Apios priceana is a native endangered species plant found in the Southeast United States. It is characterized as a leguminous species that bears wisteria like clusters with pea like flowers, a large tuberous root and four to six inch long seed pods. It is believed the Native Americans and early European settlers relied on this species as a source of protein and utilized the seeds for cultivation of the tuberous “potato” which formed. Apios priceana contains an average of 13% fiber, 6.9% protein, 71% carbohydrate and 9 of the 11 essential amino acids needed in human diets (Walter et al.,1986). In addition, A. priceanatuberous roots contain anti-carcinogenic properties known to be used to treat prostate and breast cancer as well as lowering blood pressure and cholesterol with an added use for diabetes. If removed from the endangered species list it could prove to be a valuable agronomic crop. Its use spans human and animal consumption, bio fuel, medicinal and horticultural purposes. This research was initiated to investigate a biological symbiosis between A. priceana and known beneficial soil bacteria which may indicate growth potential of known colonies. Experimental treatments were 1) no inoculation 2) Azospirillum brasilense inoculate 3) Bradyrhizobium japonicum inoculate and 4) Rhizobium leguminosarum biovar viceae inoculate. Specimens were evaluated at 30, 60 and 90 day’s growth from emergence for taproot length, number of lateral roots and taproot girth. Due to non-germination of seeds, data presented is for treatments 2 and 3. The correlation coefficient for average taproot length, number of lateral roots developed and taproot girth per treatment was as follows: taproot length to number of lateral roots, positive correlation coefficient 0.996; taproot length to taproot girth, positive correlation coefficient 0.999; and number of lateral roots to taproot girth, positive correlation coefficient 0.991. All correlation coefficients are significant at the 0.01 level.

Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies.

Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-08-22Bitstream added on 2014-06-13T19:03:37Z : No. of bitstreams: 1 souza_jam_dr_jabo.pdf: 4083420 bytes, checksum: cb86ca179e1196f514509e27854de1c3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O nitrogênio é o nutriente requerido em maior quantidade para a cultura da soja. Avanços nas pesquisas de melhoramento genético vegetal e microbiologia do solo permitiram expandir o uso de inoculantes comerciais contendo estirpes de Bradyrhizobium japonicum e Bradyrhizobium elkanii. Estas bactérias infectam as raízes da planta e induzem a formação de nódulos, que abrigam a forma bacterióide, diferenciada da bactéria, responsável pela fixação simbiótica do nitrogênio. Informações sobre processos bioquímicos envolvidos no metabolismo da relação simbiótica podem ser adquiridas através de análises globais de expressão gênica. Para esta finalidade, destaca-se a tecnologia de microarranjo de DNA para detecção de genes diferencialmente expressos em larga escala. O objetivo geral deste trabalho foi identificar genes diferencialmente expressos, por meio de microarranjos de DNA, em Bradyrhizobium elkanii SEMIA 587 cultivada em diferentes meios de cultura, RDM (Rhizobia Defined Medium), TY (Triptone-Yeast Medium) e YMB (Yeast-Mannitol Medium), e em bacterióides isolados de nódulos de soja em diferentes períodos de desenvolvimento, 13, 28 e 48 dias após inoculação. Para esta finalidade, a partir do seqüenciamento de DNA genômico de B. elkanii, um microarranjo (Be587) foi gerado contendo 2654 genes. Em meio RDM, a bactéria confrontou-se com a necessidade de se adaptar e sintetizar suas subunidades formadoras de macromoléculas a partir de uma única fonte de carbono, refletindo em um metabolismo mais ativo nas fases lag e log. Por outro lado, em meio TY, as células cultivadas na presença de uma boa fonte de carbono e energia cresceram rapidamente esgotando os recursos disponíveis no meio, 8 o que pode ter causado uma situação de estresse que se refletiu na identificação... / Nitrogen is the most required nutrient by soybean culture. Advanced researches in genetic plant breeding and soil microbiology allowed the expansion in commercial inoculants applications containing strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii. These bacteria infect plant roots and induce nodule formation which home the differentiated bacteria, named bacteroid. The bacteroid in turn is responsible for symbiotic nitrogen fixation. Biochemical knowledge about processes of symbiotic regulation can be acquired by global analysis of gene expression. To achieve such information, the DNA microarray technology, used for detection of differentially expressed genes in large scale, was used. The purpose of this work was identificate differentially expressed genes of Bradyrhizobium elkanii SEMIA 587, grown under different media conditions, such as RDM (Rhizobia Defined Medium), TY (Triptone- Yeast Medium) and YMB (Yeast-Mannitol Medium), and in bacteroids from soybean nodules at different developmental stages, 13, 28 e 49 days after inoculation. For this purpose, the DNA microarray Be587 with 2654 genes was generated from B. elkanii genomic DNA. In RDM medium the bacterium was confronted with the need of adaptation and building of macromolecules subunits from a single carbon source, what was reflected in a more active metabolism in lag and log phases. In turn, in TY medium with good carbon and energy sources the cells grew fastly and exhaust the medium sources available. Such condition can submitted the bacterial cells to a stress condition that reflected in the identification of higher number differentially expressed genes. At different bacteroids stages, the analysis detected genes related to nodulation and 10 nitrogen fixation regulation more than structural genes. Inasmuch, an organic nitrogen recycle might be involved... (Complete abstract, click electronic access below)

The objective of this work was to characterize promising isolated rhizobium of L. albescens in the nitrogen biological fixation. Two hundred and four isolates were obtained from three species of lupine (L. albescens, L. lanatos e Lupinus sp.). Thirty-eight isolates chosen randomly were characterized through their phenotype and submitted to an authenticity test done in the greenhouse. In order to select symbiotic strains that synthesizes leghaemoglobin. The promising isolates were identified by their phenotypic characteristics by comparing twenty-one strains of rhizobium. The physiological characteristics of the promising isolated were determined and evaluated about the growing capacity in different temperatures, NaCl concentrations and pH, and a molecular characterization by polymerase chain reaction (PCR) using the primer BOX. An experiment, in the greenhouse, was made to determine the symbiosis efficiency by the total nitrogen, and the dried mass in the aerial part and in the roots, and the number of nodules. The phenotypic characterization of the thirty-eight isolated enabled the development of 11 groups with distinct characteristics. The UFSM L1.3 strain developed nodules and leghaemoglobin when associated to L. Albescens and was identified in the genus Bradyrhizobium. The UFSM L1.3 strain presented growing in the temperature from 28 to 32°C, in a NaCl concentration of 0,1% and in a pH from 4,0 to 8,0. The standard of polymorphism generated by the primer BOX-PCR enabled to distinguish the UFSM L1.3 isolated from the others. The symbiosis efficiency of the UFSM L1.3 isolated was of 94,2% when compared to the without nitrogen. / O objetivo deste trabalho foi isolar e caracterizar populações promissoras de rizóbio de L. albescens na fixação biológica de nitrogênio. Duzentos e quatro isolados foram obtidos a partir de três espécies de tremoço (L. albescens, L. lanatus e Lupinus sp.). Trinta e oito isolados escolhidos após a purificação foram caracterizados fenotipicamente e submetidos a um teste de autenticação, realizado em casa de vegetação. Estes testes objetivaram selecionar estirpes simbiontes formadoras de leghemoglobina. Os isolados promissores foram identificados com base nas características fenotípicas através da comparação com vinte e duas estirpes de rizóbio. Determinou-se as características fisiológicas dos isolados promissores, sendo avaliado a capacidade de crescer em: diferentes temperaturas, concentrações de NaCl e pH, e a uma caracterização molecular com base em reação de polimerase em cadeia (PCR) utilizando o oligonucleotídeo BOX A1-R. Foi realizada a determinação da eficiência simbiôntica com base no teor de nitrogênio total da parte aérea, a massa seca da parte aérea e das raízes, e número de nódulos. A caracterização fenotípica dos trinta e oito isolados possibilitou a formação de 11 grupos com características distintas. O isolado UFSM L1.3 formou nódulos e leghemoglobina quando associado a L. albescens, tendo sido identificado no gênero Bradyrhizobium. A estirpe UFSM L1.3 apresentou crescimento em temperatura de 28 e 32°C, na concentração de NaCl de 0,1% e na faixa de pH 4,0 a 8,0. O padrão de polimorfismo gerado pelo primer BOX-PCR permitiu diferenciar o isolado UFSM L1.3 dos demais estudados. A eficiência simbiôntica do isolado de rizóbio UFSM L1.3 foi de 94,2% em comparação com a testemunha nitrogenada.

Submitted by (lucia.rodrigues@ufrpe.br) on 2016-08-10T13:48:00Z No. of bitstreams: 1 Maria de Fatima da Silva.pdf: 302537 bytes, checksum: 7c6d2b448ec2b8c92bc437c2f3c61538 (MD5) / Made available in DSpace on 2016-08-10T13:48:00Z (GMT). No. of bitstreams: 1 Maria de Fatima da Silva.pdf: 302537 bytes, checksum: 7c6d2b448ec2b8c92bc437c2f3c61538 (MD5) Previous issue date: 2007-02-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Peanut (Arachis hypogaea L.) Is legume used in the world with a high percentage of comestible oil and protein and in Brazil is cropped in different soil and climatic conditions. Due to its adaptability to tropical conditions and with high economic value peanut is very important for typically small, family based properties involved in food production in the Northeastern Brazilian. With the objective to evaluate the effectiveness of peanut inoculation with selected strains of Bradyrhizobium spp. bacteria it was carried out a field experiment in not irrigated conditions, in a soil located at the Itapirema Experimental Station (IPA), tropical rainforest zone of Pernambuco state. The experiment was conducted in a factorial 2 x10, in randomized block design, with four replicates. Were used two peanut cultivars (BR 1 and BRS Havana) comparing 8 peanut strains, including the controls treatments with N fertilization as ammonium sulphate (200 kg N ha-1) and without nitrogen fertilizers. In the periods of flowering and grain maturity plants were collected (6 per plot) for evaluation of nitrogen fixation (dry nodules biomass, dry plant biomass, total N in shoots and relative efficiency of strains). At the final of the experiment were determined the following components of plant productivity: dry biomass of 100 grains(g); dry biomass of 100 pods (g); grain and pod yields (kg ha-1). Nitrogen fertilization increased dry biomass and total N accumulation in shoots in the period of grain maturation (p≤ 0,05). The relative efficiency of the strains was greater on cultivar BRS Havana. Bradyrhizobium spp. inoculation showed no significant difference (p≤0,05) to the others treatments in reference to dry biomass of nodules. It was not observed significant difference between treatments on grain and pod yields. In the soil used the rhizobia native from soil was so effective than rhizobia applied by inoculation and they supply nitrogen to attend the N demand by peanut cropped inthe used Brazilian rainforest soil. / O amendoim (Arachis hypogaea L.) é uma oleaginosa consumida mundialmente. No Brasil é cultivada nas mais diferentes condições edafoclimáticas. Devido sua boa adaptabilidade às condições tropicais e por ser uma cultura de valor econômico, o amendoim pode ter um papel importante para a capitalização de pequenos agricultores na região Nordeste. Com o objetivo de avaliar a efetividade da inoculação com Bradyrhizobium spp. em solo da Zona de Mata de Pernambuco na cultura do amendoim, foi realizado um experimento no campo, em condições de sequeiro, na Estação Experimental de Itapirema (IPA), Goiana, Pernambuco. O delineamento experimental adotado foi o de blocos casualizados, com 4 repetições, no arranjo fatorial 2 X 10. Foram usadas duas cultivares de amendoim (BR 1 e BRS Havana) e 10 tratamentos como fontes de nitrogênio, 8 tratamentos com inoculação com estirpes de Bradyrhizobium; 1 tratamento sem inoculação e com fertilizante nitrogenado, aplicado na forma de Sulfato de amônio (200 kg ha-1 de N); e 1tratamento sem inoculação e sem fertilizante com N. Nos períodos de florescimento e de maturação dos frutos 6 plantas por parcela foram coletadas ao acaso, para avaliação da fixação biológica do nitrogênio (biomassa seca de nódulos, biomassa seca da parte aérea, N total acumulado na parte aérea e eficiência relativa dasestirpes). No final do ciclo foram determinados: biomassa seca de 100 grãos (g);biomassa seca de 100 vagens (g), rendimento de grãos (kg ha-1) e rendimento de vagens (kg ha-1). Houve diferença significativa (p≤ 0,05) para acúmulo de biomassa seca e de N da parte aérea no período de maturação dos frutos, onde o tratamento com N fertilizante foi superior aos demais. A eficiência das estirpes foi mais acentuada na cultivar BRS Havana. Não houve diferença significativa (p≤ 0,05) entre os tratamentos para biomassa seca dos nódulos e para o rendimento de vagens e de grãos. Nas condições em que o experimento foi desenvolvido a população rizobiana nativa foi eficiente no suprimento de nitrogênio para atender a demanda deste nutriente pela cultura do amendoim.