recA Gene Sequence Analysis Research Articles

Phylogenetic diversity of Rhizobium species recovered from nodules of common beans (Phaseolus vulgaris L.) in fields in Uganda: R. phaseoli, R. etli, and R. hidalgonense.

A total of 75 bacterial isolates were obtained from nodules of beans cultivated across 10 sites in six agro-ecological zones in Uganda. Using recA gene sequence analysis, 66 isolates were identified as members of the genus Rhizobium, while nine were related to Agrobacterium species. In the recA gene tree, most Rhizobium strains were classified into five recognized species. Phylogenetic analysis based on six concatenated sequences (recA-rpoB-dnaK-glnII-gyrB-atpD) placed 32 representative strains into five distinct Rhizobium species, consistent with the species groups observed in the recA gene tree: R. phaseoli, R. etli, R. hidalgonense, R. ecuadorense, and R. sophoriradicis, with the first three being the predominant. The rhizobial strains grouped into three nodC subclades within the symbiovar phaseoli clade, encompassing strains from distinct phylogenetic groups. This pattern reflects the conservation of symbiotic genes, likely acquired through horizontal gene transfer among diverse rhizobial species. The 32 representative strains formed symbiotic relationships with host beans, while the Agrobacterium strains did not form nodules and lacked symbiotic genes. Multivariate analysis revealed that species distribution was influenced by the environmental factors of the sampling sites, emphasizing the need to consider these factors in future effectiveness studies to identify effective nitrogen-fixing strains for specific locations.

Volatile organic compounds emitted by Burkholderia pyrrocinia CNUC9 trigger induced systemic salt tolerance in Arabidopsis thaliana.

Salinity is among the most significant abiotic stresses that negatively affects plant growth and agricultural productivity worldwide. One ecofriendly tool for broadly improving plant tolerance to salt stress is the use of bio-inoculum with plant growth-promoting rhizobacteria (PGPR). In this study, a bacterium strain CNUC9, which was isolated from maize rhizosphere, showed several plant growth-promoting characteristics including the production of 1-aminocyclopropane-1-carboxylate deaminase, indole acetic acid, siderophore, and phosphate solubilization. Based on 16S rRNA and recA gene sequence analysis, we identified strain CNUC9 as Burkholderia pyrrocinia. Out of bacterial determinants to elicit plant physiological changes, we investigated the effects of volatile organic compounds (VOCs) produced by B. pyrrocinia CNUC9 on growth promotion and salinity tolerance in Arabidopsis thaliana. Higher germination and survival rates were observed after CNUC9 VOCs exposure under 100 mM NaCl stress. CNUC9 VOCs altered the root system architecture and total leaf area of A. thaliana compared to the control. A. thaliana exposed to VOCs induced salt tolerance by increasing its total soluble sugar and chlorophyll content. In addition, lower levels of reactive oxygen species, proline, and malondialdehyde were detected in CNUC9 VOCs-treated A. thaliana seedlings under stress conditions, indicating that VOCs emitted by CNUC9 protected the plant from oxidative damage induced by salt stress. VOC profiles were obtained through solid-phase microextraction and analyzed by gas chromatography coupled with mass spectrometry. Dimethyl disulfide (DMDS), methyl thioacetate, and 2-undecanone were identified as products of CNUC9. Our results indicate that optimal concentrations of DMDS and 2-undecanone promoted growth in A. thaliana seedlings. Our findings provide greater insight into the salt stress alleviation of VOCs produced by B. pyrrocinia CNUC9, as well as potential sustainable agriculture applications.

Paraburkholderia gardini sp. nov. and Paraburkholderia saeva sp. nov.: Novel aromatic compound degrading bacteria isolated from garden and forest soil samples

Three forest and four botanical garden top soil isolates with unique MALDI-TOF mass spectra were identified as Paraburkholderia strains closely related to Paraburkholderia sartisoli through recA gene sequence analysis. OrthoANIu, digital DNA-DNA hybridization analyses and phylogenomic analyses demonstrated that the five strains represented two new Paraburkholderia species closely related to P. sartisoli. The genome of strain LMG 31841T had a cumulative size of 6.3 Mb and a G + C content of 62.64 mol%; strain LMG 32171T had a genome size of 5.8 Mb and a G + C content of 62.91 mol%. Hemolysis on horse blood agar, beta-galactosidase and phosphoamidase activity, and assimilation of adipic acid and trisodium citrate allowed phenotypic differentiation of strains LMG 31841T, LMG 32171T and P. sartisoli LMG 24000T. An analysis of the genomic potential for aromatic compound degradation yielded additional differences among strains representing these three species, but also highlighted some discrepancies between the presence of genes and pathways, and the phenotype revealed through growth experiments using a mineral salts medium supplemented with single aromatic compounds as carbon sources. We propose to classify all isolates from the present study into two novel Paraburkholderia species, for which we propose the names Paraburkholderia gardini with LMG 32171T (=CECT 30344T) as the type strain, and Paraburkholderia saeva with LMG 31841T (=CECT 30338T) as the type strain.

First report of Agrobacterium tumefaciens causing crown gall disease of roselle (Hibiscus sabdariffa L.) in Taiwan.

Roselle (Hibiscus sabdariffa L.) plants, whose calyces are used for production of beverages or jams, are mainly cultivated in Taitung County of eastern Taiwan. Since 2016, large crown galls were observed on the roselle plants in the commercial plantations at Taimali and Jinfong Townships of Taitung County. A follow-up survey in July and August of 2017 revealed spreading of this disease to the neighboring areas including Beinan and Dawu Townships. Disease incidence was estimated to be 0.6-10%. Galls of varying sizes (2-15 cm in diameter) were usually found on the roots and crowns of the roselle plants, starting with small swellings at the infection sites. Galls were light-colored, and smooth and tender in texture at the early stage, but later turned dark-colored, and appeared rough and woody. In some cases, adventitious roots extruding from the larger crown galls could be seen. Isolation of the causal agent was performed by quadrantally streaking bacterial suspension made from surface-sterilized, macerated galls on trypticase soy agar (TSA). After incubating at 28°C for 5 days, single colonies were transferred onto new TSA plates for further cultivation at 28°C. Finally, circular, convex, viscous and milky white colonies with smooth surface similar to colony morphology of Agrobacterium tumefaciens C58 were obtained for further identification. First, all six candidate isolates (TZ-1, TL1-2, TL2-1, TD1-1, TD1-24 and TD2-1) were identified as Agrobacterium spp. using the partial sequences of the 16S rRNA gene (accession numbers MW205820 to MW205825 in the GenBank database). The selected isolates also showed some biochemical and physiological characteristics similar to A. tumefaciens, including oxidase positive, growth at 35°C and in 2% NaCl, and alkalinity from litmus milk. Moreover, they were tested negative for utilization of citrate and acid production on potato dextrose agar (PDA) supplemented with calcium carbonate. Under a transmission electron microscope, the bacterium was rod-shaped and possessed peritrichous flagella. By means of multiplex PCR using primers designed for differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2, a 184 bp product was detected in all six isolates, indicating that they all belong to Agrobacterium biovar 1. Furthermore, the recA allele of each isolate was PCR amplified using primers F2898/F2899, and recA sequence analysis assigned all six isolates to A. tumefaciens genomospecies G7 (GenBank accession numbers MZ570905-MZ570910). Pathogenicity assay was carried out by inoculating the stems of 2-week-old roselle seedlings through wounds made with a sterile needle with bacteria on it. The inoculated seedlings were kept in plastic bags to maintain high humidity. Symptoms similar to those observed in the field developed at the inoculation sites after 7 days, and Koch's postulates were fulfilled when the bacteria re-isolated from the galls were also identified as A. tumefaciens genomospecies G7 using recA gene sequence analysis. To our knowledge, this is the first report of crown gall disease caused by A. tumefaciens on Hibiscus sabdariffa in Taiwan. This disease may potentially damage the roselle industry if no action is taken to stop its spreading. Identification of the causal agent of roselle crown gall disease could help us further investigate its ecology and develop integrated pest management strategies for prevention of this disease in the future.

Rejection of the reclassification of Leuconostoc gasicomitatum as Leuconostoc gelidum subsp. gasicomitatum based on whole genome analysis.

In 2014, Rahkila et al. transferred Leuconostoc gasicomitatum to Leuconostoc gelidum as L. gelidum subsp. gasicomitatum comb. nov. based on a 75 % DNA-DNA hybridization value. In the present study, the taxonomic status of L. gelidum subsp. gasicomitatum is re-evaluated by a polyphasic approach, including 16S rRNA, pheS, rpoA, recA, and atpA gene sequence analyses, phylogenomic treeing, analyses of ANI (average nucleotide identity) and dDDH (digital DNA-DNA hybridization), fatty acid methyl ester analysis and a phenotypic characterization. On the basis of the ANI and dDDH values, we propose to reject the proposal of Rahkila et al. to reclassify L. gasicomitatum as L. gelidum subsp. gasicomitatum.

Distribution of Burkholderia cepacia complex species isolated from industrial processes and contaminated products in Argentina.

Burkholderia cepacia complex (Bcc) members have clinical relevance as opportunistic pathogens in patients with cystic fibrosis and are responsible of numerous nosocomial infections. These closely related bacteria are also reported as frequent contaminants of industrial products. In this retrospective study, we use PCR and recA gene sequence analysis to identify at species level Bcc isolates recovered from massive consumption products and industrial processes in Argentina during the last 25years. The sequences obtained were also compared with recA sequences from clinical Bcc isolates deposited in GenBank database. We detected Bcc in purified water and preserved products from pharmaceutics, cosmetics, household cleaning articles, and beverages industries. B. contaminans (which is prevalent among people with cystic fibrosis in Argentina) was the most frequent Bcc species identified (42% of the Bcc isolates studied). B. cepacia (10%), B. cenocepacia (5%), B. vietnamiensis (16%), B. arboris (3%), and the recently defined B. aenigmatica (24%) were also detected. Rec A sequences from all B. cepacia and most B. contaminans industrial isolates obtained in this study displayed 100% identity with recA sequences from isolates infecting Argentinean patients. This information brings evidence for considering industrial massive consumption products as a potential source of Bcc infections. In addition, identification at species level in industrial microbiological laboratories is necessary for a better epidemiological surveillance. Particularly in Argentina, more studies are required in order to reveal the role of these products in the acquisition of B. contaminans infections.

Identification and characterization of Dickeya zeae strains associated with maize stalk soft-rot in northern Serbia

Bacterial stalk soft rot have been repeatedly observed on maize plants in several commercial fields in northern part of Serbia in the period of 1990 to 2014. The occurrence of the disease corresponded with warm weather and increased humidity. Etiological studies of the diseased tissue constantly resulted in isolation of pectolytic bacterial strains. In order to identify the isolated bacteria, twenty-three strains were characterized by morphological, physiolcal, biochemical, and molecular assays. Pathogenicity of the strains was confirmed by Koch’s postulates on 1-week-old maize seedlings. The strains were Gram and oxidase-negative, non-fluorescent, pectolytic, facultative anaerobic and caused hypersensitive response (HR) in tobacco leaves. They produced catalase and lecithinase, but did not produce oxidase and arginine dehydrolase. All strains reduced nitrate and grew at 37 °C, while variable growth was observed in medium containing 5% NaCl. Phenotypic tests and amplification of the specific 420-bp fragment in PCR assay showed that the strains belong to genus Dickeya. Using ERIC-PCR analysis seven different genetic profiles were obtained, suggesting the presence of genetic diversity in the population of this pathogen in Serbia. Additionally, phylogenetic analysis based on the recA gene sequence analysis indicated that the strains isolated from soft rotted maize belong to Dickeya zeae, leading to the conclusion that this bacterium was the causal agent of stalk soft rot of maize in Serbia.

Proposal of Lactobacillus kosoi Chiou et al. 2018 as a later heterotypic synonym of Lactobacillus micheneri McFrederick et al. 2018, elevation of Lactobacillus plantarum subsp. argentoratensis to the species level as Lactobacillus argentoratensis sp. nov., and Lactobacillus zhaodongensis sp. nov., isolated from traditional Chinese pickle and the intestinal tract of a honey bee (Apis mellifera).

Lactobacillus kosoi Chiou et al. 2018 and Lactobacillus micheneri McFrederick et al. 2018 are closely related, and they share 100 % 16S rRNA gene sequence similarity, 99.6 % pheS gene sequence similarity, 100 % rpoA gene sequence similarity, 97.3 % average nucleotide identity (ANI) value and 76.6 % in silico DNA-DNA hybridization (isDDH) value, indicating that they represent the same species. Fatty acid methyl esters (FAME) analysis and phenotypic characterization also indicated that L. kosoi and L. micheneri are very similar. We propose L. kosoi Chiou et al. 2018 as a later heterotypic synonym of L. micheneri McFrederick et al. 2018. The taxonomic position of Lactobacillus plantarum subsp. argentoratensis in the L. plantarum group was re-examined using a polyphasic approach, including sequence analyses of 16S rRNA, pheS, rpoA and recA genes, average nucleotide identity analysis, in silico DNA-DNA hybridization, fatty acid methyl ester analysis and phenotypic characterization. Results of 16S rRNA gene sequence analysis indicated that L. plantarum subsp. argentoratensis was closely related to L. plantarum subsp. plantarum, L. pentosus and L. paraplantarum in the L. plantarum group, sharing 99.6-99.7 % 16S rRNA gene sequence similarities. Results of pheS, rpoA and recA gene sequence analyses indicated that L. plantarum subsp. argentoratensis was most closely related to L. plantarum subsp. plantarum, having 91.8 % pheS gene sequence similarity, 98.9 % rpoA gene sequence similarity and 93.1 % recA gene sequence similarity. L. plantarum subsp. argentoratensis DSM 16365T shared 95.6 % ANI value and 62.9 % isDDH value with L. plantarum subsp. plantarum ATCC 14917T. The low isDDH value confirmed that L. plantarum subsp. argentoratensis and L. plantarum subsp. plantarum represent two different species, rather than two different subspecies in the L. plantarum group. On the basis of the data from polyphasic characterization obtained in the present study and in previous studies, L. plantarum subsp. argentoratensis is elevated to the species level and represents a novel species of the genus Lactobacillus, for which the name Lactobacillus argentoratensis sp. nov. is proposed and the type strain is DKO 22T (=CIP 108320T=DSM 16365T=JCM 16169T). Two novel Gram-stain-positive bacterial strains, designated 1206-1T and F027-1-2, were isolated from traditional pickle in Heilongjiang Province, PR China, and from the intestinal tract of a honey bee (Apis mellifera) in Hubei Province, PR China, respectively. The two bacteria were characterized by a polyphasic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, fatty acid methyl ester analysis, average nucleotide identity analysis, in silico DNA-DNA hybridization analysis and an analysis of phenotypic features. The results of 16S rRNA gene sequence analysis indicated that strains 1206-1T and F027-1-2 were distantly related to Lactobacillus sharpeae, Lactobacillus hulanensis, Lactobacillus songhuajiangensis, Lactobacillus pantheris, Lactobacillus thailandensis, Lactobacillus camelliae, Lactobacillus jixianensis, Lactobacillus nasuensis, Lactobacillus baoqingensis, Lactobacillus manihotivorans and Lactobacillus porcinae. Strain 1206-1T exhibited 94.2-96.4 % 16S rRNA gene sequence similarities, 69.5-83.3 % pheS gene sequence similarities and 73.1-90.3 % rpoA gene sequence similarities to type strains of phylogenetically related species. ANI and isDDH values between strain 1206-1T and the type strains of phylogenetically related species were 52.7-73.7 % and 21.1-30.1 %, respectively. On the basis of the data obtained in the present study, a novel species, Lactobacillus zhaodongensis sp. nov. is proposed and the type strain is 1206-1T (=CCM 8981T=CCTCC AB 2019200T=LMG 31620T).

Streptobacillus canis sp. nov. isolated from a dog.

From a phlegmon in a dog an aerobic and facultatively anaerobic, indole-, oxidase- and catalase-negative, non-motile bacterium was isolated in 2019 in Germany that stained Gram-negative and showed a pleomorphic, rod-shaped, non-spore-forming appearance. Based on the results of 16S rRNA gene sequence analyses, strain IHIT1603-19T was assigned to the genus Streptobacillus with sequence similarities of 98.6, 98.0, 97.9, 97.1 and 94.4 % to the type strains of Streptobacillus felis, Streptobacillus notomytis, Streptobacillus ratti, Streptobacillus moniliformis and Streptobacillus hongkongensis, respectively. Strain IHIT1603-19T could also clearly be differentiated from other Streptobacillus species by rpoB, groEL and recA gene, nucleotide and amino acid sequence analyses as well as by core genome phylogeny. Regarding DNA-DNA relatedness, strain IHIT1603-19T demonstrated an average nucleotide identity of 83.00 and 82.28 % compared to S. felis 131000547T and S. moniliformis DSM 12112T, respectively. Chemotaxonomic and physiological data of strain IHIT1603-19T were in congruence with other closely related members of the family Leptotrichiaceae, represented by highly similar enzyme profiles and fatty acid patterns. MALDI-TOF MS analysis also proved suitable in unequivocally discriminating strain IHIT1603-19T from all currently described taxa of the genus Streptobacillus. On the basis of these data, we propose the novel species Streptobacillus canis sp. nov. with the type strain IHIT1603-19T (=DSM 110501T=CCUG 74118T=CIP 111795T). The G+C content of the DNA of the type strain is 26.6 mol%, genome size is 1.60 Mbp.

Genetically diverse lentil- and faba bean-nodulating rhizobia are present in soils across Central and Southern Ethiopia.

In total 196 bacterial isolates were obtained from root nodules of lentil (Lens culinaris) and faba bean (Vicia faba) grown on soil samples collected from 10 different sites in central and southern parts of Ethiopia. All isolates were identified as members of the genus Rhizobium by using recA gene sequence analysis. In the recA phylogenetic tree 195 rhizobial strains were classified into nine genospecies. The phylogeny of symbiotic genes nodC and nifH revealed five and six distinct groups respectively, largely dominated by symbiovar viciae. A multivariate analysis showed that environmental variables of the sampling sites considered in this study had more effect on the distribution and composition of the genospecies than the host legumes of the strains. Twenty representative strains, selected based on their isolation site, host plant and nodC group, were able to nodulate all lentil, faba bean, field pea (Pisum abyssinicum) and grass pea (Lathyrus sativus) plants in a greenhouse test in axenic conditions. The majority of the rhizobial strains were effective nitrogen-fixing symbionts for all tested legumes, indicating their potential to serve as broad host-range inoculants in agriculture. The present work suggests the presence of taxonomically and symbiotically diverse rhizobial species for legumes in the Viciae tribe in Ethiopia.

Genetic and Phenotypic Characteristics of a Multi-strain Probiotic for Broilers.

Bacteria isolated from different segments of the gastro-intestinal tract (GIT) of healthy free-range broilers were screened for probiotic properties. Six strains were selected and identified as Lactobacillus gallinarum, Lactobacillus johnsonii, Lactobacillus salivarius, Lactobacillus crispatus, Enterococcus faecalis and Bacillus amyloliquefaciens based on 16S rRNA, gyrB and recA gene sequence analyses. All six strains produced exopolysaccharides (EPS) and formed biofilms under conditions simulating the broiler GIT. Lactobacillus johnsonii DPN184 and L. salivarius DPN181 produced hydrogen peroxide, and L. crispatus DPN167 and E. faecalis DPN94 produced bile salt hydrolase (BSH) and phytase. Bacillus amyloliquefaciens DPN123 produced phytase, amylase, surfactin and iturin A1. No abnormalities were observed when broilers were fed the multi-strain combination, suggesting that it could be used as a probiotic.

Comparative Genomics of Pandoraea, a Genus Enriched in Xenobiotic Biodegradation and Metabolism.

Comparative analysis of partial gyrB, recA, and gltB gene sequences of 84 Pandoraea reference strains and field isolates revealed several clusters that included no taxonomic reference strains. The gyrB, recA, and gltB phylogenetic trees were used to select 27 strains for whole-genome sequence analysis and for a comparative genomics study that also included 41 publicly available Pandoraea genome sequences. The phylogenomic analyses included a Genome BLAST Distance Phylogeny approach to calculate pairwise digital DNA–DNA hybridization values and their confidence intervals, average nucleotide identity analyses using the OrthoANIu algorithm, and a whole-genome phylogeny reconstruction based on 107 single-copy core genes using bcgTree. These analyses, along with subsequent chemotaxonomic and traditional phenotypic analyses, revealed the presence of 17 novel Pandoraea species among the strains analyzed, and allowed the identification of several unclassified Pandoraea strains reported in the literature. The genus Pandoraea has an open pan genome that includes many orthogroups in the ‘Xenobiotics biodegradation and metabolism’ KEGG pathway, which likely explains the enrichment of these species in polluted soils and participation in the biodegradation of complex organic substances. We propose to formally classify the 17 novel Pandoraea species as P. anapnoica sp. nov. (type strain LMG 31117T = CCUG 73385T), P. anhela sp. nov. (type strain LMG 31108T = CCUG 73386T), P. aquatica sp. nov. (type strain LMG 31011T = CCUG 73384T), P. bronchicola sp. nov. (type strain LMG 20603T = ATCC BAA-110T), P. capi sp. nov. (type strain LMG 20602T = ATCC BAA-109T), P. captiosa sp. nov. (type strain LMG 31118T = CCUG 73387T), P. cepalis sp. nov. (type strain LMG 31106T = CCUG 39680T), P. commovens sp. nov. (type strain LMG 31010T = CCUG 73378T), P. communis sp. nov. (type strain LMG 31110T = CCUG 73383T), P. eparura sp. nov. (type strain LMG 31012T = CCUG 73380T), P. horticolens sp. nov. (type strain LMG 31112T = CCUG 73379T), P. iniqua sp. nov. (type strain LMG 31009T = CCUG 73377T), P. morbifera sp. nov. (type strain LMG 31116T = CCUG 73389T), P. nosoerga sp. nov. (type strain LMG 31109T = CCUG 73390T), P. pneumonica sp. nov. (type strain LMG 31114T = CCUG 73388T), P. soli sp. nov. (type strain LMG 31014T = CCUG 73382T), and P. terrigena sp. nov. (type strain LMG 31013T = CCUG 73381T).

Pathogenicity and molecular phylogenetic analysis reveal a distinct position of the banana fingertip rot pathogen among the Burkholderia cenocepacia genomovars

Banana (Musa spp.) is one of the most widely cultivated subtropical fruits around the globe. Banana cultivation has been extensively increased in southeastern Iran over the last two decades. Recently, banana fruits possessing rotten and blackened fingertip symptoms were observed in Sistan‐Baluchestan, Iran. Isolation and characterization of the causal agent showed that the pathogen belongs to the multifaceted bacterial species Burkholderia cenocepacia. Pathogenicity tests and host range assays showed that the strains were pathogenic on banana, as well as carrot, onion and potato. All the strains were resistant to 50 mg L−1 rifampicin and 200 mg L−1 copper sulphate. Phylogenetic analysis of 16S rRNA and recA gene sequences showed that the strains belong to two different genomovars of B. cenocepacia (III‐A and III‐B), which also include environmental and cystic fibrosis associated strains of the species. The results obtained from recA phylogeny were confirmed using multilocus sequence analysis (MLSA), although MLSA showed that the banana strains were clustered as a novel phylogroup among the members of both genomovars. Banana‐pathogenic B. cenocepacia strains isolated in Iran were different from the strains isolated in Taiwan, as the ‘B. cepacia epidemic strain marker’ reported in the Taiwanese strains was absent from Iranian strains. To the authors’ knowledge, this is the first MLSA‐based study on the banana‐pathogenic strains of B. cenocepacia. However, further in‐depth molecular studies are needed to decipher the relationships between the banana fingertip rot pathogen and the clinical strains of B. cenocepacia.

The molecular and phenotypic characterization of fructophilic lactic acid bacteria isolated from the guts of Apis mellifera L. derived from a Polish apiary

This paper describes taxonomic position, phylogeny, and phenotypic properties of 14 lactic acid bacteria (LAB) originating from an Apis mellifera guts. Based on the 16S rDNA and recA gene sequence analyses, 12 lactic acid bacteria were assigned to Lactobacillus kunkeei and two others were classified as Fructobacillus fructosus. Biochemically, all isolated lactic acid bacteria showed typical fructophilic features and under anaerobic conditions grew well on fructose, but poorly on glucose. Fast growth of bacteria on glucose was noted in the presence of oxygen or fructose as external electron acceptors. The residents of honeybee guts were classified as heterofermentative lactic acid bacteria. From glucose, they produced almost equimolar amounts of lactic acid, acetic acid, and trace amounts of ethanol. Furthermore, they inhibited the growth of the major honeybee pathogen, Paenibacillus larvae, meaning that the LAB studied may have the health-conferring properties of probiotics.

Dyella halodurans sp. nov., isolated from lower subtropical forest soil.

A Gram-stain-negative, aerobic, non-endospore-forming, motile by a polar flagellum, rod-shaped bacterium, designated strain DHOG02T, which produced yellow-pigmented colonies, was isolated from a soil sample collected from the lower subtropical forest of the Dinghushan Biosphere Reserve, Guangdong Province, PR China. Strain DHOG02T grew at 12-37 °C, pH 4-9 and 0-4 % (w/v) NaCl, with optima at 28 °C, pH 6-7 and 0.5 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that this strain formed a clade with Dyella lipolytica DHOB07T and Dyella jejuensis JP1T, with sequence similarities of 98.0 and 97.4 %, respectively. The result of the concatenated partial gyrB, lepA and recA gene sequence analysis confirmed that strain DHOG02T belongs to the genus Dyella, but is distinct from all currently known species of the genus. The G+C content of the genomic DNA was 62 mol%. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid and phospholipid. Ubiquinone-8 was the only respiratory quinone detected, and iso-C15 : 0, iso-C17 : 1ω9c and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) were the major fatty acids, all of which supported the affiliation of strain DHOG02T to the genus Dyella. On the basis of the evidence presented here, strain DHOG02T represents a novel species of the genus Dyella, for which the name Dyella halodurans sp. nov. is proposed. The type strain is DHOG02T (=NBRC 111474T=CGMCC 1.15435T).

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

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

Diversity and symbiotic divergence of endophytic and non-endophytic rhizobia of Medicago sativa

Knowledge of rhizobium diversity is helping to enable the utilization of rhizobial resources. To analyze the phenotypic and genetic diversity and the symbiotic divergence of rhizobia of Medicago sativa, 30 endophytic and non-endophytic isolates were collected from different parts of five alfalfa varieties in three geographic locations in Gansu, China. Numerical analyses based on 72 phenotypic properties and restriction fragment length polymorphism (RFLP) fingerprinting indicated the abundant phenotypic and genetic diversity of the tested strains. According to the phylogenetic analysis of 16S RNA, atpD, glnII, and recA gene sequences, Rhizobium and Ensifer were further classified into four different genotypes: Rhizobium radiobacter, Rhizobium sp., Rhizobium rosettiformans, and Ensifer meliloti. The differences in architecture and functioning of the rhizobial genomes and, to a lesser extent, environment diversification helped explain the diversity of tested strains. The tested strains exhibited similar symbiotic feature when inoculated onto M. sativa cvs. Gannong Nos. 3 and 9 and Qingshui plants for the clustering feature of their parameter values. An obvious symbiotic divergence of rhizobial strains was observed in M. sativa cvs. Longzhong and WL168HQ plants because of the scattered parameter values. Their symbiotic divergence differed according to alfalfa varieties, which indicated that the sensitivity of different alfalfa varieties to rhizobial strains may differ. Most of the tested strains exhibited plant growth-promoting traits including phosphate solubilization and production of indole-3-acetic acid (IAA) when colonizing plant tissues and soil.

Burkholderia puraquae sp. nov., a novel species of the Burkholderia cepacia complex isolated from hospital settings and agricultural soils

Bacteria from the Burkholderia cepacia complex (Bcc) are capable of causing severe infections in patients with cystic fibrosis (CF). These opportunistic pathogens are also widely distributed in natural and man-made environments. After a 12-year epidemiological surveillance involving Bcc bacteria from respiratory secretions of Argentinean patients with CF and from hospital settings, we found six isolates of the Bcc with a concatenated species-specific allele sequence that differed by more than 3 % from those of the Bcc with validly published names. According to the multilocus sequence analysis (MLSA), these isolates clustered with the agricultural soil strain, Burkholderia sp. PBP 78, which was already deposited in the PubMLST database. The isolates were examined using a polyphasic approach, which included 16S rRNA, recA, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), DNA base composition, average nucleotide identities (ANIs), fatty acid profiles, and biochemical characterizations. The results of the present study demonstrate that the seven isolates represent a single novel species within the Bcc, for which the name Burkholderia puraquae sp. nov. is proposed. Burkholderia puraquae sp. nov. CAMPA 1040T (=LMG 29660T=DSM 103137T) was designated the type strain of the novel species, which can be differentiated from other species of the Bcc mainly from recA gene sequence analysis, MLSA, ANIb, MALDI-TOF MS analysis, and some biochemical tests, including the ability to grow at 42 °C, aesculin hydrolysis, and lysine decarboxylase and β-galactosidase activities.

Molecular and symbiotic characterization of peanut bradyrhizobia from the semi-arid region of Brazil

Peanut (Arachis hypogaea L.) is a legume crop native from South America and grown worldwide. This is well adapted to the unfavorable edaphoclimatic conditions found in Brazilian Semi-Arid region, where is cultivated mainly by smallholders. The selection of new rhizobial isolates is important to increase the rates of biological nitrogen fixation and yield. Furthermore, the diversity evaluation of new bacterial isolates may help us to understand the ecology of these groups in regions where little information are available. In this context, this study aimed to evaluate the phylogenetic relationships of peanut bradyrhizobia from Brazilian Semi-Arid soils by analysis of 16S rRNA, recA, nodC and nifD gene sequences, as well as their symbiotic performance. A trap-host experiment was performed using two soil samples from the municipality of Barbalha, Ceará, Brazil, taken under peanut cropping and one soil sample from the municipality of Juazeiro, Bahia, Brazil, covered by the native Arachis triseminata. Seeds of peanut cv. BR1 were sown and the plants were grown until 52days after emergence. The nodules were inoculated in YMA medium for isolation of slow-growing bacteria. The Box-PCR fingerprinting and the 16S rRNA gene sequences was evaluated for all bacteria and recA, nodC and nifD were sequenced for the six selected isolates. Selected bacteria were evaluated according to their symbiotic characteristics in a greenhouse experiment. Ten slow-growing isolates were obtained and classified within the genus Bradyrhizobium, by the 16S rRNA sequence analysis and showed divergent Box-PCR profiles. All bacteria were clustered within the B. japonicum clade. The bacteria from Juazeiro were closely related to B. yuanmingense, and those from Barbalha showed similarity to B. yuanmingense, B. kavangense and B. guangxiense. The sequences of recA showed partial congruence to the 16S rRNA gene analysis but the phylogeny trees based on the symbiotic genes were not related, indicating horizontal gene transfer. All bacteria were able to efficiently nodulate the peanut and the isolate ESA 83, pointed out regarding the nitrogen fixation ability and were selected for further analysis in non-sterile soils and field conditions.

Dyella caseinilytica sp. nov., Dyella flava sp. nov. and Dyella mobilis sp. nov., isolated from forest soil.

Three strains, DHOB09T, DHOC52T and DHON07T, were isolated from the forest soil of Dinghushan Biosphere Reserve, Guangdong Province, PR China. They were all Gram-stain-negative, aerobic, rod-shaped cells. The ranges (optimum) for the temperature, pH and NaCl concentration for growth of DHOB09T, DHOC52T and DHON07T were 10-42 (25-28) °C, pH 5.5-9.0 (7.0-7.5) and 0-4.0 (0-0.5) % (w/v); 10-42 (28) °C, pH 4.0-7.0 (4.5-6.5) and 0-2.0 (0) % (w/v) and 10-37 (25-28) °C, pH 4.0-7.5 (5.5-6.0) and 0-2.5 (0) % (w/v), respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that DHOB09T, DHOC52T and DHON07T formed a phyletic cluster with seven species of the genus Dyella within the major clade of Dyella with sequence similarities ranged from 96.9 to 98.6 %. This indicated that the three strains may represent three novel species of the genus Dyella. This result was also strongly supported by the concatenated analysis of partial gyrB, lepA and recA gene sequences. DNA-DNA hybridization between strains DHON07T and DHOB09T, as well as DHON07T and Dyella koreensis BB4T was much lower than 70 %. The G+C content of strains DHOB09T, DHOC52T and DHON07T were 59.4, 60.7 and 59.5 %, respectively. The major fatty acids of the three strains were iso-C15 : 0, iso-C16 : 0 and iso-C17 : 0 and the predominant respiratory lipoquinone was ubiquinone-8. All of the physiological, phylogenetic and chemotaxonomic data showed that strains DHOB09T, DHOC52T and DHON07T are distinctive from each other and from all species of the genus Dyellawith validly published names. Therefore, we suggest that they represent three novel species of the genus, for which the names Dyella caseinilytica sp. nov. (type strain DHOB09T=CGMCC 1.15434T=LMG 29202T), Dyella flava sp. nov. (type strain DHOC52T=NBRC 111979T=KCTC 52128T) and Dyella mobilis sp. nov. (type strain DHON07T=CGMCC 1.15400T=NBRC 111475T) are proposed.