Multilocus sequence analysis of the actinobacterial genus Kribbella

Multilocus sequence analysis (MLSA) was used to refine the phylogenetic analysis of the genus Kribbella, which currently contains 17 species with validly-published names. Sequences were obtained for the 16S rRNA, gyrB, rpoB, recA, relA and atpD genes for 16 of the 17 type strains of the genus plus seven non-type strains. A five-gene concatenated sequence of 4099 nt was used to examine the phylogenetic relationships between the species of the genus Kribbella. Using the concatenated sequence of the gyrB-rpoB-recA-relA and atpD genes, most Kribbella type strains can be distinguished by a genetic distance of >0.04. Each single-gene tree had an overall topology similar to that of the concatenated sequence tree. The single-gene relA tree, used here for the first time in MLSA of actinobacteria, had good bootstrap support, comparable to the rpoB and atpD gene trees, which had topologies closest to that of the concatenated sequence tree. This illustrates that relA is a useful addition in MLSA studies of the genus Kribbella. We propose that concatenated gyrB-rpoB-recA-relA-atpD gene sequences be used for examining the phylogenetic relationships within the genus Kribbella and for determining the closest phylogenetic relatives to be used for taxonomic comparisons.     

黑木相思根瘤菌的系统发育分析及其结瘤效果研究

【目的】针对采集自福建、广东的34株黑木相思根瘤菌进行分类研究,进一步确定其分类地位,丰富我国黑木相思根瘤菌种质资源。【方法】对选取的34株菌株测定了16S rRNA基因、持家基因atpD和glnII序列,以14株菌为代表菌株分析其系统发育情况。而且选取了部分菌株进行结瘤实验。【结果】16S rRNA基因以及持家基因atpD和glnII的系统发育分析结果与16S rRNA PCR-RFLP分型结果基本一致,14株代表菌株被分为10个不同的类群,其中有2个群组属于中慢生根瘤菌属(Mesorhizobium),其余群组属于慢生根瘤菌属(Bradyrhizobium)。结瘤试验证明,相关的供试根瘤菌能与黑木相思、银合欢、南洋楹和网脉相思结瘤共生,显示出较广的宿主范围,且对黑木相思和银合欢的促生效果较明显。【结论】研究发现黑木相思根瘤菌具有丰富的遗传多样性和共生多样性。     

MultiLocus Sequence Analysis- and Amplified Fragment Length Polymorphism-based characterization of xanthomonads associated with bacterial spot of tomato and pepper and their relatedness to Xanthomonas species

MultiLocus Sequence Analysis (MLSA) and Amplified Fragment Length Polymorphism (AFLP) were used to measure the genetic relatedness of a comprehensive collection of xanthomonads pathogenic to solaneous hosts to Xanthomonas species. The MLSA scheme was based on partial sequences of four housekeeping genes (atpD, dnaK, efp and gyrB). Globally, MLSA data unambiguously identified strains causing bacterial spot of tomato and pepper at the species level and was consistent with AFLP data. Genetic distances derived from both techniques showed a close relatedness of (i) X. euvesicatoria, X. perforans and X. alfalfae and (ii) X. gardneri and X. cynarae. Maximum likelihood tree topologies derived from each gene portion and the concatenated data set for species in the X. campestris 16S rRNA core (i.e. the species cluster comprising all strains causing bacterial spot of tomato and pepper) were not congruent, consistent with the detection of several putative recombination events in our data sets by several recombination search algorithms. One recombinant region in atpD was identified in most strains of X. euvesicatoria including the type strain.     

Genetic analysis of housekeeping genes of members of the genus Acholeplasma: phylogeny and complementary molecular markers to the 16S rRNA gene

The partial nucleotide sequences of the rpoB and gyrB genes as well as the complete sequence of the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known Acholeplasma species. The same genes of Mesoplasma and Entomoplasma species were also sequenced and used to infer phylogenetic relationships among the species within the orders Entomoplasmatales and Acholeplasmatales. The comparison of the ITS, rpoB, and gyrB phylogenetic trees with the 16S rRNA phylogenetic tree revealed a similar branch topology suggesting that the ITS, rpoB, and gyrB could be useful complementary phylogenetic markers for investigation of evolutionary relationships among Acholeplasma species. Thus, the multilocus phylogenetic analysis of Acholeplasma multilocale sequence data (ATCC 49900 (T) = PN525 (NCTC 11723)) strongly indicated that this organism is most closely related to the genera Mesoplasma and Entomoplasma (family Entomoplasmataceae) and form the branch with Mesoplasma seiffertii, Mesoplasma syrphidae, and Mesoplasma photuris. The closest genetic relatedness of this species to the order Entomoplasmatales was additionally supported by the finding that A. multilocale uses UGA as the tryptophan codon in its gyrB and gyrA sequences. Use of the UGA codon for encoding tryptophan was previously reported as a unique genetic feature of Entomoplasmatales and Mycoplasmatales but not of Acholeplasmatales. These data, as well as previously published data on metabolic features of A. multilocale, leads to the proposal to reclassify A. multilocale as a member of the family Entomoplasmataceae.     

攀枝花市银合欢根瘤菌遗传多样性

【目的】研究分离自四川攀枝花的银合欢根瘤菌的遗传多样性。【方法】采用联合16S rDNA RFLP和IGS RFLP的综合聚类分析(16S-IGS RFLP)、AFLP及多位点持家基因(16S rDNA,atpD,recA)序列的联合分析对供试银合欢根瘤菌进行研究。【结果】31株未知菌具有15种16S-IGS遗传图谱类型、27种AFLP类型。16S-IGS RFLP结果表明,没有未知菌与Bradyrhizobium的参比菌株聚在一起。在71.4%的相似水平上,31个未知菌按属的水平分成3个分支:S、M和R,分别分布在Sinorhizobium属(28株)、Mesorhizobium属(2株)和Rhizobium属(1株)。S分支的28个菌在84%的相似水平上,16S-IGS RFLP聚类图中构成3个群:群S1、群S2、群S3;在AFLP聚类图中构成9个AFLP群:S1–S9。多位点基因序列表明,代表菌株SCAU215、SCAU231分别与M.Plurifarium、R.huautlense亲缘关系最近。而分布于Sinorhizobium属SCAU222和SCAU228、SCAU213、SCAU216可能代表Sinorhizobium的3个新类群。【结论】攀枝花市银合欢根瘤菌遗传多样性丰富,分布于Sinorhizobium、Mesorhizobium和Rhizobium三个属,且优势类群为Sinorhizobium。     

Networks and groups within the genus Neisseria: analysis of argF, recA, rho, and 16S rRNA sequences from human Neisseria species.

To understand the pattern of nucleotide sequence variation among bacteria that frequently exchange chromosomal genes, we analyzed sequences of the recA, argF, and rho genes, as well as part of the small-subunit (16S) rRNA gene, from about 50 isolates of human commensal Neisseria species and the pathogenic N. meningitidis and N. gonorrhoeae. Almost all isolates of these species could be assigned to five phylogenetic groups that are found for all genes examined and generally are supported by high bootstrap values. In contrast, the phylogenetic relationships among groups varied according to the gene analyzed with notable incongruences involving N. cinerea and N. lactamica. Further analysis using split decomposition showed that for each gene, including 16S rRNA, the patterns of sequence divergence within N. meningitidis and closely related species were inconsistent with a bifurcating treelike phylogeny and better represented by an interconnected network. These data indicate that the human commensal Neisseria species can be separated into discrete groups of related species but that the relationships both within and among these groups, including those reconstructed using 16S rRNA, have been distorted by interspecies recombination events.     

Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys

The current perception of evolutionary relationships and the natural diversity of ammonia-oxidizing bacteria (AOB) is mainly based on comparative sequence analyses of their genes encoding the 16S rRNA and the active site polypeptide of the ammonia monooxygenase (AmoA). However, only partial 16S rRNA sequences are available for many AOB species and most AOB have not yet been analyzed on the amoA level. In this study, the 16S rDNA sequence data of 10 Nitrosomonas species and Nitrosococcus mobilis were completed. Furthermore, previously unavailable 16S rRNA sequences were determined for three Nitrosomonas sp. isolates and for the gamma-subclass proteobacterium Nitrosococcus halophilus. These data were used to revaluate the specificities of published oligonucleotide primers and probes for AOB. In addition, partial amoA sequences of 17 AOB, including the above-mentioned 15 AOB, were obtained. Comparative phylogenetic analyses suggested similar but not identical evolutionary relationships of AOB by using 16S rRNA and AmoA as marker molecules, respectively. The presented 16S rRNA and amoA and AmoA sequence data from all recognized AOB species significantly extend the currently used molecular classification schemes for AOB and now provide a more robust phylogenetic framework for molecular diversity inventories of AOB. For 16S rRNA-independent evaluation of AOB species-level diversity in environmental samples, amoA and AmoA sequence similarity threshold values were determined which can be used to tentatively identify novel species based on cloned amoA sequences. Subsequently, 122 amoA sequences were obtained from 11 nitrifying wastewater treatment plants. Phylogenetic analyses of the molecular isolates showed that in all but two plants only nitrosomonads could be detected. Although several of the obtained amoA sequences were only relatively distantly related to known AOB, none of these sequences unequivocally suggested the existence of previously unrecognized species in the wastewater treatment environments examined.     

Analysis of conserved non-rRNA genes of Tropheryma whipplei

The causative agent of Whipple's disease, Tropheryma whipplei, is a slow-growing bacterium that remains poorly-understood. Genetic characterization of this organism has relied heavily upon rRNA sequence analysis. Pending completion of a complete genome sequencing effort, we have characterized several conserved non-rRNA genes from T. whipplei directly from infected tissue using broad-range PCR and a genome-walking strategy. Our goals were to evaluate its phylogenetic relationships, and to find ways to expand the strain typing scheme, based on rDNA sequence comparisons. The genes coding for the ATP synthase beta subunit (atpD), elongation factor Tu (tuf), heat shock protein GroEL (groEL), beta subunit of DNA-dependent RNA polymerase (rpoB), and RNase P RNA (rnpB) were analyzed, as well as the regions upstream and downstream of the rRNA operon. Phylogenetic analyses with all non-rRNA marker molecules consistently placed T. whipplei within the class, Actinobacteria. The arrangement of genes in the atpD and rpoB chromosomal regions was also consistent with other actinomycete genomes. Tandem sequence repeats were found upstream and downstream of the rRNA operon, and downstream of the groEL gene. These chromosomal sites and the 16S-23S rRNA intergenic spacer regions were examined in the specimens of 11 patients, and a unique combination of tandem repeat numbers and spacer polymorphisms was found in each patient. These data provide the basis for a more discriminatory typing method for T. whipplei.     

Genetic diversity of rhizobia nodulating lentil (Lens culinaris) in Bangladesh

In order to determine the bacterial diversity and the identity of rhizobia nodulating lentil in Bangladesh, we performed a phylogenetic analysis of housekeeping genes (16S rRNA, recA, atpD and glnII) and nodulation genes (nodC, nodD and nodA) of 36 bacterial isolates from 25 localities across the country. Maximum likelihood (ML) and Bayesian analyses based on 16S rRNA sequences showed that most of the isolates (30 out of 36) were related to Rhizobium etli and Rhizobium leguminosarum. Only these thirty isolates were able to re-nodulate lentil under laboratory conditions. The protein-coding housekeeping genes of the lentil nodulating isolates showed 89.1-94.8% genetic similarity to the corresponding genes of R. etli and R. leguminosarum. The same analyses showed that they split into three distinct phylogenetic clades. The distinctness of these clades from closely related species was also supported by high resolution ERIC-PCR fingerprinting and phenotypic characteristics such as temperature tolerance, growth on acid-alkaline media (pH 5.5-10.0) and antibiotic sensitivity. Our phylogenetic analyses based on three nodulation genes (nodA, nodC and nodD) and cross-inoculation assays confirmed that the nodulation genes are related to those of R. leguminosarum biovar viciae, but clustered in a distinct group supported by high bootstrap values. Thus, our multi-locus phylogenetic analysis, DNA fingerprinting and phenotypic characterizations suggest that at least three different clades are responsible for lentil nodulation in Bangladesh. These clades differ from the R. etli-R. leguminosarum group and may correspond to novel species in the genus Rhizobium.     

Phylogenetic and systematic study of Korean <Emphasis Type="Italic">Orius</Emphasis> species (Heteroptera: Anthocoridae) on the basis of molecular and morphological data

A phylogenetic and systematic study of Orius species (Heteroptera: Anthocoridae) from Korea has been conducted using both morphological and molecular characters. Thirty morphological character states were coded for 10 strains of 9 species. Five molecular markers, partial cytochrome c oxidase I (COI), cytochrome b (CytB), 16S rRNA (16S), 18S rRNA (18S), and 28S rRNA (28S), from mitochondrial and nuclear genes, were tested. Phylogenetic analyses based on molecular data were conducted by minimum evolution, maximum parsimony, maximum likelihood, and Bayesian phylogenetic (BP) analyses. Analysis of morphological data was performed using the parsimony programs NONA, and the combined dataset of morphological and molecular data was analyzed using BP analyses. The results of this study indicate that use of COI and CytB enabled relatively effective identification of species, whereas the sequences of 16S, 18S and 28S did not enable identification of closely related species such as Orius minutus and O. strigicollis. We discuss the usefulness of the five molecular markers for determining phylogenetic relationships and identifying the species.     

Molecular phylogenetics and ecological diversification of the transisthmian fish genus Centropomus (Perciformes: Centropomidae).

Phylogenetic relationships among the 12 recognized fish species in the New World genus Centropomus (Pisces, Centropomidae) were analyzed using allozyme electrophoresis and 618 bp of the mitochondrial DNA 16S ribosomal RNA (rRNA) gene. Molecular phylogenetic trees were generally consistent with previously published partial hypotheses based on morphological evidence. However, previously undefined sister group relationships between major species groups were resolved using molecular data, and phylogenetic hypotheses for Centropomus based on 16S rRNA sequences were better supported than were allozyme-based hypotheses. The high level of congruence among the trees inferred from the nuclear and mitochondrial characters provided a firm phylogenetic basis for analysis of ecological diversification and molecular evolution in the genus. Compared to basal Centropomus species, members of the most nested species group were significantly larger in body size and occupied a marine niche only peripherally utilized by their congeners. We also observed substitution rate heterogeneity among 16S rRNA lineages; in contrast to expectations based on "metabolic rate" and "generation interval" models, relative substitution rates were faster than expected for the group of large-bodied snooks. Using the Pliocene rise of the Central American isthmian marine barrier to calibrate rates of 16S ribosomal gene evolution in Centropomus, we found that the rates for the genus were similar to those reported for higher vertebrates. Analysis of the three sets of transisthmian geminate taxa in Centropomus indicated that two of the pairs were probably formed during the Pliocene rise of the isthmus while the third pair diverged several million years earlier.     

Genetic diversity and phylogeny of rhizobia isolated from Caragana microphylla growing in desert soil in Ningxia, China

Rhizobia are soil bacteria with the capacity to induce nitrogen-fixing nodules on the roots or stems of legume plants. A total of 40 bacterial isolates from the root nodules of Caragana microphylla growing in desert soil in Ningxia, China, were analyzed for genetic diversity and phylogenetic position. These isolates were classified into 7 types of 16S ribosomal DNA (rDNA) using polymerase chain reaction-restriction fragment length polymorphism analysis. They were grouped into 4 clades, Rhizobium-Agrobacterium, Sinorhizobium, Phyllobacterium, and Bradyrhizobium, when the phylogenies of 16S rDNA, recA, and atpD genes were applied. Phylogenetic analysis showed that the tree generated from the 16S rDNA sequencing agreed with that produced from the recA and atpD genes. By analyzing phylogenetic relationship using the 3 loci, the isolates in the branches of Phyllobacterium and Sinorhizobium could be identified as P. brassicacearum and S. meliloti. The isolates in the branch of Rhizobium-Agrobacterium were the most abundant microsymbiont of C. microphylla and were designated R. leguminosarum, R. galegae, R. alamii, and A. tumefaciens. Two isolates with low sequence similarity to the known species of Bradyrhizobium might be novel species in this genus.     

Utility of nuclear DNA intron markers at lower taxonomic levels: phylogenetic resolution among nine Tragelaphus spp

Phylogenetic relationships among the nine spiral-horn antelope species of the African bovid tribe Tragelaphini are controversial. In particular, mitochondrial DNA sequencing studies are not congruent with previous morphological investigations. To test the utility of nuclear DNA intron markers at lower taxonomic levels and to provide additional data pertinent to tragelaphid evolution, we sequenced four nuclear DNA segments (MGF, PRKCI, SPTBN, and THY) and combined these data with mitochondrial DNA sequences from three genes (cytochrome b, 12S rRNA, and 16S rRNA). Our molecular supermatrix comprised 4682 characters which were analyzed independently and in combination. Parsimony and model based phylogenetic analyses of the combined nuclear DNA data are congruent with those derived from the analysis of mitochondrial gene sequences. The corroboration between nuclear and mtDNA gene trees reject the possibility that genetic processes such as lineage sorting, gene duplication/deletion and hybrid speciation account for the conflict evident in the previously published phylogenies. It suggests rather that the morphological characters used to delimit the Tragelaphid species are subject to convergent evolution. Divergence times among species, calculated using a relaxed Bayesian molecular clock, are consistent with hypotheses proposing that climatic oscillations and their impact on habitats were the major forces driving speciation in the tribe Tragelaphini.     

RNA polymerase beta subunit (rpoB) gene and the 16S-23S rRNA intergenic transcribed spacer region (ITS) as complementary molecular markers in addition to the 16S rRNA gene for phylogenetic analysis and identification of the species of the family Mycoplasmataceae

Conventional classification of the species in the family Mycoplasmataceae is mainly based on phenotypic criteria, which are complicated, can be difficult to measure, and have the potential to be hampered by phenotypic deviations among the isolates. The number of biochemical reactions suitable for phenotypic characterization of the Mycoplasmataceae is also very limited and therefore the strategy for the final identification of the Mycoplasmataceae species is based on comparative serological results. However, serological testing of the Mycoplasmataceae species requires a performance panel of hyperimmune sera which contains anti-serum to each known species of the family, a high level of technical expertise, and can only be properly performed by mycoplasma-reference laboratories. In addition, the existence of uncultivated and fastidious Mycoplasmataceae species/isolates in clinical materials significantly complicates, or even makes impossible, the application of conventional bacteriological tests. The analysis of available genetic markers is an additional approach for the primary identification and phylogenetic classification of cultivable species and uncultivable or fastidious organisms in standard microbiological laboratories. The partial nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known type strains and the available non-type strains of the Mycoplasmataceae species. In addition to the available 16S rRNA gene data, the ITS and rpoB sequences were used to infer phylogenetic relationships among these species and to enable identification of the Mycoplasmataceae isolates to the species level. The comparison of the ITS and rpoB phylogenetic trees with the 16S rRNA reference phylogenetic tree revealed a similar clustering patterns for the Mycoplasmataceae species, with minor discrepancies for a few species that demonstrated higher divergence of their ITS and rpoB in comparison to their neighbor species. Overall, our results demonstrated that the ITS and rpoB gene could be useful complementary phylogenetic markers to infer phylogenetic relationships among the Mycoplasmataceae species and provide useful background information for the choice of appropriate metabolic and serological tests for the final classification of isolates. In summary, three-target sequence analysis, which includes the ITS, rpoB, and 16S rRNA genes, was demonstrated to be a reliable and useful taxonomic tool for the species differentiation within the family Mycoplasmataceae based on their phylogenetic relatedness and pairwise sequence similarities. We believe that this approach might also become a valuable tool for routine analysis and primary identification of new isolates in medical and veterinary microbiological laboratories.     

Evaluation of a novel 16S rRNA/tRNA mitochondrial marker for the identification and phylogenetic analysis of shrimp species belonging to the superfamily Penaeoidea

In this study, we infer the phylogenetic relationships within commercial shrimp using sequence data from a novel mitochondrial marker consisting of an approximately 530-bp region of the 16S ribosomal RNA (rRNA)/transfer RNA (tRNA)^Val genes compared with two other mitochondrial genes: 16S rRNA and cytochrome c oxidase I (COI). All three mitochondrial markers were considerably AT rich, exhibiting values up to 78.2% for the species Penaeus monodon in the 16S rRNA/tRNA^Val genes, notably higher than the average among other Malacostracan mitochondrial genomes. Unlike the 16S rRNA and COI genes, the 16S rRNA/tRNA^Val marker evidenced that Parapenaeus is more closely related to Metapenaeus than to Solenocera, a result that seems to be more in agreement with the taxonomic status of these genera. To our knowledge, our study using the 16S rRNA/tRNA^Val gene as a marker for phylogenetic analysis offers the first genetic evidence to confirm that Pleoticus muelleri and Solenocera agassizi constitute a separate group and that they are more related to each other than to genera belonging to the family Penaeidae. The 16S rRNA/tRNA^Val region was also found to contain more variable sites (56%) than the other two regions studied (33.4% for the 16S rRNA region and 42.7% for the COI region). The presence of more variable sites in the 16S rRNA/tRNA^Val marker allowed the interspecific differentiation of all 19 species examined. This is especially useful at the commercial level for the identification of a large number of shrimp species, particularly when the lack of morphological characteristics prevents their differentiation.     

Towards a genome-based taxonomy for prokaryotes

The ranks higher than the species in the prokaryotic taxonomy are primarily designated based on phylogenetic analysis of the 16S rRNA gene sequences, but no definite standards exist for the absolute relatedness (measured by 16S rRNA or other means) between the ranks. Accordingly, it remains unknown how comparable the ranks are between different organisms. To gain insights into this question, we studied the relationship between shared gene content and genetic relatedness for 175 fully sequenced strains, using as a robust measure of relatedness the average amino acid identity (AAI) of the shared genes. Our results reveal that adjacent ranks (e.g., phylum versus class) frequently show extensive overlap in terms of genetic and gene content relatedness of the grouped organisms, and hence, the current system is of limited predictive power in this respect. The overlap between nonadjacent ranks (e.g., phylum versus family) is generally limited and attributable to clear inconsistencies of the taxonomy. In addition to providing means for standardizing taxonomy, our AAI-based approach provides a means to evaluate the robustness of alternative genetic markers for phylogenetic purposes. For instance, the 23S rRNA gene was found to be as good a marker as the 16S rRNA gene, while several of the widely distributed protein-coding genes, such as the RNA polymerase and gyrase subunits, show a strong phylogenetic signal, albeit less strong than the rRNA genes (0.78 > R2 > 0.69 for the protein-coding genes versus R2 = 0.84 for the rRNA genes). The AAI approach outlined here could contribute significantly to a genome-based taxonomy for all microbial organisms.     

Mesorhizobial strains nodulating Anagyris latifolia and Lotus berthelotii in Tamadaya ravine (Tenerife, Canary Islands) are two symbiovars of the same species, Mesorhizobium tamadayense sp. nov

Barranco de Tamadaya is a deep ravine located in southern Tenerife, which is included within a protected area where several endemic plants grow. Among them, two legumes are catalogued as critically endangered, Anagyris latifolia and Lotus berthelotii. Rhizobial strains isolated from their root nodules grown in soil samples from this ravine harboured symbiotic genes belonging to two distant symbiovars, but they shared identical 16S rRNA gene sequences (rrs). The phylogeny based on the rrs sequences placed these isolates in a separate subbranch that did not include any of the currently recognised Mesorhizobium species, but the resolution of the ribosomal tree did not permit further taxonomic conclusions. Nevertheless, multilocus sequence analysis (MLSA) of four housekeeping genes (atpD, recA, glnII and dnaK) and the rrs gene generated a highly supported Bayesian phylogeny, identifying these isolates as a new Mesorhizobium lineage. DNA-DNA hybridisation homology percentages were lower than 30% compared to type strains of the closest related species, and supported the phylogenetic data. Phenotypic characterisation also distinguished this lineage from the other closest Mesorhizobium species. The polyphasic approach thus confirmed that the isolates represented a novel species for which we propose the name Mesorhizobium tamadayense sp. nov. The type strain is Ala-3(T) (CECT 8040(T), LMG 26736(T)).     

Phylogenetic diversity of non-nodulating Rhizobium associated with pine ectomycorrhizae

Most Rhizobium species described are symbionts that form nodules on legume roots; however, non-nodulating strains of Rhizobium are also widespread in nature. Unfortunately, knowledge of non-nodulating Rhizobium is quite limited compared with nodulating Rhizobium . Here, we studied the phylogenetic diversity of Rhizobium species that inhabit Japanese red pine roots ( Pinus densiflora ). Because fine roots of pine trees are usually colonized by ectomycorrhizal fungi in nature, we mainly used ectomycorrhizal root tips for bacterial isolation. Out of 1195 bacteria isolated from 75 independent root samples from the field and greenhouse experiments, 102 isolates were confirmed to be Rhizobium following partial 16S rRNA gene analysis. Rhizobium species were occasionally dominant in culturable bacterial communities, whereas no Rhizobium species were isolated from the soil itself. Molecular phylogenetic analyses using 16S rRNA, atpD , and recA gene sequences revealed that isolated Rhizobium strains were phylogenetically diverse and that several were distantly related to known Rhizobium species. Considering that a single species of pine is associated with unique and phylogenetically diverse Rhizobium populations, we should pay more attention to non-nodulating strains to better understand the diversity, ecology, and evolution of the genus Rhizobium and plant– Rhizobium associations.     

The phylogeny of the genus Nitrobacter based on comparative rep-PCR, 16S rRNA and nitrite oxidoreductase gene sequence analysis

Strains of Nitrobacter mediate the second step in the nitrification process by oxidizing nitrite to nitrate. The phylogenetic diversity of the genus is currently not well investigated. In this study, a rep-PCR profile and the nearly complete 16S rRNA gene sequence of 30 strains, comprising a wide physiological as well as ecological diversity and encompassing representatives of the four species, were determined. The sequence diversity of the 16S rRNA gene between different species was low, indicating the need for additional phylogenetic markers. Therefore, primers were developed for amplifying the complete nxrX gene and a 380bp fragment of the nxrB1 gene, which are both genes involved in the nitrite oxidation process. These genes confirmed the division into phylogenetic groups revealed by the 16S rRNA gene but showed a better discriminatory power. They can be a valuable additional tool for phylogenetic analysis within the genus Nitrobacter and can assist in the identification of new Nitrobacter isolates.