Carotenoids in petals of Medicago falcata

Petals in three strains of M. falcata L. contain lutein 50–52%, lutein-5,6-epoxide 16–18%, chrysanthemaxanthin 9–10%, flavoxanthin 7–8%, and in smaller amounts β-carotene, ζ-carotene, a hydroxy-α-carotene-like pigment, two neoxanthins, auroxanthin and a flavoxanthin-like pigment. Almost 98% of all carotenoids were xanthophylls esterified with fatty acids. Uniformity of major carotenoid content in the three strains is consistent with their all belonging to the same species.     

Dormancy of Medicago marina (L.) seed

Medicago marina (L.) is a Mediterranean species whose seeds show strong dormancy that prevents germination. We used an integrated approach of physiological analyses and proteomics to investigate the mechanisms that control M. marina dormancy/germination and that underlie stress tolerance. First, we evaluated the effects on dormancy breaking of the following treatments: mechanical scarification, freezing at −20 °C, storage for 4 months and heating at 100 °C for 1 h. Mechanical scarification and freezing were the most effective treatments in overcoming dormancy. The role of abscisic acid (ABA) in M. marina dormancy was studied by ELISA immuno-enzymatic assay. The ABA content of germinated and non-germinated mature (control) and treated seeds was determined. The level of ABA was higher in treated seeds than in control seeds; the most significant increase occurred in the heated seeds. A comparison of the ABA level in the germinated, control and treated seeds suggests that different mechanisms modulate ABA content in response to different stresses, and that a specific ABA-signalling pathway regulates germination. Proteomic analysis revealed 46 proteins differentially expressed between treated and untreated seeds; 14 of these proteins were subsequently identified by mass spectrometry. Several of the proteins identified are important factors in the stress response, and are involved in such diverse functions as lipid metabolism, protein folding and chromatin protection. Lastly, an analysis of the phosphoproteome maps showed that the function of many proteins in seeds subjected to temperature treatment is modulated through post-translational modifications.     

Mycoplasma feriruminatoris sp. nov., a fast growing Mycoplasma species isolated from wild Caprinae

Five Mycoplasma strains from wild Caprinae were analyzed: four from Alpine ibex (Capra ibex) which died at the Berlin Zoo between 1993 and 1994, one from a Rocky Mountain goat collected in the USA prior to 1987. These five strains represented a population different from the populations belonging to the ‘Mycoplasma mycoides cluster’ as tested using multi locus sequence typing, Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis and DNA–DNA hybridization. Analysis of the 16S rRNA gene (rrs), genomic sequence based in silico as well as laboratory DNA–DNA hybridization, and the analysis of phenotypic traits in particular their exceptionally rapid growth all confirmed that they do not belong to any Mycoplasma species described to date. We therefore suggest these strains represent a novel species, for which we propose the name Mycoplasma feriruminatoris sp. nov. The type strain is G5847^T (= DSM 26019^T = NCTC 1362^T).     

No loss of genetic diversity in small and isolated populations of Medicago sativa subsp. falcata

Molecular allozyme markers of three polymorphic isozymes were used to estimate the genetic diversity among the seed progeny in fragmented Estonian populations of sickle medic Medicago sativa ssp. falcata L. depending on the population size and the isolation degree. Genetic diversity H_e was high in all populations, ranging between 0.795 and 0.893. No correlation between the genetic diversity measures and population size or isolation distance was found. Even the smallest population had equally high genetic diversity as about a hundred times larger population. Genetic differentiation of populations into two major groups was associated with the geographic position of populations, except one remote population. Elimination of seed progeny of reduced fitness by embryo abortion and continuous yearlong contribution of the highly heterozygous progeny through the soil seed bank are considered as important supplementary factors that have contributed to maintaining high levels of genetic diversity in populations of sickle medic in addition to its autotetraploid nature and perennial life form.     

Genetic diversity and symbiotic evolution of rhizobia from root nodules of Coronilla varia

Ninety symbiotic rhizobial isolates from root nodules of Coronilla varia growing in the Shaanxi province of China were characterized. Combined with the results of RFLP patterns, six genotypes were defined among the rhizobial strains and they were divided into three genomic genera. These included Mesorhizobium sp., M. alhagi, M. amorphae, M. metallidurans/M. gobiense as the dominant group (86.7%), and Rhizobium yanglingense and Agrobacterium tumefaciens as the minor groups, according to analysis of the corresponding 16S rRNA, nodC and nifH genes. Five nodC types, which mainly grouped into the Mesorhizobium genus, were obtained from all the isolates examined, implying that nodC genes probably occurred from the native habitat through lateral transfer and long-term adaptation, finally evolving toward M. alhagi. Four different nifH types, displaying obvious differences compared to those of 16S rRNA and nodC, implied that possible lateral transfer of the symbiotic genes occurred between different genera. The association between soil components and the genetic diversity of the rhizobial population demonstrated that combined genotypes were positively correlated with the pH of soil samples.     

南苜蓿高效共生根瘤菌土壤的筛选

利用云南德宏盈江县3种类型的土壤,对分离自云南楚雄、德宏等地的SWF67523、SWF67409、SWF67456等12株根瘤菌菌株进行了南苜蓿接种效果的研究。结果表明,在所有供试菌株中,菌株SWF67523、SWF67409、SWF67394表现最为优良,结瘤率较高的菌株是SWF67523、SWF67501、SWF67394和SWF67350,结瘤率均达到95％以上;而菌株SWF67523、SWF67409和SWF67394对植株株高影响最大;菌株SWF67409、SWF67523和SWF67394对提高植株干重贡献最大,其中菌株SWF67409比空白对照增产106.5%;供试菌株对含氮量的影响也很显著,接种菌株SWF67409、SWF67523和SWF67394的苜蓿植物含氮量相比其他菌株较高,影响显著。综合以上结果,发现来源于盈江南苜蓿的根瘤菌菌株SWF67523较其他菌株表现突出,同时发现根瘤菌菌株的接种效果受土壤因子的影响,在含有效钾中等的土壤平原镇Ⅱ上,接种根瘤菌的南苜蓿地上部分干重普遍高于其他两种有效钾含量为较缺的土壤,说明根瘤菌菌株与宿主和土壤环境存在密切关系,在适合的宿主和生活环境中将会发挥最大的共生固氮作用。     

Identification and characterization of the pathogenic effect of a Vibrio parahaemolyticus-related bacterium isolated from clam Meretrix meretrix with mass mortality

A mass mortality of clam, Meretrix meretrix, occurred in Jiangsu Province of China in the late September of 2007. Of the isolates obtained from the diseased clams, MM21 had the strongest virulence to the clam in the virulence test, with a LD50 value of ∼6 × 10⁶ CFU ml⁻¹. MM21 was identified as Vibrio parahaemolyticus by the VITEK 2 Compact system and 16S rDNA sequencing. Detection of virulence-associated genes by PCR indicated that MM21 was positive for toxR and tlh, and negative for tdh. Compared with control group, histiocytes from MM21-infected clams displayed a variety of cytopathological changes by transmission electron microscopy examination, which included increased lipid droplets in hepatocytes, deposition of granules in the mantle, excessive secretion in the gill. The results of our study suggested that MM21 may have been an etiological element in the mass mortalities of hard clam (M. meretrix) in Jiangsu Province of China in 2007.     

Using alfalfa (Medicago sativa) to ameliorate salt-affected soils in Yingda irrigation district in Northwest China

Secondary salinization of soil is a major limiting factor of agricultural sustainability and recovery of functional environments in irrigated agriculture in arid and semi-arid regions. The ameliorating effect of planting alfalfa (Medicago sativa) on salt-affected soils was assessed in field experiments in the irrigated region of Qingwangchuan basin for 5 years. The results showed that salt content in the soil profile gradually decreased with duration of alfalfa cultivation. Soil EC in the layer of 0-20 cm decreased significantly. The concentrations of soluble anions were found to be in the order of Cl^- > SO₄^2- > HCO₃^- in the soil profile of the study area. After alfalfa planting, Cl^- concentration in the soil profile notably decreased. In contrast, HCO₃^- concentration was significantly higher in the topsoil planted to alfalfa than in unplanted soil, especially after the first and the second year of cultivation, but markedly decreased after 3 years of alfalfa growth. With the extension of cultivation ages, total N content in different soil layers gradually increased through N2 fixation. Organic matter content in the soil profile was not enhanced significantly until the later stages of alfalfa cultivation. Available P accumulated in the topsoil in dependence on the length of cultivation. Soil pH was significantly higher in the planted than unplanted treatment, but was gradually decreased with increased duration of cultivation, especially in topsoil. Significant differences in ash content of alfalfa shoot were found between the different cultivation ages. Alfalfa shoot Na concentration showed slightly decrease, whereas shoot Cl^- concentration decreased with the duration of cultivation. The ameliorating effect of alfalfa cultivation on salt-affected soil showed a spatial and temporal variability due to the interactions between soil and plants. This positive effect resulted in either the salt leaching from the root zone to below 80-cm depth by irrigation water or the removal of less salts through harvest of alfalfa shoots.     

Centrosema is a promiscuous legume nodulated by several new putative species and symbiovars of Bradyrhizobium in various American countries

Centrosema is an American indigenous legume that can be used in agroecosystems for recovery of acidic and degraded soils. In this study, a Centrosema-nodulating rhizobial collection of strains isolated in a poor acid savanna soil from Venezuela was characterized, and the members of the collection were compared to other Centrosema strains from America. The analysis of the rrs gene showed that the strains nodulating Centrosema in American countries were closely related to different species of the genus Bradyrhizobium. However, the analysis of the atpD and recA genes, as well as the 16S–23S ITS region, showed that they formed several new phylogenetic lineages within this genus. The Venezuela strains formed three lineages that were divergent among themselves and with respect to those formed by Centrosema strains isolated in other countries, as well as to the currently described species and genospecies of Bradyrhizobium. In addition, the symbiotic genes nodC and nifH carried by Centrosema-nodulating strains were analyzed for the first time, and it was shown that they belonged to three new phylogenetic lineages within Bradyrhizobium. The nodC genes of the Centrosema strains were divergent among themselves and with respect to the genistearum and glycinearum symbiovars, indicating that Centrosema is a promiscuous legume. According to these results, the currently known Centrosema-nodulating strains represent several new putative species and symbiovars of the genus Bradyrhizobium.     

Isolation and structural characterization of biosurfactant produced by an alkaliphilic bacterium Cronobacter sakazakii isolated from oil contaminated wastewater

Production of biosurfactant from an alkaliphilic bacterium Cronobacter sakazakii (accession no. JN398668) was screened by haemolytic assay, emulsifying activity and surface tension measurement. Biosurfactant, comprised of total sugars (73.3%), reducing sugars (1.464%), protein (11.9%), uronic acid (15.98%) and sulfate (6.015%), showed low viscosity with pseudoplastic rheological behavior and exhibited significant emulsification activity with oils and hydrocarbons. A series of low and mid range mass peaks (m/z) corresponding to mono-, di-, tri- and oligosaccharides were detected in the positive ion reflector mode of MALDI TOF-TOF MS. GC-MS analysis revealed composition of monosaccharide moieties (w/w) viz. glucose (14%), mannose (24%), galactose (14%), xylose (20%) and arabinose (1.9%). ¹H NMR, FT-IR and EDX analyses confirmed the characteristic various functional groups, bonds and elements respectively. Thermostability (up to 260 °C) and CI (0.456) were determined by TG and DSC analyses. Inherent properties of biosurfactant make it a potential candidate for bioremediation of oil and hydrocarbons.     

Geodermatophilus tzadiensis sp. nov., a UV radiation-resistant bacterium isolated from sand of the Saharan desert

Three novel Gram-positive, aerobic, actinobacterial strains, CF5/2^T, CF5/1 and CF7/1, were isolated in 2007 during environmental screening of arid desert soil in the Sahara desert, Chad. Results from riboprinting, MALDI-TOF protein spectra and 16S rRNA sequence analysis confirmed that all three strains belonged to the same species. Phylogenetic analysis of 16S rRNA sequences with the strains’ closest relatives indicated that they represented a distinct species. The three novel strains also shared a number of physiological and biochemical characteristics distinct from previously named Geodermatophilus species. The novel strains’ peptidoglycan contained meso-diaminopimelic acid; their main phospholipids were phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and a small amount of phosphatidylglycerol; MK-9(H₄) was the dominant menaquinone. The major cellular fatty acids were the branched-chain saturated acids iso-C_16:0 and iso-C_15:0. Galactose was detected as diagnostic sugar. Based on these chemotaxonomic results, 16S rRNA gene sequence analysis and DNA–DNA hybridization between strain CF5/2^T and the type strains of Geodermatophilus saharensis, Geodermatophilus arenarius, Geodermatophilus nigrescens, Geodermatophilus telluris and Geodermatophilus siccatus, the isolates CF5/2^T, CF5/1 and CF7/1 are proposed to represent a novel species, Geodermatophilus tzadiensis, with type strain CF5/2^T = DSM 45416 = MTCC 11411 and two reference strains, CF5/1 (DSM 45415) and CF7/1 (DSM 45420).     

Photobacterium marinum sp. nov., a marine bacterium isolated from a sediment sample from Palk Bay,India

The novel, cream colored, Gram-staining-negative, rod-shaped, motile bacteria, designated strains AK15^T and AK18, were isolated from sediment samples collected from Palk Bay, India. Both strains were positive for arginine dihydrolase, lysine decarboxylase, oxidase, nitrate reduction and methyl red test. The major fatty acids were C_16:0, C_{18:1 ω7c}, C_{16:1 ω7c} and/or C_{16:1 ω6c} and/or iso-C_15:0 2-OH (summed feature 3). Polar lipids content of strains AK15^T and AK18 were found to bephosphatidylethanolamine (PE), two unidentified phospholipids (PL1 and PL2) and three unidentified lipids (L1-L3). The 16S rRNA gene sequence analysis indicated strains AK15^T and AK18 as the members of the genus Photobacterium and closely related to the type strain Photobacterium jeanii with pair-wise sequence similarity of 96.7%. DNA–DNA hybridization between strain AK15^T and AK18 showed a relatedness of 87%. Based on data from the current polyphasic study, strains AK15^T and AK18 are proposed as novel species of the genus Photobacterium, for which the name Photobacterium marinum sp. nov. is proposed. The type strain of Photobacterium marinum is AK15^T (=MTCC 11066^T = DSM 25368^T).     

Alternative oxidase 1 (Aox1) gene expression in roots of Medicago truncatula is a genotype-specific component of salt stress tolerance

Alternative oxidase (AOX) is the central component of the non-phosphorylating alternative respiratory pathway in plants and may be important for mitochondrial function during environmental stresses. Recently it has been proposed that Aox can be used as a functional marker for breeding stress tolerant plant varieties. This requires characterization of Aox alleles in plants with different degree of tolerance in a certain stress, affecting plant phenotype in a recognizable way. In this study we examined Aox1 gene expression levels in Medicago truncatula genotypes differing in salt stress tolerance, in order to uncover any correlation between Aox expression and tolerance to salt stress. Results demonstrated a specific induction of Aox1 gene expression in roots of the tolerant genotype that presented the lowest modulation in phenotypic and biochemical stress indices such as morphologic changes, protein level, lipid peroxidation and ROS generation. Similarly, in a previous study we reported that induction of antioxidant gene expression in the tolerant genotype contributed to the support of the antioxidant cellular machinery and stress tolerance. Correlation between expression patterns of the two groups of genes was revealed mainly in 48 h treated roots. Taken together, results from both experiments suggest that M. truncatula tolerance to salt stress may in part due to an efficient control of oxidative balance thanks to (i) induction of antioxidant systems and (ii) involvement of the AOX pathway. This reinforces the conclusion that differences in antioxidant mechanisms can be essential for salt stress tolerance in M. truncatula and possibly the corresponding genes, especially Aox, could be utilized as functional marker.     

The endemic Genista versicolor from Sierra Nevada National Park in Spain is nodulated by putative new Bradyrhizobium species and a novel symbiovar (sierranevadense)

Genista versicolor is an endemic legume from Sierra Nevada National Park which constitutes one of the UNESCO-recognized Biosphere Reserves. In the present study, a collection of strains nodulating this legume was analysed in characteristic soils of this ecosystem. Most strains nodulating G. versicolor belonged to rrs group I within the genus Bradyrhizobium and only one strain, named GV137, belonged to rrs group II from which only a single species, B. retamae, has been described in Europe to date. Strain GV137, and some strains from rrs group I, belonged to putative new species of Bradyrhizobium, although most strains from group I belonged to B. canariense, according to the ITS fragment and atpD gene analysis. This result contrasted with those obtained in Genista tinctoria in Northeast Europe whose endosymbionts were identified as B. japonicum. The analysis of the symbiotic nodC and nifH genes carried by G. versicolor-nodulating strains showed that most of them belonged to symbiovar genistearum, as did those isolated from G. tinctoria. Nevertheless, strain GV137, belonging to rrs group II, formed a divergent lineage that constituted a novel symbiovar within the genus Bradyrhizobium for which the name sierranevadense is proposed. This finding showed that the Genisteae are not restrictive legumes only nodulated by symbiovar genistearum, since Genista is a promiscuous legume nodulated by at least two symbiovars of Bradyrhizobium, as occurs in Retama species.     

Characterization of a new periplasmic single-domain rhodanese encoded by a sulfur-regulated gene in a hyperthermophilic bacterium Aquifex aeolicus

Rhodaneses (thiosulfate cyanide sulfurtransferases) are enzymes involved in the production of the sulfur in sulfane form, which has been suggested to be the relevant biologically active sulfur species. Rhodanese domains occur in the three major domains of life. We have characterized a new periplasmic single-domain rhodanese from a hyperthermophile bacterium, Aquifex aeolicus, with thiosulfate:cyanide transferase activity, Aq-1599. The oligomeric organization of the enzyme is stabilized by a disulfide bridge. To date this is the first characterization from a hyperthermophilic bacterium of a periplasmic sulfurtransferase with a disulfide bridge. The aq-1599 gene belongs to an operon that also contains a gene for a prepilin peptidase and that is up-regulated when sulfur is used as electron acceptor. Finally, we have observed a sulfur-dependent bacterial adherence linked to an absence of flagellin suggesting a possible role for sulfur detection by A. aeolicus.     

Stimulation of root acid phosphatase by phosphorus deficiency is regulated by ethylene in Medicago falcata

Plants have developed numerous strategies to cope with phosphorus (P) deficiency resulting from low availability in soils. Evolution of ethylene and up-regulation of root secreted acid phosphatase activity are common for plants in response to P deficiency. To determine the role of ethylene in response of plants to P deficiency, we investigated the effects of ethylene precursor (1-amino cyclopropane-1-carboxylic acid, ACC) and ethylene synthesis antagonists (aminoethoxyvinylglycine AVG, cobalt, Co²⁺) on P concentrations in roots and shoots of Medicago falcata seedlings grown in P-sufficient (500 μM H₂PO₄⁻) and P-deficient (5 μM H₂PO₄⁻) solution. After transferring M. falcata seedlings from P-sufficient to P-deficient solution for 2 days, root P concentration was significantly reduced. The reduction in root P concentration was reversed by AVG and Co²⁺, and a similar reduction in root P concentration of seedlings exposed to P-sufficient solution was observed by ACC. Expression of high-affinity phosphate transporters (MfPT1, MfPT5) was enhanced by P-deficiency and this process was reversed by AVG and Co²⁺. There was a marked increase in activity of root acid phosphatase (APase) and expression of gene encoding APase (MfPAP1) under P-deficient conditions, and the increase in APAse activity and expression of MfPAP1 was inhibited by AVG and Co²⁺. APase activity and expression of MfPAP1 expression in seedlings grown in P-sufficient solution were enhanced by ACC. Root and shoot P concentrations were increased when organic phosphorus was added to the P-deficient solution, and the increase in P concentration was significantly inhibited by AVG and Co²⁺. These results indicate that ethylene plays an important role in modulation of P acquisition by possibly mobilizing organic P via up-regulating root APase activity and high-affinity phosphate transporters.     

MALDI-TOF mass spectrometry as a tool for differentiation of Bradyrhizobium species: Application to the identification of Lupinus nodulating strains

Genus Bradyrhizobium includes slow growing bacteria able to nodulate different legumes as well as species isolated from plant tumours. The slow growth presented by the members of this genus and the phylogenetic closeness of most of its species difficults their identification. In the present work we applied for the first time Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) to the analysis of Bradyrhizobium species after the extension of MALDI Biotyper 2.0 database with the currently valid species of this genus. With this methodology it was possible to identify strains belonging to phylogenetically closely related species of genus Bradyrhizobium allowing the discrimination among species with rrs gene identities higher than 99%. The application of MALDI-TOF MS to strains isolated from nodules of different Lupinus species in diverse geographical locations allowed their correct identification when comparing with the results of rrs gene and ITS analyses. The nodulation of Lupinus gredensis, an endemic species of the west of Spain, by B. canariense supports the European origin of this species.     

一株溶藻细菌对海洋原甲藻的溶藻效应

从深圳大鹏湾南澳赤潮爆发海域的表层海水中分离得到1株对海洋原甲藻(Prorocentrum micans)具有溶藻活性的海洋细菌,菌株编号为N10。利用液相感染法研究了该溶藻细菌的溶藻效果和溶藻作用方式。结果表明,菌株N10能使藻细胞失去运动活性,并膨胀变形,细胞膜内物质聚集于一端,藻细胞最终破裂死亡。菌悬液接种到藻液中的量越大,初始细菌密度越高,其溶藻效果越强。菌悬液以1∶10的体积比接种到藻液中时,藻细胞在24 h的死亡率为83%,至72 h全部溶解死亡;体积比为1∶20的藻细胞在24 h的死亡率为71%,之后藻细胞密度略有波动,120 h时死亡率达77%;而体积比为1∶100的藻细胞密度在前24 h有所下降,死亡率达39%,之后藻细胞密度又开始明显上升;对照组的藻细胞密度均呈明显上升趋势。菌悬液过滤液和高温加热处理后的菌悬液过滤液对海洋原甲藻均无溶藻活性,表明菌株N10的溶藻方式为直接溶藻。通过16S rRNA序列分析并与GenBank数据进行同源性检索,并结合细菌形态及生理生化特征,菌株N10隶属于黄杆菌科(Flavobacteriaceae)中的Muricauda sp.。