Chemical constituents from the endophytic fungus Aspergillus sp. S3 of Hibiscus tiliaceus.

A new sterol, aspersterol E (1), a newly discovered alkaloid, asperginine A (2), and five known compounds (3-7) were obtained from the endophytic fungus Aspergillus sp. S3 of Hibiscus tiliaceus Linn. The compounds were extracted from their fermentation products using silica gel, ODS C18, and semi-preparative HPLC. The structure of each compound was determined through spectroscopic analysis. All the obtained compounds (1-7) were evaluated for their cytotoxic activity against the mouse pre-gastric cancer cell line MFC by using the MTT assay. The IC50 values of compounds 1, 2, 3, and 5 were found to be 153.43 µM, 61.25 µM, 73.19 µM, and 181.69 µM respectively.

Ame-miR-980-3p participates in autophagy-mediated midgut remodelling in Apis mellifera via targeting Atg2B.

Autophagy is a process that serves to degrade damaged proteins and organelles, thereby promoting cell homeostasis, differentiation, development and survival. Many miRNAs have been found to have regulatory roles in autophagy. In insects, it has been shown that autophagy is involved in hormone-regulated programmed cell death during metamorphic midgut remodelling. However, whether this is also true during the remodelling of the honey bee midgut is unclear. In the present study, we explored the relationship between autophagy and midgut remodelling and sought to identify miRNAs involved in this physiological process. We found that autophagy occurred during midgut remodelling and that the inhibition of autophagy resulted in midgut dysplasia in prepupae. Differentially expressed miRNAs enriched in the autophagy signalling pathway during midgut remodelling were identified by small RNA-seq. Ame-miR-980-3p, which targets the autophagy-related gene Atg2B, was screened out. Furthermore, abnormal expression of ame-miR-980-3p in the pupal stage led to the thinning of the midgut wall of newly emerged bees (NE). When ame-miR-980-3p expression was inhibited, the intestinal villi of NE bees became significantly shorter and sparse, and the lipid signal in the peritrophic matrix of Pb almost disappeared, indicating that the adult midgut was underdeveloped and the lipid absorption ability was weakened. Taken together, ame-miR-980-3p targeted Atg2B to participate in the regulation of midgut autophagy in the pupae, and the abnormal expression of ame-miR-980-3p would interfere with cell proliferation and death in the process of midgut remodelling, hinder the formation of adult midgut and eventually lead to adult midgut dysplasia and affect the lipid absorption function of the midgut in Apis mellifera.

A polyphasic taxonomy analysis reveals the presence of an ecotype of Rahnella contaminans associated with the gut of Dendroctonus-bark beetles.

Species belonging to the genus Rahnella are dominant members of the core gut bacteriome of Dendroctonus-bark beetles, a group of insects that includes the most destructive agents of pine forest in North and Central America, and Eurasia. From 300 isolates recovered from the gut of these beetles, 10 were selected to describe an ecotype of Rahnella contaminans. The polyphasic approach conducted with these isolates included phenotypic characteristics, fatty acid analysis, 16S rRNA gene, multilocus sequence analyses (gyrB, rpoB, infB, and atpD genes), and complete genome sequencing of two isolates, ChDrAdgB13 and JaDmexAd06, representative of the studied set. Phenotypic characterization, chemotaxonomic analysis, phylogenetic analyses of the 16S rRNA gene, and multilocus sequence analysis showed that these isolates belonged to Rahnella contaminans. The G + C content of the genome of ChDrAdgB13 (52.8%) and JaDmexAd06 (52.9%) was similar to those from other Rahnella species. The ANI between ChdrAdgB13 and JaDmexAd06 and Rahnella species including R. contaminans, varied from 84.02 to 99.18%. The phylogenomic analysis showed that both strains integrated a consistent and well-defined cluster, together with R. contaminans. A noteworthy observation is the presence of peritrichous flagella and fimbriae in the strains ChDrAdgB13 and JaDmexAd06. The in silico analysis of genes encoding the flagellar system of these strains and Rahnella species showed the presence of flag-1 primary system encoding peritrichous flagella, as well as fimbriae genes from the families type 1, α, ß and σ mainly encoding chaperone/usher fimbriae and other uncharacterized families. All this evidence indicates that isolates from the gut of Dendroctonus-bark beetles are an ecotype of R. contaminans, which is dominant and persistent in all developmental stages of these bark beetles and one of the main members of their core gut bacteriome.

Siccirubricoccus phaeus sp. nov., isolated from oil reservoir water and emended description of the genus Siccirubricoccus.

A Gram-staining-negative, non-motile, aerobic bacterium, designated 1-3T, was isolated from oil reservoir water collected from Liaohe oilfield, north-east of China. Growth was observed at 15-40 °C (optimum 37 °C) and pH 6-10 (optimum 7). The strain can grow under nitrogen-limiting condition. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel isolate was most closely related to Siccirubricoccus deserti SYSU D8009T (96.7%), followed by Paracraurococcus ruber NS89T (95.7%) and Belnapia rosea CPCC 100156T (94.9%). Genome sequencing revealed a genome size of 6.43 Mbp and a G+C content of 71.3 mol%. The average nucleotide identity values and digital DNA-DNA hybridization between 1-3T and the reference strains were all below the cut-off level (95-96% and 70%, respectively) for species delineation. The strain possessed the cytochrome P450 enzyme, which has the potential to degrade oil. The respiratory quinone was Q-10 and the major fatty acids were summed feature 8 (C18:1 ω7c/C18:1 ω6c, 38.8%), C16:0 (25.6%) and C19:0 cyclo ω8c (22.5%). The polar lipids of strain 1-3T comprised diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and three unidentified aminolipids. Based on the genotypic and phenotypic characteristics, strain 1-3T represents a novel species of genus Siccirubricoccus, for which the name Siccirubricoccus phaeus sp. nov. is proposed. The type strain of Siccirubricoccus phaeus is 1-3T (= CGMCC 1.16799T = LMG 31398T).

Methionine as a methyl donor regulates caste differentiation in the European honey bee (Apis mellifera).

Nutrition contributes to honey bee caste differentiation, but the role of individual nutrients is still unclear. Most essential amino acid contents, except that of methionine (Met), are greater in royal jelly than worker jelly. After ∼3.5 d, the Met content in the latter was slightly greater than in the former. Met is the major raw material used in the synthesis of S-adenosyl-L-methionine, an active methyl donor for DNA methylation, which is an epigenetic driver of caste differentiation. Here, we tested whether Met regulates caste differentiation in honey bees by determining its effects on the caste development of bees receiving four diets: the basic, basic + 0.2% Met, basic + 0.2% Met + 20 mg/kg 5-azacytidine, and basic + 20 mg/kg 5-azacytidine. The presence of Met decreased the adult bee body length and the numbers of ovarioles, indicating that Met may direct the development of female larvae toward worker bees. The upregulated expression of SAMS, Dnmt1, and Dnmt3 caused by Met exposure in 4-d-old larvae indicated that the worker-inductive effects of Met may occur through the promotion of DNA methylation. We investigated the co-effects of Met and glucose on bee development, and found that the effects of an increased glucose level on the number of ovarioles and body length did not strengthen the worker-inductive effects caused by Met. Our results contribute to caste development theory and suggest that Met-as a methyl donor-plays a regulatory, but not decisive, role in caste differentiation.

Frigidibacter oleivorans sp. nov., isolated from a deep well with oil reservoir water.

A Gram-stain-negative, rod-shaped bacterium, designated XJ4T, was isolated from oil-contaminated water, collected from Xinjiang Province, north-west PR China (45° 1' 27″ N, 85° 6' 14″ E). Growth occurred at 20-45 °C (optimum, 30 °C) and pH 6.0-10.0 (optimum, pH 6.0-7.0). Strain XJ4T could tolerate up to 7 % (w/v) NaCl and grow optimally in the absence of NaCl. Phylogenetic analysis based on comparative sequence analysis of 16S rRNA gene sequences indicated that strain XJ4T belonged to the genus Frigidibacter, and that was closely related to Frigidibacter mobilis cai42T (97.2 %), Frigidibacter albus SP32T (97.0 %) and Rhodobacter aestuarii JA296T (97.0 %). The average nucleotide identity values between XJ4T and three type strains were 77.9, 77.6 and 71.9 %, respectively. The DNA G+C content of strain XJ4T was 69.5 mol%. The sole respiratory quinone was Q-10. The major cellular fatty acid was summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C18 : 0 and 11-methyl C18 : 1 ω7c. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, unidentified phospholipids, an unidentified aminolipid and unidentified lipids. On the basis of phenotypic, chemotaxonomic and phylogenetic analyses, strain XJ4T represents a novel species of the genus Frigidibacter, for which the name Frigidibacter oleivorans sp. nov. is proposed. The type strain is XJ4T (=CGMCC 1.13778T=LMG 30952T).

Paracoccus alkanivorans sp. nov., isolated from a deep well with oil reservoir water.

A Gram-stain-negative, non-motile and ovoid bacterial strain, designated 4-2T, was isolated from oil-contaminated water which was collected from Xinjiang Province, north-west PR China. The 16S rRNA gene sequence analysis showed that strain 4-2T belonged to the genus Paracoccus. The species with highest similarity to strain 4-2T was Paracoccus saliphilus YIM 90738T (97.83 %), followed by 'Paracoccus siganidrum' M26 (97.83 %) and Paracoccus endophyticus SYSUP0003T (97.25 %). The average nucleotide identity values between 4-2T and three type strains were 84.69, 77.88 and 74.07 %, respectively. The genomic DNA G+C content of strain 4-2T was 61.4 mol%. Chemotaxonomical characteristic results showed that the respiratory quinone was ubiquinone Q-10 and the major fatty acids were summed feature 8 (C18 : 1 ω7c or C18 : 1 ω6c) and C19 : 0 cyclo ω8c. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, unidentified phospholipids, an unidentified aminolipid and an unidentified polar lipid. The predominant polyamines were putrescine, cadaverine and spermidine. On the basis of phenotypic, chemotaxonomic and phylogenetic inferences, strain 4-2T represents a novel species of the genus Paracoccus, for which the name Paracoccus alkanivorans sp. nov. is proposed. The type strain is 4-2T (=CGMCC 1.13669T=LMG 30882T).

Bradyrhizobium nanningense sp. nov., Bradyrhizobium guangzhouense sp. nov. and Bradyrhizobium zhanjiangense sp. nov., isolated from effective nodules of peanut in Southeast China.

Nine slow-growing rhizobia isolated from effective nodules on peanut (Arachis hypogaea) were characterized to clarify the taxonomic status using a polyphasic approach. They were assigned to the genus Bradyrhizobium on the basis of 16S rRNA sequences. MLSA of concatenated glnII-recA-dnaK genes classified them into three species represented by CCBAU 53390T, CCBAU 51670T and CCBAU 51778T, which presented the closest similarity to B. guangxiense CCBAU 53363T, B. guangdongense CCBAU 51649T and B. manausense BR 3351T, B. vignae 7-2T and B. forestalis INPA 54BT, respectively. The dDDH (digital DNA-DNA hybridization) and ANI (Average Nucleotide Identity) between the genomes of the three representative strains and type strains for the closest Bradyrhizobium species were less than 42.1% and 91.98%, respectively, below the threshold of species circumscription. Effective nodules could be induced on peanut and Lablab purpureus by all representative strains, while Vigna radiata formed effective nodules only with CCBAU 53390T and CCBAU 51778T. Phenotypic characteristics including sole carbon sources and growth features supported the phylogenetic results. Based on the genotypic and phenotypic features, strains CCBAU 53390T, CCBAU 51670T and CCBAU 51778T are designated the type strains of three novel species, for which the names Bradyrhizobium nanningense sp. nov., Bradyrhizobium guangzhouense sp. nov. and Bradyrhizobium zhanjiangense sp. nov. are proposed, respectively.

The experimental study of shunt-decompression arterialized vein flap.

BACKGROUND: Arterialized vein flap is a kind of unphysiological flap. Unphysiological reconstruction of blood circulation leads to higher load than that supported by physiological flap and is the culprit of flap swelling, blood stasis, skin blistering, and necrosis after flap grafting. To resolve the multiple disadvantages of traditional flap grafting, by introducing the principles of fluid mechanics, shunt-decompression surgery is prepared to decline the circulation preload and improve the prognosis of arterialized vein flap grafting. METHODS: By introducing the principles of fluid mechanics, we established the model of shunt-decompression arterialized vein flap, which satisfied the common properties of general fluid that the interface pressure between object and fluid is reduced when the velocity of fluid is increased and vice versa-the effect of Bernoulli. Under this rule, we anastomose the arterialized vein to the branch of main artery of recipient region or make end-to-side anastomosis, which can maintain the blood flow of main artery, decrease the perfusion of flap, and preserve the decompressive effect of main artery to branches. From March, 2016 to September, 2016, we performed animal experiments on ten male bama mini pigs with average weight of 28 ± 2.35 kg. Superior epigastric artery of pig was used for feeding artery to arterialize the superficial epigastric veins. The total area of flap is 8 cm × 6 cm. End-to-side anastomosis and end-to-end anastomosis were established in experimental group and control group, respectively. Doppler speckle perfusion imaging apparatus was used to monitor the alterations of flap perfusion, blood flow of flap, tissue swelling and survival of flaps. RESULTS: The average flap perfusion (PU) at 1 week after surgery is 83.62 ± 3.14 in experimental group and 98.14 ± 6.54 in control group, respectively (P < 0.05), indicating the significant reduction of flap blood perfusion in experimental group as compared with control group. As to the survival of flaps, 7 flaps completely survived, 3 showed partial necrosis, and no one was found as complete necrosis in experimental group, while only 3 flaps survived, and 4 flaps and 3 flaps showed partial necrosis and complete necrosis in control group, respectively (P < 0.05). CONCLUSION: Based on the physiological features of arterialized vein flap and its problems in clinical application, we improved the anastomosis strategy of flap grafting and obtained excellent experimental outcomes, which provides an insight for the clinical application of arterialized vein flaps.

Sphingomonas oleivorans sp. nov., isolated from oil-contaminated soil.

Strain FW-11T was isolated from oil-contaminated soil from Panjin in Liaoning, China. It was a Gram-stain-negative, aerobic and rod-shaped bacterium. The strain was confirmed to be a member of the genus Sphingomonas based on phylogenetic inference and phenotypic characteristics. The best growth of strain FW-11T occurred at 30 °C and pH 6.0-7.0. The strain was non-spore-forming, catalase-negative and oxidase-negative. The main polar lipids were sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and unidentified lipids. The cell-wall peptidoglycan of strain FW-11T included alanine, glycine, glutamic acid, aspartic acid and meso-diaminopimelate. The predominant isoprenoid quinones were ubiquinone Q-10 (93.2 %) and Q-9 (6.8 %). The fatty acid profile (>5 %) included C18 : 1ω6c (43.1 %), C16 : 0 (14.6 %), C17 : 1ω6c (14.0 %) and C14 : 0 2-OH (11.1 %). The most similar neighbours of FW-11T were Sphingomonas fennica K101T (97.4 %) and Sphingomonas haloaromaticamans A175T (97.0 %). The average nucleotide identity relatedness values between strain FW-11T and the two type strains (S. fennica K101T and S. haloaromaticamans A175T) were 73.2 and 75.3 %, respectively. The genome size of FW-11T was 3.8 Mbp, comprising 3735 predicted genes with a DNA G+C content of 64.0 mol%. Based on phenotypic, chemotaxonomic and phylogenetic data, strain FW-11T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas oleivorans sp. nov. is proposed. The type strain is FW-11T (=LMG 29274T=HAMBI 3659T).

Symbiotic characteristics of Bradyrhizobium diazoefficiens USDA 110 mutants associated with shrubby sophora (Sophora flavescens) and soybean (Glycine max).

Site-specific insertion plasmid pVO155 was used to knockout the genes involved in the alternation of host range of strain Bradyrhizobium diazoefficiens USDA 110 from its original determinate-nodule-forming host soybean (Glycine max), to promiscuous and indeterminate-nodule-forming shrubby legume sophora (Sophora flavescens). Symbiotic phenotypes of these mutants inoculated to these two legumes, were compared to those infected by wild-type strain USDA 110. Six genes of the total fourteen Tn5 transposon mutated genes were broken using the pVO155 plasmid. Both Tn5 and pVO155-inserted mutants could nodulate S. flavescens with different morphologies of low-efficient indeterminate nodules. One to several rod or irregular bacteroids, containing different contents of poly-ß-hydroxybutyrate or polyphosphate were found within the symbiosomes in nodulated cells of S. flavescens infected by the pVO155-inserted mutants. Moreover, none of bacteroids were observed in the pseudonodules of S. flavescens, infected by wild-type strain USDA 110. These mutants had the nodulation ability with soybean but the symbiotic efficiency reduced to diverse extents. These findings enlighten the complicated interactions between rhizobia and legumes, i. e., mutation of genes involved in metabolic pathways, transporters, chemotaxis and mobility could alter the rhizobial entry and development of the bacteroid inside the nodules of a new host legume.

Ensifer shofinae sp. nov., a novel rhizobial species isolated from root nodules of soybean (Glycine max).

Two bacterial strains isolated from root nodules of soybean were characterized phylogenetically as members of a distinct group in the genus Ensifer based on 16S rRNA gene comparisons. They were also verified as a separated group by the concatenated sequence analyses of recA, atpD and glnII (with similarities ≤93.9% to the type strains for defined species), and by the average nucleotide identities (ANI) between the whole genome sequence of the representative strain CCBAU 251167T and those of the closely related strains in Ensifer glycinis and Ensifer fredii (90.5% and 90.3%, respectively). Phylogeny of symbiotic genes (nodC and nifH) grouped these two strains together with some soybean-nodulating strains of E. fredii, E. glycinis and Ensifer sojae. Nodulation tests indicated that the representative strain CCBAU 251167T could form root nodules with capability of nitrogen fixing on its host plant and Glycine soja, Cajanus cajan, Vigna unguiculata, Phaseolus vulgaris and Astragalus membranaceus, and it formed ineffective nodules on Leucaena leucocephala. Strain CCBAU 251167T contained fatty acids 18:1 ω9c, 18:0 iso and 20:0, differing from other related strains. Utilization of l-threonine and d-serine as carbon source, growth at pH 6.0 and intolerance of 1% (w/v) NaCl distinguished strain CCBAU 251167T from other type strains of the related species. The genome size of CCBAU 251167T was 6.2Mbp, comprising 7,581 predicted genes with DNA G+C content of 59.9mol% and 970 unique genes. Therefore, a novel species, Ensifer shofinae sp. nov., is proposed, with CCBAU 251167T (=ACCC 19939T=LMG 29645T) as type strain.

[Relationship between PMI and relative expression of myocardial various RNAs in rats died of different causes].

OBJECTIVE: To observe the changes of relative expression of myocardial various RNAs in rats died of different causes and their relationship with PMI. METHODS: The rat models were established in which the rats were sacrificed by broken neck, asphyxia, and hemorrhagic shock. Total RNAs were extracted from myocardium. The quantitative real time PCR was used to calculate threshold cycle values of RNAs including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, inducible nitric oxide synthase (iNOS), hypoxia-inducible factor-1 (HIF-1), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and U6 small nuclear RNA (U6 snRNA) and to study the changes of the relative expressions of various indexes with PMI. RESULTS: U6 snRNA with stable expression level could be used as appropriate internal control. In the early PMI, the relative expression of GAPDH, HIF-1, iNOS, TNF-alpha, and IL-6 more characteristically increased in groups of asphyxia and hemorrhagic shock than in group of broken neck, but the quantity of beta-actin decreased in all groups. In the late PMI, all the relative expressions significantly declined in correlation with the degradation of RNA. CONCLUSION: The characteristic changes of each RNA expression can be used as references to estimate PMI in deaths by different causes.

Day-night cycles and the sleep-promoting factor, Sleepless, affect stem cell activity in the Drosophila testis.

Adult stem cells maintain tissue integrity and function by renewing cellular content of the organism through regulated mitotic divisions. Previous studies showed that stem cell activity is affected by local, systemic, and environmental cues. Here, we explore a role of environmental day-night cycles in modulating cell cycle progression in populations of adult stem cells. Using a classic stem cell system, the Drosophila spermatogonial stem cell niche, we reveal daily rhythms in division frequencies of germ-line and somatic stem cells that act cooperatively to produce male gametes. We also examine whether behavioral sleep-wake cycles, which are driven by the environmental day-night cycles, regulate stem cell function. We find that flies lacking the sleep-promoting factor Sleepless, which maintains normal sleep in Drosophila, have increased germ-line stem cell (GSC) division rates, and this effect is mediated, in part, through a GABAergic signaling pathway. We suggest that alterations in sleep can influence the daily dynamics of GSC divisions.

[Effect of glutathione on blood coagulation function].

The purpose of this study was to investigate the effect of glutathione (GSH) on blood coagulation. The normal plasma samples and mixed plasma samples were taken randomly, and into which the normal dose and different concentration of GSH were added. The prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB) and thrombin time (TT) were detected by using coagulation method before and after treatment with GSH. The detection results of normal plasma and mixed plasma containing GSH of different concentration were compared and analyzed with linear regression. The results showed that the APTT and FIB values of the plasma containing 2.5 mg/L glutathione or more, PT values of the plasma containing 10 mg/L glutathione or more, and TT values of the plasma containing 1250 mg/L glutathione or more were significantly different from those results of normal plasma or mixed plasma (P < 0.01) . There was a linear relation between all of the detection results of PT,APTT, FIB, TT and glutathione concentrations. The results of TT, APTT, PT and FIB detection in patient plasma were statistically different (P < 0.01) before and after treatment with normal concentration GSH. It is concluded that glutathione can influence detection results of coagulation function.

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.

Mesorhizobium silamurunense sp. nov., isolated from root nodules of Astragalus species.

Four rhizobial strains representing a previously defined novel group in the genus Mesorhizobium and isolated from Astragalus species in China were further characterized using a polyphasic approach. Phylogenetic analysis of 16S rRNA gene sequences showed that these Gram-negative bacteria belonged to the genus Mesorhizobium, with Mesorhizobium plurifarium LMG 11892(T) as the closest neighbour sharing a sequence similarity of 99.8 %. Comparative sequence analysis of the atpD, recA, glnII, rpoB, nodC and nifH genes, SDS-PAGE of whole-cell soluble proteins, DNA-DNA hybridization, fatty acid profiles and a series of phenotypic and physiological tests differentiated the novel group from all recognized species of the genus Mesorhizobium. Based on the data obtained in the present and previous studies, this group represents a novel species within the genus Mesorhizobium, for which the name Mesorhizobium silamurunense sp. nov. is proposed. The type strain is CCBAU 01550(T) (= HAMBI 3029(T) = LMG 24822(T)), and could form effective nodules on Astragalus membranaceus, Astragalus adsurgens and Caragana intermedia, and ineffective nodules on Phaseolus vulgaris in cross-nodulation tests.

Rhizobium vignae sp. nov., a symbiotic bacterium isolated from multiple legume species.

A group of rhizobial strains isolated from nodules of multiple legume species grown in different geographical regions of China had identical 16S rRNA genes. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the novel strains formed a subclade in the genus Rhizobium together with Rhizobium galegae, Rhizobium huautlense and Rhizobium alkalisoli, with 99.8  % gene sequence similarity between the strains. The DNA-DNA relatedness values between the representative strain CCBAU 05176(T) and R. galegae ATCC 43677(T), R. huautlense S02(T) and R. alkalisoli CCBAU 01393(T) were 22.6  %, 8.9  % and 15.9  %, respectively. The novel strains were distinguished from recognized species of the genus Rhizobium by using a polyphasic approach, including PCR-based restriction fragment length polymorphism analysis (RFLP) of the 16S-23S intergenic spacer (IGS), phenotypic and physiological tests, sequence comparisons of housekeeping genes and cellular fatty acid profiles. Therefore, it is suggested that this group of strains represents a novel species for which the name Rhizobium vignae sp. nov. is proposed. The type strain is CCBAU 05176(T) (=HAMBI 3039(T)=LMG 25447(T)).

Mesorhizobium shangrilense sp. nov., isolated from root nodules of Caragana species.

Five strains of bacteria isolated from nodules of Caragana bicolor and Caragana erinacea in Yunnan Province of China were classified within the genus Mesorhizobium in the class Alphaproteobacteria. The highest degree of 16S rRNA gene sequence similarity was determined to be to Mesorhizobium loti LMG 6125(T) (99.7 %) and Mesorhizobium ciceri UPM-Ca7(T) (99.7 %). Polyphasic taxonomic methods including SDS-PAGE of whole-cell soluble proteins, comparative housekeeping sequence analysis of atpD, glnII and recA, fatty acid profiles and a series of phenotypic and physiological tests allowed us to cluster the five strains into a coherent group while differentiating them from all previously established Mesorhizobium species. The DNA-DNA relatedness between the representative strain CCBAU 65327(T) and the type strains of M. loti and M. ciceri was 26.5 and 23.4 %, respectively, clearly indicating that strain CCBAU 65327(T) represents a novel species for which we propose the name Mesorhizobium shangrilense sp. nov. Strain CCBAU 65327(T) (=LMG 24762(T) =HAMBI 3050(T)) is designated as the type strain, and could nodulate Caragana microphylla, Caragana intermedia, Glycyrrhiza uralensis, Astragalus adsurgens, Vigna unguiculata, Vigna radiata and Phaseolus vulgaris in cross-nodulation tests.

Mesorhizobium gobiense sp. nov. and Mesorhizobium tarimense sp. nov., isolated from wild legumes growing in desert soils of Xinjiang, China.

Twenty-four Mesorhizobium strains were isolated from desert soils in the Xinjiang region of China and were characterized by a polyphasic approach. These strains grouped into three clusters in IGS-RFLP, SDS-PAGE analysis of whole-cell proteins and BOX-PCR analysis, corresponding to genomic species V, VI and VII as found in a previous study. The results were supported by sequencing analyses of rrs, IGS, atpD and recA genes. Genospecies VII was most related to Mesorhizobium septentrionale, while genospecies V and VI were both most closely related to Mesorhizobium tianshanense, but were distinct from each other and from M. tianshanense. The DNA-DNA hybridization value between the representative strain CCBAU 83284 (genospecies VII) and the type strain of M. septentrionale was 90.1 %. Genospecies VII was thus defined as M. septentrionale. The DNA-DNA relatedness value for representative strains of genospecies V or VI with the related reference strains of recognized species were always lower than 60 %. Low values of DNA-DNA hybridization (32.79 %) between representative strains of genospecies V (CCBAU 83330(T)) and of VI (CCBAU 83306(T)) were also observed. Based upon these results, two novel species are proposed: Mesorhizobium gobiense sp. nov. represented by genospecies V (type strain, CCBAU 83330(T)=LMG 23949(T)=HAMBI 2974(T)) and Mesorhizobium tarimense sp. nov. represented by genospecies VI (type strain, CCBAU 83306(T)=LMG 24338(T)=HAMBI 2973(T)). Strain CCBAU 83278 grouped as the most peripheral member with genospecies VI in SDS-PAGE of whole-cell proteins and BOX-PCR analysis and in the phylogenetic tree of 16S-23S rRNA intergenic spacer (IGS) sequences. The results of analyses of rrs, atpD and recA gene sequences, as well as those of DNA-DNA hybridization studies, strongly supported the suggestion that this strain belonged to a species quite different from genospecies V and VI and from any other recognized species of the genus Mesorhizobium. As only one strain has been isolated to date, strain CCBAU 83278 was not proposed as a novel species in this study. Mesorhizobium gobiense sp. nov. and Mesorhizobium tarimense sp. nov. could be differentiated from each other as well as from recognized species of the genus Mesorhizobium on the basis of phenotypic characteristics. The symbiotic loci (nodC and nifH) of the two novel species formed two phylogenetic branches related to Mesorhizobium loti and M. tianshanense. The type strains of the two novel species were able to nodulate Glycyrrhiza uralensis, Lotus corniculatus, Oxytropis glabra and Robinia pseudoacacia but not Astragalus membranaceus, Leucaena leucocephala, Phaseolus vulgaris, Pisum sativum or Medicago sativa.