Pseudomonas edaphica sp. nov., isolated from rhizospheric soil of Cistus ladanifer L. in Spain.

During a study on biodiversity of bacteria inhabiting rhizospheric soil of rockrose (Cistus ladanifer L.), we isolated a strain coded RD25T in a soil from Northern Spain. The 16S rRNA gene sequence showed 99.5 % identity with respect to the closest related species Pseudomonas brenneri DSM15294T, and 99.4 % with respect to P. paralactis WS4672T. The following related Pseudomonas species showed 99.3 % or less identity, and therefore RD25T was classified within genus Pseudomonas. The phylogenetic analysis of 16S rRNA and the housekeeping genes rpoB, rpoD and gyrB suggested that this strain could be a novel species. The strain RD25T has several polar-subpolar flagella. It can grow at 36 °C, at 0-6 % NaCl concentration and a range of pH 5-9. Positive for arginine dihydrolase and urease production, and negative for reduction of nitrate. The strain is catalase and oxidase positive. Major fatty acids are C16 : 1 ω7c / C16 : 1 ω6c in summed feature 3, C16 : 0, and C18 : 1 ω7c / C18 : 1 ω6c in summed feature 8. The respiratory ubiquinone is Q9. The DNA G+C content was 59.9 mol%. The digital DNA-DNA hybridisation average values (dDDH) ranged between 30-61.2 % relatedness and the ANIb values ranged between 93.9-80.5 % with respect to the type strains of the closely related species. Therefore, the genotypic, genomic, phenotypic and chemotaxonomic data support the classification of strain RD25 as a novel species of genus Pseudomonas, for which the name P. edaphica sp. nov. is proposed. The type strain is RD25T (=LMG 30152T=CECT 9373T).

Ultrastructure of compatible and incompatible interactions in phloem sieve elements during the stylet penetration by cotton aphids in melon.

Resistance of the melon line TGR-1551 to the aphid Aphis gossypii is based on preventing aphids from ingesting phloem sap. In electrical penetration graphs (EPGs), this resistance has been characterized with A. gossypii showing unusually long phloem salivation periods (waveform E1) mostly followed by pathway activities (waveform C) or if followed by phloem ingestion (waveform E2), ingestion was not sustained for more than 10 min. Stylectomy with aphids on susceptible and resistant plants was performed during EPG recording while the stylet tips were phloem inserted. This was followed by dissection of the penetrated leaf section, plant tissue fixation, resin embedding, and ultrathin sectioning for transmission electron microscopic observation in order to study the resistance mechanism in the TGR. The most obvious aspect appeared to be the coagulation of phloem proteins inside the stylet canals and the punctured sieve elements. Stylets of 5 aphids per genotype were amputated during sieve element (SE) salivation (E1) and SE ingestion (E2). Cross-sections of stylet bundles in susceptible melon plants showed that the contents of the stylet canals were totally clear and also, no coagulated phloem proteins occurred in their punctured sieve elements. In contrast, electron-dense coagulations were found in both locations in the resistant plants. Due to calcium binding, aphid saliva has been hypothesized to play an essential role in preventing/suppressing such coagulations that cause occlusion of sieves plate and in the food canal of the aphid's stylets. Doubts about this role of E1 salivation are discussed on the basis of our results.

Paenibacillus tritici sp. nov., isolated from wheat roots.

A bacterial strain designated RTAE36T was isolated from wheat roots in northern Spain. Phylogenetic analyses based on 16S rRNA gene sequence placed the isolate into the genus Paenibacillus with its closest relative being Paenibacillus borealis DSM 13188T with 97.7 % sequence similarity. Cells of the isolate were facultatively anaerobic, Gram-stain-positive, motile and sporulating rods. Catalase and oxidase were positive. Gelatin, casein and starch were not hydrolysed. Growth was supported by many carbohydrates and organic acids as carbon sources. MK-7 was the only menaquinone detected, and anteiso-C15 : 0, C16 : 0, iso-C14 : 0 and iso-C16 : 0 were the major fatty acids. The polar lipids profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid, two unidentified phospholipids, three unidentified phosphoaminolipids, one unidentified glycolipid and one unidentified lipid. meso-Diaminopimelic acid was detected in the cell-wall peptidoglycan. Strains RTAE36T and P. borealis DSM 13188T had an mean DNA-DNA relatedness of 39 % and differed in several phenotypic and chemotaxonomic characteristics, confirming that strain RTAE36T should be considered as a representative of a novel species of the genus Paenibacillus, for which the name Paenibacillus tritici sp. nov. is proposed. The type strain is RTAE36T (=LMG 29502T=CECT 9125T).

The role of native lichens in the biomonitoring of gaseous mercury at contaminated sites.

Contamination by atmospheric mercury has been assessed in two different areas from Spain (Las Cuevas, Ciudad Real and Flix, Tarragona) using lichens as biomonitors. The relationship established between mercury contents in the soils and the gaseous mercury (GM) was also observed. It was found that the GM is highest in the vicinity of the source and it is dispersed depending on of the distance to the source and the wind directions. The mercury concentration in the gas phase in Flix was higher than that found in Las Cuevas and also higher than the value that the US EPA recommended. The mercury bioaccumulation in the native lichens from genders Ramalina and Xanthoria were used as biomonitors for absorbing mercury in Las Cuevas and Flix, respectively. The mercury uptake by Ramalina was higher than the amount accumulated by Xanthoria, a difference that was mainly due to the lichen characteristics. The content of mercury in lichens in relation to the mercury in gas was fitted by a Freundlich type equation, indicating that the equilibrium between both phases was established. Besides, transplanted Ramalina lichen in Las Cuevas allowed to obtain the kinetic of mercury uptake. A kinetic model of first order based on the equilibrium was proposed and the mass transfer constants for each sampling station were estimated. As it was expected, these values increased with the predominant wind flow direction.

Brevundimonas canariensis sp. nov., isolated from roots of Triticum aestivum.

A bacterial strain designated GTAE24T was isolated from a root of wheat growing in soil from the Canary Islands, Spain. Phylogenetic analyses based on 16S rRNA gene sequences placed the isolate in the genus Brevundimonas with Brevundimonas abyssalisTAR-001T as its closest relative at 99.4 % similarity. DNA-DNA hybridization studies showed an average of 38 % relatedness between strain GTAE24T and the type strain of B. abyssalis. Cells were Gram-stain-negative and motile by polar flagella. The strain was positive for oxidase and weakly positive for catalase. Gelatin, starch and casein were not hydrolysed. Growth was supported by many carbohydrates and organic acids as carbon source. Ubiquinone Q-10 was the predominant isoprenoid quinone and C18 : 1ω7c/C18 : 1ω6c (summed feature 8) and C16 : 0 were the major fatty acids. The major polar lipids were phosphatidylglycerol, 1,2-di-O-acyl-3-O-[d-glucopyranosyl-(1,4)-α-d-glucopyranuronosyl] glycerol, 1,2-diacyl-3-O-[6'-phosphatidyl-α-d-glucopyranosyl] glycerol, 1,2-di-O-acyl-3-O-α-d-glucopyranosyl glycerol, and 1,2-di-O-acyl-3-O-α-d-glucopyranuronosyl glycerol. The DNA G+C content was 63.9 mol%. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain GTAE24T should be considered as representing a novel species of the genus Brevundimonas, for which the name Brevundimonas canariensis sp. nov. is proposed. The type strain is GTAE24T (=LMG 29500T=CECT 9126T).

Paenibacillus hispanicus sp. nov. isolated from Triticum aestivum roots.

A bacterial strain designated AMTAE16T was isolated from a root of wheat in Spain. Phylogenetic analyses based on 16S rRNA gene sequences placed the isolate in the genus Paenibacilluswith its closest relative being Paenibacillus daejeonensis AP-20T with 99.0 % 16S rRNA gene sequence similarity. DNA-DNA hybridization studies showed a mean of 30 % DNADNA relatedness between strain AMTAE16T and the type strain of P. daejeonensis. The isolate was a Gram-stainvariable, motile and sporulating rod. Catalase and oxidase activities were positive. Gelatin and starch were hydrolysed but not casein. Growth was supported by many carbohydrates and organic acids as carbon source. MK-7 was the only menaquinone detected and anteiso-C15 : 0, C16 : 0 and iso-C16 : 0 were the major fatty acids. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminophospholipids, four unidentified phospholipids and two unidentified lipids. meso-Diaminopimelic acid was detected in the peptidoglycan. The DNA G+C content was 55.4 mol%. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain AMTAE16T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus hispanicus sp. nov. is proposed. The type strain is AMTAE16T(=LMG 29501T=CECT 9124T).

Paenibacillus periandrae sp. nov., isolated from nodules of Periandra mediterranea.

A bacterial strain designated PM10T was isolated from root nodules of Periandra mediterranea in Brazil. Phylogenetic analyses based on 16S rRNA gene sequences placed the isolate in the genus Paenibacillus with its closest relatives being Paenibacillus vulneris CCUG 53270T and Paenibacillus yunnanensis YN2T with 95.6 and 95.9% 16S rRNA gene sequence similarity, respectively. The isolate was a Gram-stain-variable, motile, sporulating rod that was catalase-negative and oxidase-positive. Caseinase was positive, amylase was weakly positive and gelatinase was negative. Growth was supported by many carbohydrates and organic acids as carbon sources. MK-7 was the only menaquinone detected and anteiso-C15 : 0 was the major fatty acid. Major polar lipids were diphosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol and two unidentified lipids. meso-Diaminopimelic acid was detected in the peptidoglycan. The DNA G+C content was 52.9 mol%. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain PM10T should be considered representative of a novel species of the genus Paenibacillus, for which the name Paenibacillus periandrae sp. nov. is proposed. The type strain is PM10T (=LMG 28691T=CECT 8827T).

Erwinia endophytica sp. nov., isolated from potato (Solanum tuberosum L.) stems.

We analysed, using a polyphasic taxonomic approach, two bacterial strains coded BSTT30T and BSTT40, isolated in the course of a study of endophytic bacteria occurring in the stems and roots of potatoes growing in soil from Salamanca, Spain. The 16S rRNA gene sequence was identical in both strains and had 98.4 % identity with respect to the closest relatives Erwinia tasmaniensis Et1/99T and Erwinia rhapontici ATCC29283T. Erwinia billingiae E63T and Erwinia toletana A37T were also closely related with 98.2 % sequence similarities, so the novel strains were classified within the genus Erwinia. The analysis of the housekeeping genes gpd, gyrB and rpoD confirmed the phylogenetic affiliation of strains BSTT30T and BSTT40 with similarities of lower than 90 % in all cases with respect to the closest relatives mentioned above. The respiratory quinone of strain BSTT30T was Q8. The major fatty acids were C16 : 0, C16 : 1ω7c/16 : 1ω6c in summed feature 3 and C18 : 1ω7c/18 : 2ω6,9c in summed feature 8. The novel strains were oxidase-negative and catalase-positive. Glucose was fermented without gas production. They were negative for arginine dihydrolase, urease and indole production. The strains could grow at 35 °C and at pH 10. DNA G+C content was 50.1 mol%. DNA-DNA hybridization results showed values of lower than 29 % relatedness with respect to the type strains of the four most closely related species. Therefore, the combined genotypic, phenotypic and chemotaxonomic data support the classification of strains BSTT30T and BSTT40 into a novel species of the genus Erwinia, for which the name Erwinia endophytica sp. nov. is proposed. The type strain is BSTT30T ( = LMG 28457T, CECT 8692T).

Photosynthetic and Ultrastructure Parameters of Maize Plants are Affected During the Phyto-Rhizoremediation Process of Degraded Metal Working Fluids.

A phyto-rhizoremediation system using corn and esparto fiber as rooting support to remediate degraded metal working fluids (dMWFs) has been developed in the present study. In order to improve the process, plants were inoculated at the root level with bacteria either individually, and with a consortium of strains. All strains used were able to grow with MWFs. The results show that this system significantly lowers the Chemical Oxygen Demand below legal limits within 5 days. However, results were only improved with the bacterial consortium. Despite the effectiveness of the phyto-rhizoremediation process, plants are damaged at the photosynthetic level according to the photosynthetic parameters measured, as well as at the ultrastructure of the vascular cylinder and the Bundle Sheath Cells. Interestingly, the bacterial inoculation protects against this damage. Therefore, it seems that that the inoculation with bacteria can protect the plants against these harmful effects.

Pseudomonas endophytica sp. nov., isolated from stem tissue of Solanum tuberosum L. in Spain.

A bacterial strain named BSTT44(T) was isolated in the course of a study of endophytic bacteria occurring in stems and roots of potato growing in a soil from Salamanca, Spain. The 16S rRNA gene sequence had 99.7% identity with respect to that of its closest relative, Pseudomonas psychrophila E-3T, and the next most closely related type strains were those of Pseudomonas fragi, with 99.6% similarity, Pseudomonas deceptionensis, with 99.2% similarity, and Pseudomonas lundensis, with 99.0% similarity; these results indicate that BSTT44(T) should be classified within the genus Pseudomonas. Analysis of the housekeeping genes rpoB, rpoD and gyrB confirmed its phylogenetic affiliation and showed identities lower than 92% in all cases with respect to the above-mentioned closest relatives. Cells of the strain bore one polar-subpolar flagellum. The respiratory quinone was Q-9.The major fatty acids were C16:0, C18:1ω7c and summed feature 3 (C16:1ω7c and/or C16:1ω6c). The strain was oxidase-, catalase- and urease-positive and the arginine dihydrolase system was present, but tests for nitrate reduction, ß-galactosidase production and aesculin hydrolysis were negative. It could grow at 35 °C and at pH 5-9.The DNA G+C content was 60.2 mol%. DNA-DNA hybridization results showed less than 48% relatedness with respect to the type strains of the four most closely related species. Therefore, the combined results of genotypic, phenotypic and chemotaxonomic analyses support the classification of strain BSTT44 into a novel species of the genus Pseudomonas, for which the name Pseudomonas endophytica sp. nov. is proposed. The type strain is BSTT44(T) ( = LMG 28456(T) = CECT 8691(T)).

Fontibacillus solani sp. nov. isolated from potato (Solanum tuberosum L.) root.

A bacterial strain designated A4STR04(T) was isolated from the inner root tissue of potatoes in Spain. Phylogenetic analysis based on the 16S rRNA gene sequence placed the isolate into the genus Fontibacillus, being most closely related to Fontibacillus panacisegetis KCTC 13564(T) with 99% identity. The isolate was observed to form Gram-positive, motile and sporulating rods. The catalase test was found to be negative and oxidase positive. Nitrate was found to be reduced to nitrite. ß-Galactosidase and caseinase were observed to be produced but the production of gelatinase, urease, arginine dehydrolase, ornithine and lysine decarboxylase was negative. Aesculin hydrolysis was found to be positive and acetoin production was negative. Growth was found to be supported by many carbohydrates and organic acids as carbon source. MK-7 was the only menaquinone detected and the major fatty acid (61.5%) was identified as anteiso-C(15:0), as occurs in the other species of genus Fontibacillus. The strain A4STR04(T) was found to display a complex lipid profile consisting of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, a glycolipid, two phospholipids, a lipid and two aminophospholipids. Mesodiaminopimelic acid was detected in the peptidoglycan. The G+C content was determined to be 50.5 mol% (Tm). Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain A4STR04(T) (=LMG 28458 (T) = CECT 8693(T)) should be classified as representing a novel species of genus Fontibacillus, for which the name Fontibacillus solani sp. nov. is proposed.

Azoarcus sp. CIB, an anaerobic biodegrader of aromatic compounds shows an endophytic lifestyle.

BACKGROUND: Endophytic bacteria that have plant growth promoting traits are of great interest in green biotechnology. The previous thought that the Azoarcus genus comprises bacteria that fit into one of two major eco-physiological groups, either free-living anaerobic biodegraders of aromatic compounds or obligate endophytes unable to degrade aromatics under anaerobic conditions, is revisited here. METHODOLOGY/PRINCIPAL FINDINGS: Light, confocal and electron microscopy reveal that Azoarcus sp. CIB, a facultative anaerobe ß-proteobacterium able to degrade aromatic hydrocarbons under anoxic conditions, is also able to colonize the intercellular spaces of the rice roots. In addition, the strain CIB displays plant growth promoting traits such nitrogen fixation, uptake of insoluble phosphorus and production of indoleacetic acid. Therefore, this work demonstrates by the first time that a free-living bacterium able to degrade aromatic compounds under aerobic and anoxic conditions can share also an endophytic lifestyle. The phylogenetic analyses based on the 16S rDNA and nifH genes confirmed that obligate endophytes of the Azoarcus genus and facultative endophytes, such as Azoarcus sp. CIB, locate into different evolutionary branches. CONCLUSIONS/SIGNIFICANCE: This is the first report of a bacterium, Azoarcus sp. CIB, able to degrade anaerobically a significant number of aromatic compounds, some of them of great environmental concern, and to colonize the rice as a facultative endophyte. Thus, Azoarcus sp. CIB becomes a suitable candidate for a more sustainable agricultural practice and phytoremediation technology.

Copper microlocalisation and changes in leaf morphology, chloroplast ultrastructure and antioxidative response in white lupin and soybean grown in copper excess.

The microlocalisation of Cu was examined in the leaves of white lupin and soybean grown hydroponically in the presence of 1.6 (control) or 192 µM (excess) Cu, along with its effect on leaf morphology, (ultra)structure and the antioxidative response. The 192 µM dose led to a reduction in the total leaf area and leaf thickness in both species, although more strongly so in white lupin. In the latter species it was also associated with smaller spongy parenchyma cells, and smaller spaces between them, while in the soybean it more strongly reduced the size of the palisade parenchyma and epidermal cells. Energy-dispersive X-ray microanalysis showed that under Cu excess the metal was mainly localised inside the spongy parenchyma cells of the white lupin leaves, and in the lower epidermis cell walls in those of the soybean. Cu excess also promoted ultrastructural chloroplast alterations, reducing the photosynthetic capacity index and the green area of the leaves, especially in the soybean. Despite this, soybean appeared to be more tolerant to Cu excess than white lupin, because soybean displayed (1) lower accumulation of Cu in the leaves, (2) enhanced microlocalisation of Cu in the cell walls and (3) greater levels of induced total -SH content and superoxide dismutase and catalase activities that are expected for better antioxidative responses.

Lupinus albus plants acquire mercury tolerance when inoculated with an Hg-resistant Bradyrhizobium strain.

One strain of Bradyrhizobium canariense (L-7AH) was selected for its metal-resistance and ability to nodulate white lupin (Lupinus albus L.) plants, from a collection of rhizobial strains previously created from soils of the Almadén mining district (Spain) with varying levels of Hg contamination. Plants were inoculated with either strain L-7AH (Hg-tolerant) or L-3 (Hg-sensitive, used as control), and watered with nutrient solutions supplemented with various concentrations (0-200 µM) of HgCl2 in a growth chamber. L. albus inoculated with L-7AH were able to nodulate even at the highest concentration of Hg while those inoculated with L-3 had virtually no nodules at Hg concentrations above 25 µM. Plants inoculated with L-7AH, but not those with the control strain, were able to accumulate large amounts of Hg in their roots and nodules. Nodulation with L-7AH allowed plants to maintain constant levels of both chlorophylls and carotenoids in their leaves and a high photosynthetic efficiency, whereas in those inoculated with L-3 both pigment content and photosynthetic efficiency decreased significantly as Hg concentration increased. Nitrogenase activity of plants nodulated with L-7AH remained fairly constant at all concentrations of Hg used. Results suggest that this symbiotic pair may be used for rhizoremediation of Hg-contaminated soils.

Spent metal working fluids produced alterations on photosynthetic parameters and cell-ultrastructure of leaves and roots of maize plants.

In this work we assess the capacity of maize (Zea mays) plants to phytoremediate spent metal working fluids (MWFs) and its effects on photosynthesis and ultrastructure of mesophyll and root cells. A corn-esparto fibre system patented by us has been used to phytoremediate MWFs in hydroponic culture. Furthermore, a plant growth promoting rhizobacteria (PGPR) has been used to improve the process. The results show that this system is capable of significantly reducing the chemical oxygen demand, under local legislation limits. However, plant systems are really damaged, mainly its photosynthetic system, as shown by the photosynthetical parameters. Nevertheless, strain inoculated improves these parameters, especially Hill reaction. The ultrastructure of photosynthetic apparatus was also affected. Chloroplast number decreased and becomes degraded in the mesophyll of MWFs treated plants. In some cases even plasmolysis of chloroplast membrane was detected. Early senescence symptoms were detected in root ultrastructural study. Severe cellular damage was observed in the parenchymal root cells of plants grown with MWFs, while vascular bundles cell remained unchanged. It seems that the inoculation minimises the damage originated by the MWFs pollutants, appearing as less degenerative organelles and higher chloroplast number than in non-inoculated ones.

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

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

Differential effectiveness of novel and old legume-rhizobia mutualisms: implications for invasion by exotic legumes.

The degree of specialization in the legume-rhizobium mutualism and the variation in the response to different potential symbionts are crucial factors for understanding the process of invasion by exotic legumes and the consequences for the native resident plants and bacteria. The enhanced novel mutualism hypothesis predicts that exotic invasive legumes would take advantage of native rhizobia present in the invaded soils. However, recent studies have shown that exotic legumes might become invasive by using exotic introduced microsymbionts, and that they could be a source of exotic bacteria for native legumes. To unravel the role of novel and old symbioses in the progress of invasion, nodulation and symbiotic effectiveness were analyzed for exotic invasive plants and native co-occurring legumes in a Mediterranean coastal dune ecosystem. Although most of the studied species nodulated with bacteria from distant origins these novel mutualisms were less effective in terms of nodulation, nitrogenase activity and plant growth than the interactions of plants and bacteria from the same origin. The relative effect of exotic bradyrhizobia was strongly positive for exotic invasive legumes and detrimental for native shrubs. We conclude that (1) the studied invasive legumes do not rely on novel mutualisms but rather need the co-introduction of compatible symbionts, and (2) since exotic rhizobia colonize native legumes in invaded areas, the lack of effectiveness of these novel symbiosis demonstrated here suggests that invasion can disrupt native belowground mutualisms and reduce native legumes fitness.

Mercury-resistant rhizobial bacteria isolated from nodules of leguminous plants growing in high Hg-contaminated soils.

A survey of symbiotic bacteria from legumes grown in high mercury-contaminated soils (Almadén, Spain) was performed to produce a collection of rhizobia which could be well adapted to the environmental conditions of this region and be used for restoration practices. Nineteen Hg-tolerant rhizobia were isolated from nodules of 11 legume species (of the genera Medicago, Trifolium, Vicia, Lupinus, Phaseolus, and Retama) and characterized. Based on their growth on Hg-supplemented media, the isolates were classified into three susceptibility groups. The minimum inhibitory concentrations (MICs) and the effective concentrations that produce 50% mortality identified the patterns of mercury tolerance and showed that 15 isolates were tolerant. The dynamics of cell growth during incubation with mercury showed that five isolates were unaffected by exposure to Hg concentrations under the MICs. Genetic analyses of the 16S rRNA gene assigned ten strains to Rhizobium leguminosarum, six to Ensifer medicae, two to Bradyrhizobium canariense, and one to Rhizobium radiobacter. Inoculation of host plants and analysis of the nodC genes revealed that most of them were symbiotically effective. Finally, three isolates were selected for bioremediation processes with restoration purposes on the basis of their levels of Hg tolerance, their response to high concentrations of this heavy metal, and their genetic affiliation and nodulation capacity.

Characterization of rhizobia from legumes of agronomic interest grown in semi-arid areas of Central Spain relates genetic differences to soil properties.

A study of symbiotic bacteria from traditional agricultural legumes from Central Spain was performed to create a collection of rhizobia from soils differing in physicochemical, analytical and/or agroecological properties which could be well-adapted to the environmental conditions of this region, and be used for sustainable agricultural practices. Thirty-six isolates were obtained from root-nodules of fifteen legume species (including Cicer arietinum, Lathyrus sativus, Lens culinaris, Lupinus spp., Medicago sativa, Phaseolus vulgaris, Pisum sativum, and Vicia spp.) from three agriculture areas with soils of different pHs and from a forest area with undisturbed soils. Phenotypical characterization revealed uniformity across the thirty-six isolates, with important exceptions in terms of environmental tolerance (three isolates survived at high temperatures, three at high salinity and three at acid pH). The molecular analysis of 16S rRNA gene showed a close relationship of twenty-nine isolates to Rhizobium leguminosarum, one to Rhizobium gallicum, one to Mesorhizobium ciceri, two to Sinorhizobium (Ensifer) meliloti and three to Bradyrhizobium canariense. The sequence analysis of a symbiosis-specific gene, nod C, showed a correlation with the plant host and grouped twenty-six isolates with Rhizobium leguminosarum bv. viciae, establishing the diversity in relation to legume-host. The 16S-23S rRNA intergenic spacer (IGS) region allowed for intraspecific differentiation, so that strains with equal 16S rRNA were grouped by means of their soil origin. These results indicated that phenotypical and genetically related strains may be widely distributed in this region and that soil abiotic characteristics could have a substantial bearing on the selection of the strains living in each environment.

Stress tolerance, genetic analysis and symbiotic properties of root-nodulating bacteria isolated from Mediterranean leguminous shrubs in Central Spain.

Nine root-nodulating bacterial isolates were obtained from the leguminous shrubs Spartium junceum, Adenocarpus hispanicus, Cytisus purgans, Cytisus laburnuum, Retama sphaerocarpa and Colutea arborescens in areas of Central Spain. A poliphasic approach analyzing phenotypic, symbiotic and genetic properties was used to study their diversity and characterize them in relation to Mediterranean conditions. Stress tolerance assays revealed marked variations in salinity, extreme pH and cadmium tolerance compared with reference strains, with the majority showing salinity, alkalinity and Cd tolerance and three of them growing at acid pH. Variation within the 16S rRNA gene was examined by amplified 16S rDNA restriction analysis (ARDRA) and direct sequencing to show genetic diversity. Phylogeny confirmed the close relationship of four isolates with Bradyrhizobium canariense, three with Phylobacterium myrsinacearum, one with Rhizobium rhizogenes and another with Mesorhizobium huakuii. The cross inoculation tests revealed wide spectra of nodulation. This is the first report of P. myrsinacearum being able to nodulate these leguminous shrubs, and also the first time reported the association between B.canariense, R. rhizogenes and M. huakuii and C. laburnuum, C. purgans and C. arborescens, respectively. These results suggested that native rhizobia could be suitable candidates as biofertilizers and/or inoculants of leguminous shrubs with restoration or revegetation purposes in Mediterranean areas.