Azoarcus halotolerans sp. nov., a novel member of Rhodocyclaceae isolated from activated sludge collected in Hong Kong.

A floc-forming bacterial strain, designated HKLI-1T, was isolated from the activated sludge of a municipal sewage treatment plant in Hong Kong SAR, PR China. Cells of this strain were Gram-stain-negative, strictly aerobic, catalase- and oxidase-positive, rod-shaped and motile by means of a single polar flagellum. Growth occurred at 18-37 °C (optimum, 28 °C), pH 5.5-9.0 (optimum, pH 7.5) and with 0-8.0 % (w/v) NaCl (optimum, 1-1.5 %) concentration. The major fatty acids of strain HKLI-1T were C16 : 0 and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c). The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and three unidentified lipids. The DNA G+C content was 63.5 mol% from whole genomic sequence analysis. Based on the results of 16S rRNA gene sequences analysis, this strain should be assigned to the genus Azoarcus and is closely related to Azoarcus olearius DQS-4T (94.93 % 16S rRNA gene sequence pairwise similarity), Azoarcus toluclasticus MF63T (94.91 %) and Azoarcus communis SWub3T (94.01 %), but separate from them by large distances in different phylogenetic trees. Based on whole genome analysis, the orthologous average nucleotide identity and in silico DNA-DNA hybridization values against four of the closest relatives were 73.03-74.83 and 17.2-23.0 %, respectively. The phylogenetic, genotypic, phenotypic and chemotaxonomic data demonstrated that strain HKLI-1T could be distinguished from its phylogenetically related species, and that this strain represented a novel species within the genus Azoarcus, for which the name Azoarcus halotolerans sp. nov. is proposed. The type strain is HKLI-1T (= 72659T=CCTCC AB 2019312T).

Description of Azoarcus nasutitermitis sp. nov. and Azoarcus rhizosphaerae sp. nov., two nitrogen-fixing species isolated from termite nest and rhizosphere of Ficus religiosa.

A polyphasic taxonomic approach was used to characterise two presumably novel bacteria, designated strains CC-YHH838T and CC-YHH848T isolated from termite nest and rhizosphere of Ficus religiosa, respectively. These two nitrogen-fixing strains were observed to be Gram-staining-negative, aerobic rod, and colonies were yellowish in color. Growth of strains was observed at 20-37 °C, pH 7-8, and in the presence of 1-2% NaCl. Phylogenetic analyses based on 16S rRNA genes revealed a distinct taxonomic position attained by strain CC-YHH838T and CC-YHH848T associated with Thauera hydrothermalis (97.1% sequence identity), and formed a separate branch with Azoarcus indigens (95.4%), Aromatoleum aromaticum (96.2%), and lower sequence similarity to other species. The calculation of OrthoANI values pointed out strains CC-YHH838T and CC-YHH848T gave 78.9% and 79.8% compared to Thauera hydrothermalis, respectively. The major fatty acids (> 5%) were C16:0, C17:0 cyclo, C10:0 3-OH, C16:1ω7c/C16:1ω6c and C18:1ω7c/C18:1ω6c. The polar lipid profile comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and unidentified aminophospholipid and phospholipids; the predominant polyamines were putrescine and spermidine. The predominant respiratory system was ubiquinone (Q-8) and the DNA G + C contents were 61.4 ± 0.1 mol% and 60.2 ± 1.3 mol%, respectively. Based on the phylogenetic and polyphasic comparisons, strains CC-YHH838T and CC-YHH848T are proposed to represent two novel species within the genus Azoarcus in the family Rhodocyclaceae, for which the name Azoarcus nasutitermitis sp. nov. (type strain CC-YHH838T = BCRC 81059T = JCM 32001T) and Azoarcus rhizosphaerae sp. nov. (type strain CC-YHH848T = BCRC 81060T = JCM 32002T) were proposed.

Azoarcus pumilus sp. nov., isolated from seawater in Sanya, China.

A novel Gram-stain-negative, motile, rod-shaped (0.4-0.5×1.0-2.0 µm) strain with one polar flagellum, designated SY39T, was isolated from seawater in Sanya, China. Strain SY39T was able to grow at 15-40 °C (optimum, 35-37 °C), pH 6.5-8.5 (pH 8.0) and 0.5-6.0 % (w/v) NaCl (3.5 %). Chemotaxonomic analysis showed that the isoprenoid quinones were Q-8 (88.6 %) and Q-7 (11.4 %). The dominant fatty acids were C16 : 0 and summed feature 3 (C16 : 1ω7c/C16 : 1ω6c). The polar lipids of strain SY39T consisted of diphosphatidyglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unknown phosphoglycolipid, one unknown glycolipid and two unknown aminophosphoglycolipids. The DNA G+C content of the genomic DNA was 66.5 mol%. The phylogenetic analysis of 16S rRNA gene sequences showed that strain SY39T belongs to the genus Azoarcus with similarity ranging from 92.3 to 95.2 %. Based on the phenotypic, chemotaxonomic and phylogenetic features, strain SY39T is concluded to represent a novel species of the genus Azoarcus, for which the name Azoarcus pumilus sp. nov. is proposed. The type strain is SY39T (=KCTC 62157T=MCCC 1K03430T).

Global expression analysis of the response to microaerobiosis reveals an important cue for endophytic establishment of Azoarcus sp. BH72.

The endophyte Azoarcus sp. BH72, fixing nitrogen microaerobically, encounters low O2 tensions in flooded roots. Therefore, its transcriptome upon shift to microaerobiosis was analyzed using oligonucleotide microarrays. A total of 8.7% of the protein-coding genes were significantly modulated. Aerobic conditions induced expression of genes involved in oxidative stress protection, while under microaerobiosis, 233 genes were upregulated, encoding hypothetical proteins, transcriptional regulators, and proteins involved in energy metabolism, among them a cbb3 -type terminal oxidase contributing to but not essential for N2 fixation. A newly established sensitive transcriptional reporter system using tdTomato allowed to visualize even relatively low bacterial gene expression in association with roots. Beyond metabolic changes, low oxygen concentrations seemed to prime transcription for plant colonization: Several genes known to be required for endophytic rice interaction were induced, and novel bacterial colonization factors were identified, such as azo1653. The cargo of the type V autotransporter Azo1653 had similarities to the attachment factor pertactin. Although for short term swarming-dependent colonization, it conferred a competitive disadvantage, it contributed to endophytic long-term establishment inside roots. Proteins sharing such opposing roles in the colonization process appear to occur more generally, as we demonstrated a very similar phenotype for another attachment protein, Azo1684. This suggests distinct cellular strategies for endophyte establishment.

Azoarcus taiwanensis sp. nov., a denitrifying species isolated from a hot spring.

The strain NSC3(T), a novel, facultative, chemolithotrophic, denitrifying, alkaliphilic, sulfide-oxidizing bacterium isolated from a hot spring in Yang-Ming Mountain, Taiwan, was Gram negative, rod shaped, and motile by single polar flagella and grew facultatively by adopting a denitrifying metabolism. The 16S rRNA sequence analysis revealed that strain NSC3(T) belongs to beta subclass of the Proteobacteria and most closely related to Azoarcus evansii KB740(T) (95.44 %), Azoarcus toluvorans Td-21(T) (95.21 %), Azoarcus tolulyticus Tol-4(T) (95.08 %), and Azoarcus toluclasticus MF63(T) (94.94 %). The phylogenetic analyses based on 16S rRNA gene sequences indicated that the strain NSC3(T) formed a distinct lineage in the Betaproteobacteria and that it exhibited the highest level of sequence similarity with species of the genera Azoarcus (95.28-93.13 %). The major fatty acids of the type strain were C16:0 (26.9 %), C16:1w7c (28.9 %), C18:0 (9.6 %), and C18:1w7c/w6c (29.9 %). The DNA G+C content of genomic DNA was 63.7 mol%. On the basis of the 16S rRNA sequence similarity, phenotypic and genotypic characteristics, and chemotaxonomic data, the strain NSC3(T) could be differentiated from other species of the genus Azoarcus. Therefore, strain NSC3(T) (equal to BCRC 80111(T) and DSM 24109(T)) is proposed as a novel species in genus Azoarcus, for which the name Azoarcus taiwanensis sp. nov. is proposed. The strain NSC3(T) is deposited in Bioresource Collection and Research Center, Taiwan, under the reference number BCRC 80111(T), and German Collection of Microorganisms and Cell Cultures, Germany (DSMZ), with DSM 24109(T).

Microbial community in packed bed bioreactor involved in nitrate remediation from low level radioactive waste.

Nitrate is the second largest contaminant of agriculture soil after pesticides. It also is a major pollutant from nuclear and metallurgical operations. Conventional methods for nitrate removal suffers from high cost and complexity leaving bioremediation as a viable alternative strategy. A pilot plant of 2.5 m(3)/day capacity has been functioning since 2005 based on microbial consortia treating actual effluent from nuclear power plant having pH of 7-8.5 (optimum) with N:C ratio of 1:1.7. The maximum biodegradable nitrate concentration of 3000 ppm could be reduced to below permissible limit (44.2 ppm) within 24 h in presence of sodium acetate as carbon source. Culture independent analysis (16S rDNA based) revealed clones having closest identity with uncultured bacterium, Pseudomonas stutzeri and Azoarcus sp. The existence of dissimilatory pathway of nitrate reduction in the community DNA is indicated by presence of nirS and nirK gene. Though the microbial mass was developed using municipal sewage, absence of Mycobacterium sp was confirmed using PCR. The understanding of the molecular identification of the consortium would help in developing the preservation strategy of the microbial mass for replication and perpetuation of the system.

Enhancement of denitrification in a down-flow hanging sponge reactor by effluent recirculation.

A down-flow hanging sponge reactor, constructed by connecting three identical units in series, was applied to the treatment of artificial wastewater containing phenol and ammonia under high salinity conditions (10.9 g-Cl(-)/L). The theoretical hydraulic retention time (HRT) of each unit was 4 h (total HRT = 12 h). To enhance denitrification by effluent recirculation, the effluent recirculation ratio was increased in increments ranging from 0.0 to 2.0. The concentration of total ammonia nitrogen (TAN), NO2-N, and NO3-N in the final effluent as a proportion of the TAN in the influent was determined to calculate the unrecovered, or denitrification, proportion. The denitrification proportion of the reactor was equivalent to 19.1 ± 14.1% with no effluent recirculation; however, this was increased to 58.6 ± 6.2% when the effluent recirculation ratio was increased to 1.5. Further increasing the effluent recirculation ratio to 2.0 resulted in a decrease in the denitrification proportion to 50.9 ± 9.3%. Activity assays of nitrification and denitrification, as well as 16S rRNA gene sequence analysis, revealed that denitrification occurred primarily in the upper sections of the reactor, while nitrification increased in the lower sections of the reactor. Gene sequence analysis revealed that denitrification by Azoarcus-like species using phenol as an electron donor was dominant.

Azoarcus olearius sp. nov., a nitrogen-fixing bacterium isolated from oil-contaminated soil.

A novel nitrogen-fixing strain, designated DQS-4(T), was isolated from oil-contaminated soil in Taiwan and was characterized using a polyphasic taxonomic approach. Cells of strain DQS-4(T) stained Gram-negative, contained poly-ß-hydroxybutyrate granules and were motile rods, surrounded by a thin capsule. Cells displayed a strictly aerobic type of metabolism and fixed nitrogen microaerobically. Growth occurred at 10-45 °C (optimum, 35-40 °C), at pH 7.0-8.0 (optimum, pH 7.0) and with 0-2 % NaCl (optimum, 0.5-1 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain DQS-4(T) belonged to the genus Azoarcus, and its closest neighbours were Azoarcus indigens VB32(T) and Azoarcus communis SWub3(T), with sequence similarities of 97.4 and 96.4 %, respectively. The major cellular fatty acids of strain DQS-4(T) were summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C18 : 1ω7c. The major cellular hydroxy fatty acid was C10 : 0 3-OH. The DNA G+C content was 64.5 mol%. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several uncharacterized aminophospholipids and phospholipids. The mean level of DNA-DNA relatedness between strain DQS-4(T) and A. indigens LMG 9092(T) was 27.4 %. On the basis of the genotypic and phenotypic data, strain DQS-4(T) represents a novel species in the genus Azoarcus, for which the name Azoarcus olearius sp. nov. is proposed. The type strain is DQS-4(T) ( = BCRC 80407(T) = KCTC 23918(T) = LMG 26893(T)).

Complete genome sequence of the denitrifying and N2O-reducing bacterium Azoarcus sp. strain KH32C.

We report the finished and annotated genome sequence of a denitrifying and N(2)O-reducing betaproteobacterium, Azoarcus sp. strain KH32C. The genome is composed of one chromosome and one megaplasmid and contains genes for plant-microbe interactions and the gene clusters for aromatic-compound degradations.

Characterization of microbial populations in pilot-scale fluidized-bed reactors treating perchlorate- and nitrate-laden brine.

A salt-tolerant, perchlorate- and nitrate-reducing bacterial culture developed previously was used to inoculate two acetate-fed fluidized bed reactors (FBRs) which treated a 6% ion-exchange regenerant brine containing 500 +/- 84 mg-N/L nitrate and 4.6 +/- 0.6 mg/L perchlorate. The reactors were operated in series in continuous flow mode for 107 days after an acclimation period of 65 days. Pilot operation data suggest that complete denitrification was achieved after 70 days of operation, but significant perchlorate removal was not observed. Molecular analysis of the inoculum culture and biomass from the pilot plant samples using denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH) revealed that the composition of the biomass in the pilot-plant was evolving with time in each FBR. The total number of Azoarcus/Denitromonas decreased in the first reactor with time and position in the bioreactor during acclimation and operation. FISH analysis clearly revealed that the number of Halomonas which was the dominant nitrate-reducing organism increased in the first reactor. This indicates a shift towards nitrate reduction which corresponds to the operation data. Both DGGE and FISH demonstrated that the Azoarcus/Denitromonas was still present in the second bioreactor, which indicated that the removal of nitrate in the first reactor was allowing the perchlorate-reducing organisms to establish themselves in the second reactor. The study also suggests that FISH was more effective for analysis of the composition of these cultures and it would be a better tool for the routine monitoring of cultures.

Enrichment of marine anammox bacteria from seawater-related samples and bacterial community study.

Anaerobic ammonium oxidation (anammox) is a novel nitrogen pathway catalyzed by anammox bacteria which are obligate anaerobic chemoautotrophs. In this study, enrichment culture of marine anammox bacteria (MAAOB) from the samples related to seawater was conducted. Simultaneous removal of ammonium and nitrite was confirmed in continuous culture inoculated with sediment of a sea-based waste disposal site within 50 days. However, no simultaneous nitrogen removal was observed in cultures inoculated with seawater-acclimated denitrifying sludge or with muddy sediment of tideland even during 200 days. Nitrogen removal rate of 0.13 kg/m(3)/day was achieved at nitrogen loading rate of 0.16 kg/m(3)/day after 320th days in the culture inoculated with the sediment of waste disposal site. The nitrogen removal ratio between ammonium nitrogen and nitrite nitrogen was 1:1.07. Denaturing gradient gel electrophoresis (DGGE) analysis indicated that an abundance of the bacteria close to MAAOB and coexistence of ammonium oxidizing bacteria and denitrifying bacteria in the culture.

Degradative capacities and bioaugmentation potential of an anaerobic benzene-degrading bacterium strain DN11.

Azoarcus sp. strain DN11 is a denitrifying bacterium capable of benzene degradation under anaerobic conditions. The present study evaluated strain DN11 for its application to bioaugmentation of benzene-contaminated underground aquifers. Strain DN11 could grow on benzene, toluene, m-xylene, and benzoate as the sole carbon and energy sources under nitrate-reducing conditions, although o- and p-xylenes were transformed in the presence of toluene. Phenol was not utilized under anaerobic conditions. Kinetic analysis of anaerobic benzene degradation estimated its apparent affinity and inhibition constants to be 0.82 and 11 microM, respectively. Benzene-contaminated groundwater taken from a former coal-distillation plant site was anaerobically incubated in laboratory bottles and supplemented with either inorganic nutrients (nitrogen, phosphorus, and nitrate) alone, or the nutrients plus strain DN11, showing that benzene was significantly degraded only when DN11 was introduced. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments, and quantitative PCR revealed that DN11 decreased after benzene was degraded. Following the decrease in DN11 16S rRNA gene fragments corresponding to bacteria related to Owenweeksia hongkongensis and Pelotomaculum isophthalicum, appeared as strong bands, suggesting possible metabolic interactions in anaerobic benzene degradation. Results suggest that DN11 is potentially useful for degrading benzene that contaminates underground aquifers at relatively low concentrations.

RNA-based stable isotope probing and isolation of anaerobic benzene-degrading bacteria from gasoline-contaminated groundwater.

Stable isotope probing (SIP) of benzene-degrading bacteria in gasoline-contaminated groundwater was coupled to denaturing gradient gel electrophoresis (DGGE) of DNA fragments amplified by reverse transcription-PCR from community 16S rRNA molecules. Supplementation of the groundwater with [(13)C(6)]benzene together with an electron acceptor (nitrate, sulfate, or oxygen) showed that a phylotype affiliated with the genus Azoarcus specifically appeared in the (13)C-RNA fraction only when nitrate was supplemented. This phylotype was also observed as the major band in DGGE analysis of bacterial 16S rRNA gene fragments amplified by PCR from the gasoline-contaminated groundwater. In order to isolate the Azoarcus strains, the groundwater sample was streaked on agar plates containing nonselective diluted CGY medium, and the DGGE analysis was used to screen colonies formed on the plates. This procedure identified five bacterial isolates (from 60 colonies) that corresponded to the SIP-identified Azoarcus phylotype, among which two strains (designated DN11 and AN9) degraded benzene under denitrifying conditions. Incubation of these strains with [(14)C]benzene showed that the labeled carbon was mostly incorporated into (14)CO(2) within 14 days. These results indicate that the Azoarcus population was involved in benzene degradation in the gasoline-contaminated groundwater under denitrifying conditions. We suggest that RNA-based SIP identification coupled to phylogenetic screening of nonselective isolates facilitates the isolation of enrichment/isolation-resistant microorganisms with a specific function.

Thauera and Azoarcus as functionally important genera in a denitrifying quinoline-removal bioreactor as revealed by microbial community structure comparison.

Structural shifts associated with functional dynamics in a bacterial community may provide clues for identifying the most valuable members in an ecosystem. A laboratory-scale denitrifying reactor was adapted from use of non-efficient seeding sludge and was utilized to degrade quinoline and remove the chemical oxygen demand. Stable removal efficiencies were achieved after an adaptation period of six weeks. Both denaturing gradient gel electrophoresis profiling of the 16S rRNA gene V3 region and comparison of the 16S rRNA gene sequence clone libraries (LIBSHUFF analysis) demonstrated that microbial communities in the denitrifying reactor and seeding sludge were significantly distinct. The percentage of the clones affiliated with the genera Thauera and Azoarcus was 74% in the denitrifying reactor and 4% in the seeding sludge. Real-time quantitative PCR also indicated that species of the genera Thauera and Azoarcus increased in abundance by about one order of magnitude during the period of adaptation. The greater abundance of Thauera and Azoarcus in association with higher efficiency after adaptation suggested that these phylotypes might play an important role for quinoline and chemical oxygen demand removal under denitrifying conditions.

Microbial characterization of toluene-degrading denitrifying consortia obtained from terrestrial and marine ecosystems.

The degradation characteristics of toluene coupled to nitrate reduction were investigated in enrichment culture and the microbial communities of toluene-degrading denitrifying consortia were characterized by denaturing gradient gel electrophoresis (DGGE) technique. Anaerobic nitrate-reducing bacteria were enriched from oil-contaminated soil samples collected from terrestrial (rice field) and marine (tidal flat) ecosystems. Enriched consortia degraded toluene in the presence of nitrate as a terminal electron acceptor. The degradation rate of toluene was affected by the initial substrate concentration and co-existence of other hydrocarbons. The types of toluene-degrading denitrifying consortia depended on the type of ecosystem. The clone RS-7 obtained from the enriched consortium of the rice field was most closely related to a toluene-degrading and denitrifying bacterium, Azoarcus denitrificians (A. tolulyticus sp. nov.). The clone TS-11 detected in the tidal flat enriched consortium was affiliated to Thauera sp. strain S2 (T. aminoaromatica sp. nov.) that was able to degrade toluene under denitrifying conditions. This indicates that environmental factors greatly influence microbial communities obtained from terrestrial (rice field) and marine (tidal flat) ecosystems.

Intrinsic bioremediability of an aromatic hydrocarbon-polluted groundwater: diversity of bacterial population and toluene monoxygenase genes.

The functional and phylogenetic biodiversity of bacterial communities in a benzene, toluene, ethylbenzene and xylene (BTEX)-polluted groundwater was analysed. To evaluate the feasibility of using an air sparging treatment to enhance bacterial degradative capabilities, the presence of degrading microorganisms was monitored. The amplification of gene fragments corresponding to toluene monooxygenase (tmo), catechol 1,2-dioxygenase, catechol 2,3-dioxygenase and toluene dioxygenase genes in DNA extracted directly from the groundwater samples was associated with the presence of indigenous degrading bacteria. Five months of air injection reduced species diversity in the cultivable community (as calculated by the Shannon-Weaver index), while little change was noted in the degree of biodiversity in the total bacterial community, as characterised by denaturing gradient gel electrophoresis (DGGE) analysis. BTEX-degrading strains belonged to the genera Pseudomonas, Microbacterium, Azoarcus, Mycobacterium and Bradyrhizobium. The degrading capacities of three strains in batch liquid cultures were also studied. In some of these microorganisms different pathways for toluene degradation seemed to operate simultaneously. Pseudomonas strains of the P24 operational taxonomic unit, able to grow only on catechol and not on BTEX, were the most abundant, and were present in the groundwater community at all stages of treatment, as evidenced both by cultivation approaches and by DGGE profiles. The presence of different tmo-like genes in phylogenetically distant strains of Pseudomonas, Mycobacterium and Bradyrhizobium suggested recent horizontal gene transfer in the groundwater.

Isolation and characterization of a new denitrifying spirillum capable of anaerobic degradation of phenol.

Two kinds of phenol-degrading denitrifying bacteria, Azoarcus sp. strain CC-11 and spiral bacterial strain CC-26, were isolated from the same enrichment culture after 1 and 3 years of incubation, respectively. Both strains required ferrous ions for growth, but strain CC-26 grew better than strain CC-11 grew under iron-limited conditions, which may have resulted in the observed change in the phenol-degrading bacteria during the enrichment process. Strain CC-26 grew on phenol, benzoate, and other aromatic compounds under denitrifying conditions. Phylogenetic analysis of 16S ribosomal DNA sequences revealed that this strain is most closely related to a Magnetospirillum sp., a member of the alpha subclass of the class Proteobacteria, and is the first strain of a denitrifying aromatic compound-degrading bacterium belonging to this group. Unlike previously described Magnetospirillum strains, however, this strain did not exhibit magnetotaxis. It grew on phenol only under denitrifying conditions. Other substrates, such as acetate, supported aerobic growth, and the strain exhibited microaerophilic features.

Preferential occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza sativa in comparison with modern races.

Several diazotrophic species of Azoarcus spp. occur as endophytes in the pioneer plant Kallar grass. The purpose of this study was to screen Asian wild rice and cultivated Oryza sativa varieties for natural association with these endophytes. Populations of culturable diazotrophs in surface-sterilized roots were characterized by 16S rDNA sequence analysis, and Azoarcus species were identified by genomic fingerprints. A. indigens and Azoarcus sp. group C were detected only rarely, whereas Azoarcus sp. group D occurred frequently in samples of flooded plants: in 75% of wild rice, 80% of land races of O. sativa from Nepal and 33% of modern cultivars from Nepal and Italy. The putatively endophytic populations of diazotrophs differed with the rice genotype. The diversity of cultured diazotrophs was significantly lower in wild rice species than in modern cultivars. In Oryza officinalis (from Nepal) and O. minuta (from the Philippines), Azoarcus sp. group D were the predominant diazotrophic putative endophytes in roots. In contrast, their number was significantly lower in modern cultivars of O. sativa, whereas numbers and diversity of other diazotrophs, such as Azospirillum spp., Klebsiella sp., Sphingomonas paucimobilis, Burkholderia sp. and Azorhizobium caulinodans, were increased. In land races of O. sativa, the diazotrophic diversity was equally high; however, Azoarcus sp. was found in high apparent numbers. Similar differences in populations were also observed in a culture-independent approach comparing a wild rice (O. officinalis) and a modern-type O. sativa plant: in clone libraries of root-associated nitrogenase (nifH) gene fragments, the diazotrophic diversity was lower in the wild rice species. New lineages of nifH genes were detected, e.g. one deeply branching cluster within the anf (iron) nitrogenases. Our studies demonstrate that the natural host range of Azoarcus spp. extends to rice, wild rice species and old varieties being preferred over modern cultivars.