Bradyrhizobium arachidis sp. nov., isolated from effective nodules of Arachis hypogaea grown in China

Twenty-three bacterial strains isolated from root nodules of Arachis hypogaea and Lablab purpureus grown in five provinces of China were classified as a novel group within the genus Bradyrhizobium by analyses of PCR-based RFLP of the 16S rRNA gene and 16S–23S IGS. To determine their taxonomic position, four representative strains were further characterized. The comparative sequence analyses of 16S rRNA and six housekeeping genes clustered the four strains into a distinctive group closely related to the defined species Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense, Bradyrhizobium huanghuaihaiense, Bradyrhizobium japonicum and Bradyrhizobium daqingense. The DNA–DNA relatedness between the reference strain of the novel group, CCBAU 051107^T, and the corresponding type strains of the five mentioned species varied between 46.05% and 13.64%. The nodC and nifH genes of CCBAU 051107^T were phylogenetically divergent from those of the reference strains for the related species. The four representative strains could nodulate with A. hypogaea and L. purpureus. In addition, some phenotypic features differentiated the novel group from the related species. Based on all the results, we propose a new species Bradyrhizobium arachidis sp. nov. and designate CCBAU 051107^T (=CGMCC 1.12100^T = HAMBI 3281^T = LMG 26795^T) as the type strain, which was isolated from a root nodule of A. hypogaea and had a DNA G + C mol% of 60.1 (Tm).     

Definition of a novel symbiovar (sv. retamae) within Bradyrhizobium retamae sp. nov., nodulating Retama sphaerocarpa and Retama monosperma

In this paper we analyze through a polyphasic approach several Bradyrhizobium strains isolated in Spain and Morocco from root nodules of Retama sphaerocarpa and Retama monosperma. All the strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium lablabi CCBAU 23086^T, with 99.41% identity with respect to the strain Ro19^T. Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII were divergent in Ro19^T and B. lablabi CCBAU 23086^T, with identity values of 95.71%, 93.75% and 93.11%, respectively. These differences were congruent with DNA–DNA hybridization analysis that revealed an average of 35% relatedness between the novel species and B. lablabi CCBAU 23086^T. Also, differential phenotypic characteristics of the new species were found with respect to the already described species of Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose to classify the group of strains isolated from R. sphaerocarpa and R. monosperma as a novel species named Bradyrhizobium retamae sp. nov. (type strain Ro19^T = LMG 27393^T = CECT 8261^T). The analysis of symbiotic genes revealed that some of these strains constitute a new symbiovar within genus Bradyrhizobium for which we propose the name “retamae”, that mainly contains nodulating strains isolated from Retama species in different continents.     

Bradyrhizobium xenonodulans sp. nov. isolated from nodules of Australian Acacia species invasive to South Africa

A genealogical concordance approach was used to delineate strains isolated from Acacia dealbata and Acacia mearnsii root nodules in South Africa. These isolates form part of Bradyrhizobium based on 16S rRNA sequence similarity. Phylogenetic analysis of six housekeeping genes (atpD, dnaK, glnII, gyrB, recA and rpoB) confirmed that these isolates represent a novel species, while pairwise average nucleotide identity (ANIb) calculations with the closest type strains (B. cosmicum 58S1^T, B. betae PL7HG1^T, B. ganzhouense CCBAU 51670 ^T, B. cytisi CTAW11^T and B. rifense CTAW71^T) resulted in values well below 95–96%. We further performed phenotypic tests which revealed that there are high levels of intraspecies variation, while an additional analysis of the nodA and nifD loci indicated that the symbiotic loci of the strains are closely related to those of Bradyrhizobium isolates with an Australian origin. Strain 14AB^T (=LMG 31415 ^T = SARCC-753 ^T) is designated as the type strain of the novel species for which we propose the name Bradyrhizobium xenonodulans sp. nov.     

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

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

Phylogenetic multilocus sequence analysis of indigenous slow-growing rhizobia nodulating cowpea (Vigna unguiculata L.) in Greece

Cowpea (Vigna unguiculata) is a promiscuous grain legume, capable of establishing efficient symbiosis with diverse symbiotic bacteria, mainly slow-growing rhizobial species belonging to the genus Bradyrhizobium. Although much research has been done on cowpea-nodulating bacteria in various countries around the world, little is known about the genetic and symbiotic diversity of indigenous cowpea rhizobia in European soils. In the present study, the genetic and symbiotic diversity of indigenous rhizobia isolated from field-grown cowpea nodules in three geographically different Greek regions were studied. Forty-five authenticated strains were subjected to a polyphasic approach. ERIC-PCR based fingerprinting analysis grouped the isolates into seven groups and representative strains of each group were further analyzed. The analysis of the rrs gene showed that the strains belong to different species of the genus Bradyrhizobium. The analysis of the 16S-23S IGS region showed that the strains from each geographic region were characterized by distinct IGS types which may represent novel phylogenetic lineages, closely related to the type species of Bradyrhizobium pachyrhizi, Bradyrhizobium ferriligni and Bradyrhizobium liaoningense. MLSA analysis of three housekeeping genes (recA, glnII, and gyrB) showed the close relatedness of our strains with B. pachyrhizi PAC48^T and B. liaoningense USDA 3622^T and confirmed that the B. liaoningense-related isolate VUEP21 may constitute a novel species within Bradyrhizobium. Moreover, symbiotic gene phylogenies, based on nodC and nifH genes, showed that the B. pachyrhizi-related isolates belonged to symbiovar vignae, whereas the B. liaoningense-related isolates may represent a novel symbiovar.     

Bradyrhizobium aeschynomenes sp. nov., a root and stem nodule microsymbiont of Aeschynomene indica

Aeschynomene indica has a distinctive symbiosis with Bradyrhizobium in which nodulation is Nod factor-independent. In this study, we characterised three Gram-negative and rod-shaped strains (83002^T, 81013 and 83012) isolated from root nodules of Aeschynomene indica in Shandong Peninsula. The major cellular fatty acids of isolates were C_16:0, C_18:0, C_18:1 ω7c 11-methyl, summed feature 3 and summed feature 8. The major polar lipids were phosphatidylethanolamine (PE), aminolipids (AL) and phosphatidylcholine (PC). Phylogenetic analysis based on the 16S rRNA locus showed that they belonged to the Bradyrhizobium genus, and shared the highest similarity to the type strains Bradyrhizobium oligotrophicum S58^T and Bradyrhizobium denitrificans LMG 8443^T. As expected, analysis of concatenated sequences of six housekeeping genes (atpD, recA, glnII, dnaK, gyrB, and rpoB) and nifH gene proposed that these three strains formed a distinct clade within the genus Bradyrhizobium. The highest average nucleotide identity and DNA-DNA hybridization values of the three strains in comparison to the closest Bradyrhizobium species were 87.5% and 65.3%, respectively, which are far below the threshold of species delineation, and thus confirmed the three strains as a new species. The genome size of strain 83002^T is 7.52 Mbp, and the DNA G+C content is 65.42 mol%. Strain 83002^T (=KCTC 82266^T=MCCC 1K04775^T) was chosen as the type strain of the new species, for which the name Bradyrhizobium aeschynomenes sp. nov. was proposed.     

Rhizobium cauense sp. nov., isolated from root nodules of the herbaceous legume Kummerowia stipulacea grown in campus lawn soil

Three bacterial isolates (CCBAU 101002^T, CCBAU 101000 and CCBAU 101001) originating from root nodules of the herbaceous legume Kummerowia stipulacea grown in the campus lawn of China Agricultural University were characterized with a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence analysis showed that the isolates shared 99.85–99.92% sequence similarities and had the highest similarities to the type strains of Rhizobium mesoamericanum (99.31%), R. endophyticum (98.54%), R. tibeticum (98.38%) and R. grahamii (98.23%). Sequence similarity of four concatenated housekeeping genes (atpD, glnII, recA and rpoB) between CCBAU 101002^T and its closest neighbor (R. grahamii) was 92.05%. DNA–DNA hybridization values between strain CCBAU 101002^T and the four type strains of the most closely related Rhizobium species were less than 28.4 ± 0.8%. The G + C mol% of the genomic DNA for strain CCBAU 101002^T was 58.5% (Tm). The major respiratory quinone was ubiquinone (Q-10). Summed feature 8 (18:1ω7cis/18:1ω6cis) and 16:0 were the predominant fatty acids. Strain CCBAU 101002^T contained phosphatidylcholine and phosphatidylethanolamine as major polar lipids, and phosphatidylglycerol and cardiolipin as minor ones. No glycolipid was detected. Unlike other strains, this novel species could utilize dulcite or sodium pyruvate as sole carbon sources and it was resistant to 2% (w/v) NaCl. On the basis of the polyphasic study, a new species Rhizobium cauense sp. nov. is proposed, with CCBAU 101002^T (=LMG 26832^T = HAMBI 3288^T) as the type strain.     

Rhizobium croatiense sp. nov. and Rhizobium redzepovicii sp. nov., two new species isolated from nodules of Phaseolus vulgaris in Croatia

Phaseolus vulgaris is a legume indigenous to America which is nodulated by strains of genus Rhizobium in Croatia. Four of these strains, 13T^T, 9T, 18T^T and 8Z are phylogenetically close to the species from the Rhizobium leguminosarum phylogenetic complex in the 16S rRNA gene analysis. The results of both the analyses of the concatenated recA and atpD genes and whole genomes revealed that the strains 13T^T and 9T clustered with Rhizobium sophoriradicis CCBAU 03470^T and the strains 18T^T and 8Z with Rhizobium ecuadorense CNPSO 671^T. Whole genome average nucleotide identity blast (ANIb) and dDDH values between the strains 13T^T and the type strain of R. sophoriradicis and between the strains 18T^T and the type strain of R. ecuadorense were lower than 95% and 70%, respectively, which are the threshold values recommended for bacterial species differentiation. These results combined with those of chemotaxonomic and phenotypic analyses support the affiliation of these strains to two novel species within the genus Rhizobium for which we propose the names Rhizobium croatiense sp. nov. 13T^T (=LMG 32397^T, = HAMBI 3740^T) as type strain and Rhizobium redzepovicii sp. nov. 18T^T (=LMG 32398^T, = HAMBI 3741^T) as type strain.     

Wild peanut Arachis duranensis are nodulated by diverse and novel Bradyrhizobium species in acid soils

Aiming at learning the microsymbionts of Arachis duranensis, a diploid ancestor of cultivated peanut, genetic and symbiotic characterization of 32 isolates from root nodules of this plant grown in its new habitat Guangzhou was performed. Based upon the phylogeny of 16S rRNA, atpD and recA genes, diverse bacteria belonging to Bradyrhizobium yuanmingense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense and four new lineages of Bradyrhizobium (19 isolates), Rhizobium/Agrobacterium (9 isolates), Herbaspirillum (2 isolates) and Burkholderia (2 isolates) were defined. In the nodulation test on peanut, only the bradyrhizobial strains were able to induce effective nodules. Phylogeny of nodC divided the Bradyrhizobium isolates into four lineages corresponding to the grouping results in phylogenetic analysis of housekeeping genes, suggesting that this symbiosis gene was mainly maintained by vertical gene transfer. These results demonstrate that A. duranensis is a promiscuous host preferred the Bradyrhizobium species with different symbiotic gene background as microsymbionts, and that it might have selected some native rhizobia, especially the novel lineages Bradyrhizobium sp. I and sp. II, in its new habitat Guangzhou. These findings formed a basis for further study on adaptation and evolution of symbiosis between the introduced legumes and the indigenous rhizobia.     

Putative novel Bradyrhizobium and Phyllobacterium species isolated from root nodules of Chamaecytisus ruthenicus

In this study, the diversity and the phylogenetic relationships of bacteria isolated from root nodules of Chamaecytisus ruthenicus growing in Poland were investigated using ERIC-PCR fingerprinting and by multilocus sequence analysis (MLSA). Two major clusters comprising 13 and 3 isolates were detected which 16S rRNA gene sequencing identified as Bradyrhizobium and Phyllobacterium. The results of phylogenetic analysis of individual and concatenated atpD, gyrB and recA gene sequences showed that the studied strains may represent novel species in the genera Bradyrhizobium and Phyllobacterium. In the phylogenetic tree based on the atpD-gyrB-recA concatemers, Bradyrhizobium isolates were split into two groups closely related to Bradyrhizobium algeriense STM89^T and Bradyrhizobium valentinum LmjM3^T. The genus Phyllobacterium isolates formed a separate cluster close to Phyllobacterium ifriqiyense LMG27887^T in the atpD-gyrB-recA phylogram. Analysis of symbiotic gene sequences (nodC, nodZ, nifD, and nifH) showed that the Bradyrhizobium isolates were most closely related to Bradyrhizobium algeriense STM89^T, Bradyrhizobium valentinum LmjM3^T and Bradyrhizobium retamae Ro19^T belonging to symbiovar retamae. This is the first report on the occurrence of members of symbiovar retamae from outside the Mediterranean region. No symbiosis related genes were amplified from Phyllobacterium strains, which were also unable to induce nodules on C. ruthenicus roots. Based on these findings Phyllobacterium isolates can be regarded as endophytic bacteria inhabitating root nodules of C. ruthenicus.     

Gilliamella intestini sp. nov., Gilliamella bombicola sp. nov., Gilliamella bombi sp. nov. and Gilliamella mensalis sp. nov.: Four novel Gilliamella species isolated from the bumblebee gut

Spectra of five isolates (LMG 28358^T, LMG 29879^T, LMG 29880^T, LMG 28359^T and R-53705) obtained from gut samples of wild bumblebees of Bombus pascuorum, Bombus lapidarius and Bombus terrestris were grouped into four MALDI-TOF MS clusters. RAPD analysis revealed an identical DNA fingerprint for LMG 28359^T and R-53705 which also grouped in the same MALDI-TOF MS cluster, while different DNA fingerprints were obtained for the other isolates.Comparative 16S rRNA gene sequence analysis of the four different strains identified Gilliamella apicola NCIMB 14804^T as nearest neighbour species. Average nucleotide identity values of draft genome sequences of the four isolates and of G. apicola NCIMB 14804^T were below the 96% threshold value for species delineation and all four strains and G. apicola NCIMB 14804^T were phenotypically distinct. Together, the draft genome sequences and phylogenetic and phenotypic data indicate that the four strains represent four novel Gilliamella species for which we propose the names Gilliamella intestini sp. nov., with LMG 28358^T as the type strain, Gilliamella bombicola sp. nov., with LMG 28359^T as the type strain, Gilliamella bombi sp. nov., with LMG 29879^T as the type strain and Gilliamella mensalis sp. nov., with LMG 29880^T as the type strain.     

Genetic diversity and distribution of Bradyrhizobium and Azorhizobium strains associated with the herb legume Zornia glochidiata sampled from across Senegal

Herb legumes have great potential for rehabilitation of semi-arid degraded soils in Sahelian ecosystems as they establish mutualistic symbiosis with N₂-fixing rhizobia. A phylogenetic analysis was performed for 78 root nodule bacteria associated with the common Sahelian herb legume Zornia glochidiata Reichb ex DC in Senegal. Based on ITS (rDNA16S-23S) and recA sequences, these strains were shown to belong to the two genera Bradyrhizobium and Azorhizobium. Strains of this latter, although frequent, formed small and ineffective nodules and suggested a parasitism rather than a symbiotic association. A potential negative effect of Azorhizobium on Zornia growth was tested for when inoculated alone or in association with a Bradyrhizobium strain. Bradyrhizobium isolates were distributed in four groups. Groups A and B were two sister clades in a larger monophyletic group also including Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense, and Bradyrhizobium japonicum. Strains of cluster D fell in a sister clade of the photosynthetic Bradyrhizobium sp. group, including ORS278, whereas group C appeared to be divergent from all known Bradyrhizobium clusters. Amplified fragment length polymorphism (AFLP) clustering was congruent with ITS and recA phylogenies, but displayed much more variability. However, within the main Bradyrhizobium clades, no obvious relationship could be detected between clustering and geographical origin of the strains. Each sub-cluster included strains sampled from different locations. Conversely, Azorhizobium strains showed a tendency in the phylogeny to group together according to the site of sampling. The predominance of ineffective Azorhizobium strains in the nodules of Zornia roots, the large Bradyrhizobium genetic diversity and the geographical genetic diversity pattern are explored.     

Burkholderia sp. Induces Functional Nodules on the South African Invasive Legume Dipogon lignosus (Phaseoleae) in New Zealand Soils

The South African invasive legume Dipogon lignosus (Phaseoleae) produces nodules with both determinate and indeterminate characteristics in New Zealand (NZ) soils. Ten bacterial isolates produced functional nodules on D. lignosus. The 16S ribosomal RNA (rRNA) gene sequences identified one isolate as Bradyrhizobium sp., one isolate as Rhizobium sp. and eight isolates as Burkholderia sp. The Bradyrhizobium sp. and Rhizobium sp. 16S rRNA sequences were identical to those of strains previously isolated from crop plants and may have originated from inocula used on crops. Both 16S rRNA and DNA recombinase A (recA) gene sequences placed the eight Burkholderia isolates separate from previously described Burkholderia rhizobial species. However, the isolates showed a very close relationship to Burkholderia rhizobial strains isolated from South African plants with respect to their nitrogenase iron protein (nifH), N-acyltransferase nodulation protein A (nodA) and N-acetylglucosaminyl transferase nodulation protein C (nodC) gene sequences. Gene sequences and enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive element palindromic PCR (rep-PCR) banding patterns indicated that the eight Burkholderia isolates separated into five clones of one strain and three of another. One strain was tested and shown to produce functional nodules on a range of South African plants previously reported to be nodulated by Burkholderia tuberum STM678^T which was isolated from the Cape Region. Thus, evidence is strong that the Burkholderia strains isolated here originated in South Africa and were somehow transported with the plants from their native habitat to NZ. It is possible that the strains are of a new species capable of nodulating legumes.     

Photosynthetic Bradyrhizobia from Aeschynomene spp. Are Specific to Stem-Nodulated Species and Form a Separate 16S Ribosomal DNA Restriction Fragment Length Polymorphism Group

We obtained nine bacterial isolates from root or collar nodules of the non-stem-nodulated Aeschynomene species A. elaphroxylon, A. uniflora, or A. schimperi and 69 root or stem nodule isolates from the stem-nodulated Aeschynomene species A. afraspera, A. ciliata, A. indica, A. nilotica, A. sensitiva, and A. tambacoundensis from various places in Senegal. These isolates, together with 45 previous isolates from various Aeschynomene species, were studied for host-specific nodulation within the genus Aeschynomene, also revisiting cross-inoculation groups described previously by D. Alazard (Appl. Environ. Microbiol. 50:732–734, 1985). The whole collection of Aeschynomene nodule isolates was screened for synthesis of photosynthetic pigments by spectrometry, high-pressure liquid chromatography, and thin-layer chromatography analyses. The presence of puf genes in photosynthetic Aeschynomene isolates was evidenced both by Southern hybridization with a Rhodobacter capsulatus photosynthetic gene probe and by DNA amplification with primers defined from photosynthetic genes. In addition, amplified 16S ribosomal DNA restriction analysis was performed on 45 Aeschynomene isolates, including strain BTAi1, and 19 reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii, and other Bradyrhizobium sp. strains of uncertain taxonomic positions. The 16S rRNA gene sequence of the photosynthetic strain ORS278 (LMG 12187) was determined and compared to sequences from databases. Our main conclusion is that photosynthetic Aeschynomene nodule isolates share the ability to nodulate particular stem-nodulated species and form a separate subbranch on the Bradyrhizobium rRNA lineage, distinct from B. japonicum and B. elkanii.     

Photosynthetic Bradyrhizobia Are Natural Endophytes of the African Wild Rice Oryza breviligulata

We investigated the presence of endophytic rhizobia within the roots of the wetland wild rice Oryza breviligulata, which is the ancestor of the African cultivated rice Oryza glaberrima. This primitive rice species grows in the same wetland sites as Aeschynomene sensitiva, an aquatic stem-nodulated legume associated with photosynthetic strains of Bradyrhizobium. Twenty endophytic and aquatic isolates were obtained at three different sites in West Africa (Senegal and Guinea) from nodal roots of O. breviligulata and surrounding water by using A. sensitiva as a trap legume. Most endophytic and aquatic isolates were photosynthetic and belonged to the same phylogenetic Bradyrhizobium/Blastobacter subgroup as the typical photosynthetic Bradyrhizobium strains previously isolated from Aeschynomene stem nodules. Nitrogen-fixing activity, measured by acetylene reduction, was detected in rice plants inoculated with endophytic isolates. A 20% increase in the shoot growth and grain yield of O. breviligulata grown in a greenhouse was also observed upon inoculation with one endophytic strain and one Aeschynomene photosynthetic strain. The photosynthetic Bradyrhizobium sp. strain ORS278 extensively colonized the root surface, followed by intercellular, and rarely intracellular, bacterial invasion of the rice roots, which was determined with a lacZ-tagged mutant of ORS278. The discovery that photosynthetic Bradyrhizobium strains, which are usually known to induce nitrogen-fixing nodules on stems of the legume Aeschynomene, are also natural true endophytes of the primitive rice O. breviligulata could significantly enhance cultivated rice production.     

Possible contribution of Bradyrhizobium on nitrogen fixation in sweet potatoes

Aims

Sweet potato (Ipomoea batatas) is known for its ability to grow under nitrogen-limited conditions. To clarify the possible contribution of biological nitrogen fixation, we tried to isolate and identify diazotrophic bacteria from sweet potatoes.

Methods

By using cultivation technique, we isolated putative endophytes, which possess nifH genes, from surface-sterilized sweet potatoes. Their nitrogen-fixing abilities were demonstrated by the acetylene reduction assay in a semi-solid malate medium and sweet potato extracts. We also examined the colonization of an isolated strain (AT1) in sweet potatoes and their influence on growth and nitrogen fixation in plants as assessed by an acetylene reduction assay and ¹⁵N-isotope dilution technique.

Results

The isolates were identified as strains of Bradyrhizobium sp. AT1, Paenibacillus sp. AS2 and Pseudomonas sp. T16 based on their 16S rRNA gene sequences. They showed acetylene reduction activity (ARA) in the semi-solid malate medium. Among them, B. sp. AT1 showed ARA in sweet potato extracts under micro-aerobic conditions whereas both P. sp. AS2 and P. sp. T16 showed no ARA. The inoculation of B. sp. AT1 to the sweet potatoes resulted in increases in the fresh weights and detection of ARA in the inoculated plants. Moreover, the reduction of ¹⁵N atom % was observed in the inoculated plants compared to uninoculated controls.

Conclusions

B. sp. AT1 actively expresses nitrogenase activity in sweet potatoes and may contribute to the nitrogen nutrition of host plants.     

Photobacterium piscicola sp. nov., isolated from marine fish and spoiled packed cod

Five isolates from marine fish (W3^T, WM, W1S, S2 and S3) and three isolates misclassified as Photobacterium phosphoreum, originating from spoiled modified atmosphere packed stored cod (NCIMB 13482 and NCIMB 13483) and the intestine of skate (NCIMB 192), were subjected to a polyphasic taxonomic study. Phylogenetic analysis of 16S rRNA gene sequences showed that the isolates were members of the genus Photobacterium. Sequence analysis using the gapA, gyrB, pyrH, recA and rpoA loci showed that these isolates formed a distinct branch in the genus Photobacterium, and were most closely related to Photobacterium aquimaris, Photobacterium kishitanii, Photobacterium phosphoreum and Photobacterium iliopiscarium. The luxA gene was present in isolates W3^T, WM, W1S, S2 and S3 but not in NCIMB 13482, NCIMB 13483 and NCIMB 192. AFLP and (GTG)₅-PCR fingerprinting indicated that the eight isolates represented at least five distinct genotypes. DNA–DNA hybridizations revealed 89% relatedness between isolate W3^T and NCIMB 192, and values below 70% with the type strains of the phylogenetically closest species, P. iliopiscarium LMG 19543^T, P. kishitanii LMG 23890^T, P. aquimaris LMG 26951^T and P. phosphoreum LMG4233^T. The strains of this new taxon could also be distinguished from the latter species by phenotypic characteristics. Therefore, we propose to classify this new taxon as Photobacterium piscicola sp. nov., with W3^T (=NCCB 100098^T = LMG 27681^T) as the type strain.