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Background: Biserrula pelecinus is an annual legume native to the Mediterranean basin, found in pastureland, alone or in association with other legumes (Leguminosae) and grasses (Poaceae). It has been used in revegetation programmes of mining wastes showing phytoremediation potentials and thus becoming potentially highly attractive for plant ecology and restoration management of natural ecosystems.Aims: To characterise native root-nodule bacteria isolated from B. pelecinus from the Iberian Peninsula, and to select suitable N fixers for field-application and soil rehabilitation.Methods: Strains were isolated and molecularly identified by 16S rRNA amplification and sequencing. Strains were phenotypically characterised in different abiotic conditions (acidity, salinity and heavy metals) and tested for their ability to fix atmospheric N2. The most suitable N fixers were applied in greenhouse experiments with B. pelecinus under different fertilization levels to assess their tolerance to fertilized and polluted...

The United Nations Food and Agriculture Organization (FAO) estimates that the total demands for agricultural products will be 60 percent higher in 2030 than present time. And more than 85% of this additional demand will come from developing countries. For over half a century, the world has relied on increasing crop yields to supply an ever increasing demand for food. In order to increase world food production in a sustainable manner, farmers have to use balanced fertilizer timely. One of the most important factors in the generation of high yields is nitrogen fertilizer. That is why farmers are applying high amounts of the fertilizers which is very costly and make the environment hazardous especially when use discriminately. In addition, more than 50% of the applied N-fertilizers are somehow lost through different processes which not only represent a cash loss to the farmers and consequently polluted the environmental. International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 5 Number 4 (2016) pp. 942-956 Journal homepage: http://www.ijcmas.com

In the present study, a total of 353 nodule-associated bacteria were isolated from 220 legume plant samples belonging to Cicer arietinum (85), Glycine max (74), Vigna radiata (21) and Cajanus cajan (40). A total of 224 bacteria were identified as fast-growing Rhizobium spp. on the basis of differential staining (Gram staining and carbol fuchsin staining) and biochemical tests. All the isolates were tested for indole acetic acid production (IAA), phosphate solubilization and siderophore production on plate assay. To examine the effect of volatile organic metabolites (VOM) and water soluble soil components (WSSC) on nodule bacteria, culture conditions were optimized by observing the effects of various parameters such as pH, salt content and temperatures on the growth of bacteria. Selected rhizobia were subjected to random amplified polymorphic DNA (RAPD) and amplified ribosomal DNA restriction analysis (ARDRA) analysis to identify their species. On the basis of RAPD and ARDRA, 10 isolates were identified as Rhizobium meliloti. In this study, Rhizobium GO4, G16, G20, G77, S43, S81, M07, M37, A15 and A55 were observed as the best candidates among the tested bacteria and can be further used as potent bioinoculants.

Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 microM added iron) and tested for catechol and hydroxamate siderophore production and the excretion of malate and citrate. The growth response of the strains to iron differed markedly. For 12 strains (Bradyrhizobium strains NC92B and 32H1, B. japonicum USDA110 and CB1809, B. lupini WU8, cowpea Rhizobium NGR234, Rhizobium meliloti strains U45 and CC169, Rhizobium leguminosarum bv viciae WU235 and Rhizobium leguminosarum bv trifolii strains TA1, T1 and WU95) the mean generation time showed no variation with the 200-fold increase in iron concentration. In contrast, in Bradyrhizobium strains NC921, CB756 and TAL1000, B. japonicum strain 61A76 and R. leguminosarum bv viciae MNF300 there was a 2-5 fold decrease in growth rate at low iron. R. meliloti strains WSM419 and WSM540 showed decreased growth at high iron. All strains of root nodule bacteria tested gave a positive CAS (chrome azurol S) assay for siderophore production. No catechol-type siderophores were found in any strain, and only R. leguminosarum bv trifolii T1 and bv viciae WU235 produced hydroxamate under low iron (0.1 and 1.0 microM added iron). Malate was excreted by all strains grown under all iron regimes. Citrate was excreted by B. japonicum USDA110 and B. lupini WU8 in all iron concentrations, while Bradyrhizobium TAL1000, R. leguminosarum bv viciae MNF300 and B. japonicum 61A76 only produced citrate under low iron (0.1 and/or 1.0 microM added iron) during the stationary phase of growth.

Twelve polyhydroxyalkanoate (PHA) producing microbes were isolated from root nodules of 8 leguminous plants belonging to two phyla: Proteobacteria and Firmicutes. One of the isolate VK-12 of genus Burkholderia showed the highest PHA accumulation (42% wt/wt) as compared to other isolates in mineral medium. The effect of different cultural and growth conditions were studied on isolate VK-12 in shake flasks for highest PHA accumulation. VK-12 showed highest PHA accumulation in sucrose and ammonium sulphate amongst other carbon and nitrogen sources tested in the medium. The medium containing sucrose and ammonium sulphate having C: N ratio of 39.72 gave the highest PHA accumulation. The optimum pH, temperature, inoculum concentration and incubation time for highest PHA accumulation were 7.0, 30°C, 10% and 48 h respectively. An overall increase in PHA accumulation from 42 to 63% wt/wt was obtained under optimised conditions. The PHA was characterised using gas chromatography-mass spectrometry (GC-MS). All the isolates produced poly (3-hydroxybutyrate) (PHB) except VK-9, which produced polymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate). Keywords: Polyhydroxyalkanoate (PHA), PHB-co-HV, root nodule bacteria, carbon sources, C: N ratio, characterization of PHA

The theoretical and experimental data on salt tolerance of root nodule bacteria Sinorhizobium meliloti (Ensifer meliloti), an alfalfa symbiont, and on genetic determination of this feature are reviewed. Extensive data on the genes affecting adaptation of proteobacteria are provided, as well as on the groups of genes with activity depending on the osmolarity of the medium. Structural and functional polymorphism of the bet genes involved in betaine synthesis and transport in S. meliloti is discussed. The phenotypic and. genotypic polymorphism in 282 environmental rhizobial strains isolated from the centers of alfalfa diversity affected by aridity and salinity is discussed. The isolates from the Aral Sea area and northern Caucasus were shown to possess the betC gene represented by two types of alleles: the dominant A-type allele found in Rm 1021 and the less common divergent E-type allele, which was revealed in regions at the frequencies at the frequencies of 0.35 and 0.48, respectively. In the isolates with the salt-tolerant phenotype, which were isolated from root nodules and subsequently formed less effective symbioses with alfalfa, the frequency of E-type alleles was 2.5 times higher. Analysis of the nucleotide and amino acid sequences of the E-type allele of the betC gene revealed that establishment of this allele in the population was a result of positive selection. It is concluded that diversification of the functionally diverse bet genes occurring in S. meliloti affects the salt tolerance and symbiotic effectivity of rhizobia.

In this study, bacteria (8 species and 5 genera) belonging to the classes Betaproteobacteria, Gammaproteobacteria, and Sphingobacteria were isolated from root nodules of the multipurpose legume Clitoria ternatea L. and identified on the basis of partial 16S rRNA sequencing. The root nodule bacteria were subjected to phenotypic clustering and diversity studies using biochemical kits, including Hi-Media Carbokit™, Enterobacteriaceae™ identification kit, ERIC–PCR, and 16S ARDRA. All the strains showed growth on Ashby’s N-free media over 7 generations, indicative of presumptive nitrogen fixation and further confirmed by amplification of the nifH gene. None of the strains showed the capability to renodulate the host plant, neither alone nor in combination with standard rhizobial strains, which was further confirmed by the absence of nodC bands in PCR assay. The results clearly indicate the common existence of nonrhizobial microflora inside the root nodules of legumes, which were thought to be colonized only by rhizobia and were responsible for N2fixation in leguminous crops. However, with the recent discovery of nodule endophytes from a variety of legumes, as also observed here, it can be assumed that symbiotic rhizobia are not all alone and that these invasive endophytes belonging to various bacterial genera are more than just opportunistic colonizers of specialized nodule niche.

Forage legumes are important resources in semiarid regions due to their abilities to adapt to soils with low fertility and fix nitrogen when associated with diazotrophic bacteria. Here, we applied a polyphasic approach to characterize a set of legume nodule isolates obtained fromClitoria ternateaandStylosanthes capitatacultivated in the soils of a semiarid region of Brazil. A total of 126 bacterial isolates were obtained: 45 isolates fromC. ternateaand 81 isolates fromS. capitata. Nodulation tests revealed only ten isolates that nodulated their original host: six isolates fromC. ternateaand four isolates fromS. capitate. These ten legume nodule isolates were phenotypically and genotypically characterized. All isolates grew in fructose, glucose, sodium glutamate, maltose, xylose, and sucrose. Metabolic tests showed a relationship between tolerance to salt and high temperatures, where isolates that tolerated the highest salt concentration also tolerated the highest temperature. Three isolates showed amylolytic activity, and four isolates showed carboxymethyl cellulolytic activity. Streptomycin was the most limiting and nalidixic acid was the least limiting antibiotic to bacterial growth. Seven out of ten isolates were indol-acetic acid producers. Additionally, 16S rRNA gene partial sequencing enabled the identification of members of the generaBacillus(1), Bradyrhizobium(4),Leifsonia(3), Microvirga(1), andRhizobium(1). These data reveal phenotypically and genotypically diverse bacteria inhabiting the nodules of the forage legumesC. ternateaandS. capitatarepresent an important microbial source to protect new biotechnological products and improve forage legumes in semiarid regions., Leguminosas forrageiras são importantes recursos na região semi-árida, devido a sua capacidade de adaptação a solos de baixa fertilidade e fixação de nitrogênio quando associados a bactérias diazotróficas. Aqui, aplicamos uma abordagem polifásica com o objetivo de caracterizar bactérias isoladas de nódulos obtidos deClitoria ternateaeStylosanthes capitatacultivadas em solos do semiárido brasileiro. O isolamento das bactérias produziu uma coleção de 126 isolados: 45 isolados deC. ternateae 81 deS. capitata. Os testes de nodulação resultaram em apenas dez isolados que nodularam seu hospedeiro original: seis deC. ternateae quatro deS. capitata. Esses dez isolados de nódulos de leguminosas tiveram suas características fenotípicas e genotípicos avaliadas. Todos os isolados cresceram em frutose, glicose, glutamato de sódio, maltose, xilose e sacarose. Testes metabólicos mostraram uma relação entre a tolerância ao sal e altas temperaturas; onde o isolado que tolerou a maior concentração de sal também tolerou a temperatura mais alta. Três isolados apresentaram atividade amilolítica e quatro foram capazes de produzir carboximetil celulolítico. A estreptomicina foi o antibiótico mais limitante para o crescimento bacteriano, e o ácido nalidíxico foi o menos limitante. Sete dos dez isolados eram produtores de IAA. O sequenciamento parcial do gene 16S rRNA permitiu identificar isolados como membros dos gênerosBacillus(1),Bradyrhizobium(4),Leifsonia(3),Microvirga(1) eRhizobium(1). Esses dados revelam diversidade fenotípicas e genotípicas de bactérias que habitam nódulos das forrageirasC. ternateaeS. capitata, e representam uma importante fonte microbiana para a prospecção de novos produtos biotecnológicos para promover melhor desenvolvimento das leguminosas forrageiras na região semiárida.

Crack fertilization is a soybean cultivation technique for nodulation control in which midterm subsoiling is used to supply fertilizing materials to deep soil just before the flowering stage. This study examined the effects of fertilizing materials and the continuous application of nodulation control, on soybean yield enhancement in two field experiments. The survival of nodule bacteria in deep soil was also evaluated by a bioassay of nodule bacteria in a root box. When the nodule bacteria on biochar were continuously applied without any other chemical fertilizers for three successive years, seed weight was significantly heavier being up to 1.34 times that of the control. The application of nodulation control in the previous year but not in the experimental year did not have residual effects on seed weight. The enhancement of seed weight in a farm field converted from a paddy was much lower. This may be partly attributed to the midterm tillage practice, whichdestroys the crack structure after the ...

The isolation of root nodule bacteria (RNB) usually lead to obtainment of several non-rhizobia, mainly those fast-growing bacteria. The plant-authentication experiments to select the nodulating bacteria are time consuming and can be ruined in case of nodulation of negative controls. To speed up this step in rhizobiology research a fast, simple and efficient duplex PCR assay for the amplification of nodC and nifH genes in was developed aiming to separate rhizobia and non-rhizobia in RNB culture collections. The method was optimized with 43 reference strains and was applied to a new culture collection for validation. Considering all bacteria, 106 out of 109 nodulating rhizobia were positive for both genes. Among non-nodulating, 48 out of 48 were nodC-negative. The results demonstrated the efficiency of this new method that can be completed in a day, to save time and money in RNB isolation projects, fulfilling Koch's postulate, and separating rhizobia from non-rhizobial bacteria.

Legume-rhizobia symbiosis is a cross-kingdom association that results in large mounts of nitrogen incorporated in food webs. For the Brazilian semi-arid region, data on genetic variability and symbiotic efficiency of Papilionoidae rhizobial communities are very scarce. The aim of this study was to evaluate the genetic variability and the symbiotic efficiency of eight rhizobial isolates obtained from ?mulungu? (Erythrina velutina Willd.) nodules. For 16S rRNA gene sequencing, the genomic DNA was extracted using a commercial kit, amplified with universal primers, and subjected to sequencing reactions. For the isolate ESA 71, PCR amplifications for nodC and nodA genes were attempted. Rhizobial efficiency was assessed by two greenhouse experiments. The first assay was carried out under gnotobiotic conditions, with sterile sand as a substrate; the second experiment was conducted in a non-sterile soil. For both experiments, the inoculation treatments consisted of a single inoculation of each isolate, in addition to a treatment with Bradyrhizobium elkanii BR 5609 as a reference strain. Furthermore, two non-inoculated control treatments, supplied and not supplied with mineral N, were also evaluated. Bacterial identification indicated that both and rhizobia could be found in ?mulungu? root nodules. Three isolates where classified within the Rhizobium genus, four bacteria belonged to Bradyrhizobium and one isolate clustered with Burkholderia. Positive amplification of an intragenic fragment of the nodA gene using a primer set to ?-rhizobia could be found for ESA 71 (Burkholderia). All bacterial isolates were effective in colonizing ?mulungu? roots. In the first experiment, all inoculated treatments and N fertilization increased the N concentration in ?mulungu? shoot tissues. For total N in the shoots, the isolates ESA 70, ESA 72, and ESA 75 stood out. In the non-sterile substrate experiment, the isolates ESA 70, ESA 71, ESA 72, and ESA 75, together with the reference strains, induced increases in the shoot N concentration and total accumulation compared to the absolute control. The results indicate that ?mulungu? is able to establish associations with efficient ? and ?-rhizobia in Brazilian semi-arid soils.

The nitrogen fixation is a key factor in low-input agricultural systems to sustain long-term soil fertility. Plant–bacteria interactions in the rhizosphere are the determinants of plant health and soil fertility. The climatic conditions of arid and semiarid regions are often characterized by hot, dry summers, subhumid monsoon, and cold dry winter. The climatic conditions in this region restrict the buildup of soil organic matter and soils are generally deficient in nitrogen. Knowledge of rhizobial diversity from arid and semiarid areas is essential to improve their nutrient-poor fertility status. Soils of these regions often suffer from moisture stress, salinity, unfavorable pH, nutrient deficiency, mineral toxicity, temperature extremes, plant diseases, trace element deficiencies, etc., which inhibit nodulation and impose limitations on the vigor of the host legume. The tolerance to high levels of salinity and the survival and persistence in harsh desert conditions make these rhizobia highly valuable inocula to improve productivity of the leguminous plants cultivated under extreme environments. This functional diversity and tolerance to extreme environments displayed by efficient rhizobial isolates alone or co-inoculation with PGPR can be effectively utilized for improving legume crop production and productivity in arid and semiarid regions.