) (98.0 %) and Microvirga guangxiensis 25B(T) (97.2 %). The G+C content of the genomic DNA was 62.2 mol% for strain 5420S-12(T) and 61.5 mol% for strain 5420S-16(T). The major fatty acid was C(18 : 1)ω7c. The results of DNA-DNA hybridization and the phenotypic data showed that strains 5420S-12(T) and 5420S-16(T) could be distinguished from phylogenetically related species and represent two novel species within the genus Microvirga, for which the names Microvirga aerophila sp. nov. (type strain 5420S-12(T) =KACC 12743(T) =NBRC 106136(T)) and Microvirga aerilata sp. nov. (type strain 5420S-16(T) =KACC 12744(T) =NBRC 106137(T)) are proposed. Furthermore, the reclassification of Balneimonas flocculans as Microvirga flocculans comb. nov. (type strain TFB(T) =JCM 11936(T) =KCTC 12101(T) =IAM 15034(T) =ATCC BAA-817(T)) is proposed and an emended description of the genus Microvirga is provided.