BOX-PCR Fingerprinting Research Articles

First Report of Xanthomonas gardneri Causing Bacterial Spot of Tomato in Ohio and Michigan.

In 2009 and 2010, outbreaks of bacterial spot characterized by significant fruit spotting occurred in at least 2,000 ha of commercial processing tomatoes in northwest Ohio and southeast Michigan. Losses were estimated at $7.8 million. Diseased fruit and foliage were collected from 32 Ohio and Michigan fields in 2010. Excised lesions from fruit and leaves were dipped briefly in 70% ethanol, air dried, and chopped into pieces in 10 mM potassium phosphate buffer (KPB), pH 7.4. Ten-fold serial dilutions in KPB were plated on yeast dextrose carbonate agar medium and 83 yellow mucoid colonies were purified. All isolates were gram negative and induced a hypersensitive response in tobacco (Nicotiana tabacum) plants 24 h after inoculation with a 108 CFU/ml bacterial suspension in water. All 83 isolates were identified as Xanthomonas spp. using genus-specific primers RST65/69 (2). Of these, 11 were identified as X. euvesicatoria and 8 as X. perforans using the species-specific primers RST27/28 (1) and JJ19/22 (5'-AACCCAACTAATTTCCCTC-3' and 5'-AACGAGATTTGTTACGAACC-3'; J. B. Jones, personal communication), respectively. DNA fingerprint profiles of 62 of the 64 remaining strains generated using BOX-PCR assays (4) were identical to the profile of X. gardneri type strain XCGA2. The DNA profiles of 2 of the 64 Xanthomonas strains did not resemble those of any reference strains. The 16S rDNA and ITS1 genes from two representative strains (SM174-10 and SM230-10) were PCR amplified, direct sequenced, and aligned using nBLAST with the same gene region from XCGA2 (GenBank Accession No. AF123093). Strains SM174-10 and SM230-10 differed from XCGA2 by 2 bp (99% nucleotide similarity). Pathogenicity tests were performed twice on 6-week-old tomato seedlings (cv. Peto 696). Three tomato seedlings were sprayed until runoff with strain SM174-10 (~108 CFU/ml), three seedlings were sprayed similarly with water (control treatment), and all six plants were grown under high relative humidity (24 s of mist per 12 min) at day/night temperatures of 29/23°C for 15 days. Seedlings inoculated with SM174-10 exhibited water-soaked lesions and chlorosis on the foliage, similar to field symptoms, within 14 days. Seedlings sprayed with water did not develop symptoms. Isolates cultured as described above from all three pathogen-inoculated seedlings were similar in morphology to strain SM174-10; no cultures were recovered from water-inoculated plants. The BOX-PCR fingerprint profile of a representative reisolated colony was identical to that of SM174-10. Although bacterial spot of tomato is a common disease in Ohio and Michigan, to our knowledge this is the first report of X. gardneri infecting tomatoes in these states and provides evidence that there may have been a shift in the primary causal agent of bacterial spot from X. euvesicatoria (3) to X. gardneri.

Small cardamom (Elettaria cardamomum Maton.) and ginger (Zingiber officinale Roxb) bacterial wilt is caused by same strain of Ralstonia solanacearum: a result revealed by multilocus sequence typing (MLST)

Bacterial wilt in cardamom (Elettaria cardamomum Maton) was observed in Kerala state of India. Infected plants showed wilting wherein all leaves roll or curl upward towards the midrib centre, turn yellow, and the whole plant finally dies; the collar region shows water-soaked lesions initially and turns dark brown eventually; copious quantity of bacterial exudate is observed on the cut end of the pseudostem. The bacterium was identified as Ralstonia solanacearum based on a panel of phenotypic characters such as fluidal white colony on Kelman’s medium, biovar assay and biolog assay (BiologGN), and genotypic characters such as Multiplex-PCR based phylotyping, sequences of 16S rDNA, 16-23S intergenic region, and recN gene. Collectively these tests revealed that the R. solanacearum infecting cardamom belong to biovar 3 and phylotype 1 confirming its Asian origin. Upon soil inoculation, the bacterium caused typical wilting of the cardamom plants in three weeks and ginger plantlets in two weeks. Cross transmissibility of the bacterium was observed in cardamom and ginger wherein the plants succumbed to wilt when R. solanacearum from either of the host was inoculated. BOX-PCR fingerprinting revealed that the strain is identical (100%) to a ginger strain of R. solanacearum, which is widely prevalent in the Indian sub-continent. Furthermore, Multilocus Sequence Typing (MLST) based strain comparison confirmed that cardamom and ginger strain were identical to each other at 11 loci. Apart from striking phenotypic and genotypic (allelic) similarities, geographical origin, and cross transmissibility of the cardamom strain of R. solanacearum strongly suggest that the new occurrence of wilt of cardamom in India could have an origin in bacterial wilt of ginger. Perusal of records on Ralstonia-induced bacterial wilt in crop plants, particularly among the Zingiberaceae family, reveals that this is a new report of bacterial wilt disease in small cardamom.

BIOLOGICAL AND MOLECULAR CHARACTERISTICS OF IRANIAN BIOVARS 2A AND 2T OF RALSTONIA SOLANACEARUM

The biological and molecular characteristics of 8 strains of biovars 2A of Ralstonia solanacearum from the Iranian province of Isfahan and of 7 isolates of biovar 2T from the province of Khuzestan were investigated morphologically, physiologically, biochemically, and using pathogenicity tests, BOX-PCR, ERIC-PCR and RFLP. Biovar 2A strains showed irregular, creamy-white and highly fluid colonies with pink center on TZC medium, slow spreading necrosis in hypersensivite reaction (HR) after 48 h, produced melanin in tyrosin test, were less pectolytic and did not utilize ribose and tryptophan. By contrast, biovar 2T strains had small size, less fluid, round and deep red colonies with a thin white margin, did not produce melanin, had a higher pectolytic activity, rapid HR on tobacco leaves after 24 h and utilized ribose and tryptophan. Comparative analysis of the BOX-PCR and ERIC-PCR fingerprint of the 15 strains at the 45% similarity level, revealed two main clusters. comprising all biovar 2A strains (cluster 1) or all biovar 2T strains (cluster 2). RFLP profiles of both biovers did not show any polymorphism. Typically, biovar 2A isolates were more pathogenic and bacterial density was higher in the xylem of all hosts tested. There was no significant difference in biovar 2A and 2T survival in loamy-clay soil, which was up to 186 days under controlled conditions.

Isolates of Thermoanaerobacter thermohydrosulfuricus from decaying wood compost display genetic and phenotypic microdiversity

In this study, 12 strains of Thermoanaerobacter were isolated from a single decaying wood compost sample and subjected to genetic and phenotypic profiling. The 16S rRNA encoding gene sequences suggested that the isolates were most similar to strains of either Thermoanaerobacter pseudethanolicus or Thermoanaerobacter thermohydrosulfuricus. Examination of the lesser conserved chaperonin-60 (cpn60) universal target showed that some isolates shared the highest sequence identity with T.thermohydrosulfuricus; however, others to Thermoanaerobacter wiegelii and Thermoanaerobacter sp. Rt8.G4 (formerly Thermoanaerobacter brockii Rt8.G4). BOX-PCR fingerprinting profiles identified differences in the banding patterns not only between the isolates and the reference strains, but also among the isolates themselves. To evaluate the extent these genetic differences were manifested phenotypically, the utilization patterns of 30 carbon substrates were examined and the niche overlap indices (NOI) calculated. Despite showing a high NOI (>0.9), significant differences existed in the substrate utilization capabilities of the isolates suggesting that either a high degree of niche specialization or mechanisms allowing for non-competitive co-existence, were present within this ecological context. Growth studies showed that the isolates were physiologically distinct in both growth rate and the fermentation product ratios. Our data indicate that phenotypic diversity exists within genetically microdiverse Thermoanaerobacter isolates from a common environment.

Comparison of Genotypic and Phylogenetic Relationships of Environmental Enterococcus Isolates by BOX-PCR Typing and 16S rRNA Gene Sequencing

Environmental Enterococcus spp. were compared by BOX-PCR genotyping and 16S rRNA gene sequencing to clarify the predictive relationship of BOX-PCR fingerprints to species designation. BOX-PCR and 16S rRNA gene relationships agreed for 77% of strains. BOX-PCR provided superior intraspecies discrimination but incorrectly identified some strains to the species level and divided some species into multiple groups.

Genetic diversity and population structure of Clavibacter michiganensis subsp. nebraskensis

Clavibacter michiganensis subsp. nebraskensis (CMN) is a gram-positive bacterium and an incitant of Goss's bacterial wilt and leaf blight or "leaf freckles" in corn. A population structure of a wide temporal and geographic collection of CMN strains (n = 131), originating between 1969 and 2009, was determined using amplified fragment length polymorphism (AFLP) analysis and repetitive DNA sequence-based BOX-PCR. Analysis of the composite data set of AFLP and BOX-PCR fingerprints revealed two groups with a 60% cutoff similarity: a major group A (n = 118 strains) and a minor group B (n = 13 strains). The clustering in both groups was not correlated with strain pathogenicity. Group A contained two clusters, A1 (n = 78) and A2 (n = 40), with a linkage of 75%. Group A strains did not show any correlation with historical, geographical, morphological, or physiological properties of the strains. Group B was very heterogeneous and eight out of nine clusters were represented by a single strain. The mean similarity between clusters in group B varied from 13% to 63%. All strains in group B were isolated after 1999. The percentage of group B strains among all strains isolated after 1999 (n = 69) was 18.8%. Implications of the findings are discussed.

Diversity of gut Bifidobacterium species is not altered between allergic and non-allergic French infants

Some clinical studies have suggested a relationship between allergic diseases and gut microbiota. We aimed to study bifidobacterial colonization at species and strain levels in ten allergic French infants included at their first clinical consultation and 20 controls matching for age at sampling, mode of delivery, per partum antibiotics, type of feeding and antibiotics in the first weeks of life. The faecal microbiota was analyzed by culture methods and TTGE. Bifidobacterial species and strains were identified using multiplex PCR and Box-PCR fingerprinting. No differences were observed between groups in the number of colonized infants or in the levels of colonization by the main aerobic and anaerobic genera. All infants were colonized with high levels of Bifidobacterium except for one in each group. One to 5 Bifidobacterium species and 1 to 7 strains were observed per subject independently of allergic status and age at sampling. Our study showed the infants to be colonized by several species and strains, including several strains from the same species. This diversity in Bifidobacterium colonization was not related with the allergic status and showed that the link between Bifidobacterium colonization and allergic diseases is complex and cannot be restricted to the role attributed to Bifidobacterium species.

Rhizobium sphaerophysae sp. nov., a novel species isolated from root nodules of Sphaerophysa salsula in China

Four gram-negative, aerobic, motile, non-spore, forming rods with a wide pH and temperature range for growth (pH 7.0-11.0, optimum pH 8.0; 20-45°C, optimum 28°C) strains were isolated from root nodules of Sphaerophysa salsula and characterized by means of a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the four strains formed a new lineage related to the genus Rhizobium and the sequence similarities between the isolate and the most related type strain Rhizobium giardinii was 96.5%. These strains also formed a distinctive group from the reference strains for defined Rhizobium species based on housekeeping gene sequences (atpD and recA), BOX-PCR fingerprinting, phenotypic features and symbiotic properties. The representative strain CCNWGS0238(T) has DNA-DNA relatedness of less than 33.4% with the most closely related species R. giardinii. It is therefore proposed as a new species, Rhizobium sphaerophysae sp. nov., with isolate CCNWGS0238(T) (=ACCC17498(T)=HAMBI3074(T)) as the type strain.

Prevalence of virulence factors in Escherichia coli isolated from healthy animals and water sources in Brazil

The aim of this work was to verify the presence of seven virulence factors (ST, LT, eae, stx(1), stx(2), INV and EAEC) among Escherichia coli strains isolated from healthy humans, bovines, chickens, sheep, pigs and goats, from two sewage treatment plants and from the Tietê River. We have found a high prevalence of eae, stx(1) and stx(2) in ruminants. The EAEC gene was only found in humans and sewage. No strains presented ST, LT or INV. BOX-PCR fingerprints revealed a high diversity among the strains analysed and a non-clonal origin of strains that presented the same virulence factors. Therefore, we concluded that ruminants may constitute an important reservoir of most diarrheagenic E. coli in Brazil, except for EAEC strains. These results emphasize the importance of the identification of the animal source of fecal contamination for the correct water risk assessment.

Genetic diversity of Ralstonia solanacearum strains from China assessed by PCR-based fingerprints to unravel host plant- and site-dependent distribution patterns

Bacterial wilt caused by Ralstonia solanacearum is a serious threat to crop production in China. A collection of 319 R. solanacearum strains isolated from 14 different diseased host plants collected in 15 Chinese provinces was investigated by BOX fingerprints in order to test the influence of the site and the host plant on their genetic diversity. Phylotype, fliC-RFLP patterns and biovar were determined for all strains and the sequevar for 39 representative strains. The majority of strains belonged to the Asian phylotype I, shared identical fliC-RFLP patterns and were assigned to four biovars (bv3:123; bv4:162; bv5:3; and bv6:11). Twenty strains were phylotype II, assigned to biovar 2, and had distinct fliC-RFLP patterns. BOX-PCR fingerprints generated from the genomic DNA of each strain revealed a high diversity of the phylotype I strains, where 28 types of BOX fingerprints could be distinguished. While many BOX clusters comprised isolates from different provinces and several host plants, some groups contained isolates that were plant or site specific. All phylotype II isolates originating from 10 provinces belonged to sequevar 1 and displayed identical BOX patterns as the potato brown rot strains from various regions of the world.

Molecular diversity of thermophilic bacteria isolated from Pasinler hot spring (Erzurum, Turkey)

The present study was conducted to determine the phenotypic and genotypic characterization of thermophilic bacteria isolated from Pasinler hot spring, Erzurum, Turkey. Fatty acid profiles, BOX PCR fingerprints, and 16S rDNA sequence data were used for the phenotypic and genotypic characterization of thermophilic bacteria. Totally 9 different bacterial strains were selected based on morphological, physiological, and biochemical tests. These strains were characterized by molecular tests including fatty acid and BOX profiles, and 16S rDNA sequence. The data of fatty acid analysis showed the presence of 14 different fatty acids in the 9 bacterial strains examined. Additionally, 5 of these fatty acids, 15:0 iso, 15:0 anteiso, 16:0 iso, 17:0 iso, and 17:0 anteiso fatty acids, were found in all isolates. Based on fatty acid profiles, it was determined that the bacterial strains were classified into 2 phenotypic groups, and these data were confirmed by BOX PCR genomic fingerprint profiles and 16S rDNA sequence analysis. The first group, identified as Bacillus licheniformis, was represented by 4 strains, and the second group, identified as Aeribacillus pallidus, was represented by 5 strains.

Dynamics, Diversity and Function of Endophytic Siderophore-Producing Bacteria in Rice

Siderophore production confers to bacteria competitive advantages to colonize plant tissues and to exclude other microorganisms from the same ecological niche. This work shows that the community of endophytic siderophore-producing bacteria (SPB) associated to Oryza sativa cultivated in Uruguayan soils is dynamic and diverse. These bacteria were present in grains, roots, and leaves, and their density fluctuated between log(10) 3.44 and log(10) 5.52 cfu g(-1) fresh weight (fw) during the plant growth. Less than 10% of the heterotrophic bacteria produced siderophores in roots and leaves of young plants, but most of the heterotrophic bacteria were siderophore-producers in mature plants. According to their amplified restriction DNA ribosomal analysis (ARDRA) pattern, 54 of the 109 endophytic SPB isolated from different plant tissues or growth stages from replicate plots, were unique. Bacteria belonging to the genera Sphingomonas, Pseudomonas, Burkholderia, and Enterobacter alternated during plant growth, but the genus Pantoea was predominant in roots at tillering and in leaves at subsequent stages. Pantoea ananatis was the SPB permanently associated to any of the plant tissues, but the genetic diversity within this species-revealed by BOX-PCR fingerprinting- showed that different strains were randomly distributed along time and plant tissue, suggesting that a common trait of the species P. ananatis determined the interaction with the rice plant. Several isolates were stronger IAA producers than Azospirillum brasilense or Herbaspirillum seropedicae. In vitro inhibition assays showed that SPB of the genus Burkholderia were good antagonists of pathogenic fungi and that only one SPB isolate of the genus Pseudomonas was able to inhibit A. brasilense and H. seropedicae. These results denoted that SPB were selected into the rice plant. P. ananatis was the permanent and dominant associated species which was unable to inhibit two of the relevant plant growth-promoting bacteria.

Rhizobium vallis sp. nov., isolated from nodules of three leguminous species

Four bacterial strains isolated from root nodules of Phaseolus vulgaris, Mimosa pudica and Indigofera spicata plants grown in the Yunnan province of China were identified as a lineage within the genus Rhizobium according to the analysis of 16S rRNA gene sequences, sharing most similarity with Rhizobium lusitanum P1-7(T) (99.1 % sequence similarity) and Rhizobium rhizogenes IAM 13570(T) (99.0 %). These strains also formed a distinctive group from the reference strains for defined species of the genus Rhizobium in a polyphasic approach, including the phylogenetic analyses of the 16S rRNA gene and housekeeping genes (recA, atpD, glnII), DNA-DNA hybridization, BOX-PCR fingerprinting, phenotypic characterization, SDS-PAGE of whole-cell proteins, and cellular fatty acid profiles. All the data obtained in this study suggested that these strains represent a novel species of the genus Rhizobium, for which the name Rhizobium vallis sp. nov. is proposed. The DNA G+C content (mol%) of this species varied between 60.9 and 61.2 (T(m)). The type strain of R. vallis sp. nov. is CCBAU 65647(T) ( = LMG 25295(T) =HAMBI 3073(T)), which has a DNA G+C content of 60.9 mol% and forms effective nodules on Phaseolus vulgaris.

Diversity of Burkholderia cepacia Complex from the Moso Bamboo (Phyllostachys edulis) Rhizhosphere Soil

The purpose of this study was to determine the existence of Burkholderia cepacia complex (Bcc) at species level and the predominant species in the environment of moso bamboo plantations in Hangzhou, China. A total of 423 isolates were recovered from moso bamboo rhizhosphere soil samples of three sites on the selective medium during 2007-2008. Isolates were identified by Bcc-specific PCR assays, followed by recA-restriction fragment length polymorphism assays, species-specific PCR analysis, recA gene sequencing, multilocus sequence typing (MLST) scheme, and BOX-PCR fingerprinting for genomic diversity. Out of 423 isolates, 278 isolates were assigned to the following Bcc species, eight B. stabilis, 26 B. anthina, 193 B. pyrrocinia, and 51 B. arboris, which indicated B. pyrrocinia as the most dominant species followed by B. arboris. Moreover, false positives were observed in certain isolates of B. arboris while performing species-specific PCR test. Furthermore, the results of recA gene sequence similarity and MLST data demonstrated that nine isolates formed a single discrete cluster but were PCR negative to species-specific primers representing novel species may exist within the Bcc. In addition, BOX-PCR fingerprinting for all the Bcc isolates also showed the strain diversity. It is the first report of the existence of B. arboris and predominance of B. pyrrocinia in the moso bamboo environment.

Identification of Aeromonas salmonicida in European perch from North Lithuanian rivers during mass mortalities in 2008

Abstract The investigation was carried out to ascertain the cause of mass mortalities in European perch (Perca fluviatilis L.) in North Lithuanian rivers in 2008 that reached a maximum in mid-autumn at a water temperature of 6°C. Marked changes were detected in morphophysiological parameters (spleen-, gill-, liver- and heart-somatic indices), which corresponded to changes in haematological parameters (leukocyte count was significantly elevated or reduced) of the infected fish. The viable bacterial counts in the gill, liver and kidney of infected live fish samples ranged from 2.3±0.3 × 103 to 6.3×00B1;0.4 × 103 c.f.u./g. The BOX-PCR fingerprinting technique was used for characterization and identification of the isolated bacterial strains from liver and kidney of infected perch. The bacterial isolates were identified by sequence analysis of the 16S rRNA gene. Pathogenic Aeromonas salmonicida subsp. salmonicida has been identified as a possible causative agent of the observed mortality in European perch.

Comparative biogeography of Chromobacterium from the neotropics

The genus Chromobacterium encompasses free-living Gram-negative bacteria. Until 2007, the genus consisted of only one species but six species are now recognized. Chromobacterium violaceum is the type species of the genus and is commonly found in soil and water in tropical and sub-tropical regions. We have investigated a collection of 111 isolates displaying violet pigmentation from undisturbed aquatic and soil environments from Brazilian Cerrado ecosystem. The 16S rRNA gene phylogeny revealed that all isolates were allocated in a monophyletic cluster inside the Chromobacterium genus and formed few clusters related most closely with Chromobacterium piscinae. The two sets of isolates from water and soil were analyzed by the repetitive extragenic palindromic (rep)-PCR genomic fingerprinting technique using a BOX-AR1 primer. The antimicrobial susceptibility and the different carbon sources utilized by these isolates were also investigated. Physiological profiles of the isolates generated by BIOLOG GN2 plates showed great versatility in the substrate utilization, much higher than the C. violaceum ATCC 12472. All isolates exhibited a high minimum inhibitory concentration (MIC) to ampicillin (MIC>512μg/ml) and were inhibited by ciprofloxacin, tetracycline and mercury at the lowest concentration tested (MIC<2μg/ml). Thirteen BOX-PCR band patterns were identified from 33 individual fingerprints. Eleven patterns provided evidence for endemic distributions. Antimicrobial susceptibility and BOX-PCR fingerprint clustering showed a clear distinction between Chromobacterium isolates from the water and soil. The results suggested that microenvironment barriers such as water and soil can play an important role in the periodic selection and diversification of Chromobacterium population ecotypes.

Diversity of culturable thermo-resistant aquatic bacteria along an environmental gradient in Cuatro Ciénegas, Coahuila, México

At the desert oasis of Cuatro Ciénegas in Coahuila, México, more than 300 oligotrophic pools can be found and a large number of endemic species of plants and animals. The most divergent taxa of diatoms, snail and fishes are located in the Churince hydrological system, where we analyzed the local diversification of cultivable Firmicutes and Actinobacteria. The Churince hydrological system is surrounded by gypsum dunes and has a strong gradient for salinity, temperature, pH and dissolved oxygen. In August 2003, surface water samples were taken in 10 sites along the Churince system together with the respective environmental measurements. 417 thermo-resistant bacteria were isolated and DNA was extracted to obtain their BOX-PCR fingerprints, revealing 55 different patterns. In order to identify similarities and differences in the diversity of the various sampling sites, an Ordination Analysis was applied using Principal Component Analysis. This analysis showed that conductivity is the environmental factor that explains the distribution of most of the microbial diversity. Phylogenetic reconstruction from their 16S rRNA sequences was performed for a sample of 150 isolates. Only 17 sequences had a 100% match in the Gene Bank (NCBI), representing 10 well known cosmopolitan taxa. The rest of the sequences cluster in 22 clades for Firmicutes and another 22 clades for Actinobacteria, supporting the idea of high diversity and differentiation for this site.

Genotyping of Aeromonas hydrophila by Box elements

PCR-based DNA fingerprinting techniques were evaluated to genotype eight diseased, particularly normal and environmental isolates of Aeromonas hydrophila. PCR-based fingerprinting method has an advantage of having repetitive sequence also called Box elements that are interspersed throughout the genome in diverse bacterial species. The BOX-PCR fingerprinting technique was evaluated for the discrimination of different isolates of A. hydrophila. All the studied isolates have shown major banding patterns ranged from 500-3000 bp. These finding could be advantageous to investigate the strain level specific fingerprints of A. hydrophila as potential genotypic markers.

The genus Micromonospora is widespread in legume root nodules: the example of Lupinus angustifolius

Our current knowledge of plant-microbe interactions indicate that populations inhabiting a host plant are not restricted to a single microbial species but comprise several genera and species. No one knows if communities inside plants interact, and it has been speculated that beneficial effects are the result of their combined activities. During an ecological study of nitrogen-fixing bacterial communities from Lupinus angustifolius collected in Spain, significant numbers of orange-pigmented actinomycete colonies were isolated from surface-sterilized root nodules. The isolates were analysed by BOX-PCR fingerprinting revealing an unexpectedly high genetic variation. Selected strains were chosen for 16S rRNA gene sequencing and phylogenetic analyses confirmed that all strains isolated belonged to the genus Micromonospora and that some of them may represent new species. To determine the possibility that the isolates fixed atmospheric nitrogen, chosen strains were grown in nitrogen-free media, obtaining in some cases, significant growth when compared with the controls. These strains were further screened for the presence of the nifH gene encoding dinitrogenase reductase, a key enzyme in nitrogen fixation. The partial nifH-like gene sequences obtained showed a 99% similarity with the sequence of the nifH gene from Frankia alni ACN14a, an actinobacterium that induces nodulation and fixes nitrogen in symbiosis with Alnus. In addition, in situ hybridization was performed to determine if these microorganisms inhabit the inside of the nodules. This study strongly suggests that Micromonospora populations are natural inhabitants of nitrogen-fixing root nodules.

Ruegeria pelagia is a later heterotypic synonym of Ruegeria mobilis

The 16S rRNA genes of Ruegeria pelagia NBRC 102038T and Ruegeria mobilis NBRC 101030T were resequenced and the results confirmed that they differ by only one base in their almost full-length sequences (1425 nt). The gyrB gene sequence similarity between the two strains was also high (97.7%). The outcome of API 20NE, API ZYM and antibiotic susceptibility tests showed that the two strains show only one difference, in beta-galactosidase activity, in API tests and five differences in susceptibility among 30 tested antibiotics. In addition, similar BOX-PCR fingerprints were obtained and the DNA-DNA relatedness between the two strains was 91+/-4%. On the basis of these results, it is concluded that Ruegeria pelagia Lee et al. 2007 is a later heterotypic synonym of Ruegeria mobilis Muramatsu et al. 2007.