A leguminosa Adesmia latifolia encontrada nos campos do Rio Grande do Sul e Santa Catarina apresenta alta qualidade forrageira e pode ser utilizada em programas de melhoramento de campos nativos. Essa espécie apresenta simbiose com rizóbios do solo. Tais rizóbios podem ser utilizados na promoção do crescimento de A. latifolia e outras espécies vegetais. Assim, os objetivos deste trabalho foram estudar e selecionar rizóbios nativos, isolados de A. latifolia, para a promoção de crescimento de plantas de espécies de Adesmia e do porta-enxerto micropropagado de macieira cv Marubakaido (Malus prunifolia Bork). Para atingir estes objetivos foram realizados três estudos. O primeiro estudo avaliou a promoção de crescimento em espécies de Adesmia por rizóbios produtores de ácido indolacético. O segundo estudo caracterizou e avaliou a eficiência simbiótica dos rizóbios de A. latifolia. O terceiro avaliou a utilização dos rizóbios de A. latifolia para o enraizamento in vitro e a aclimatização do porta-enxerto micropropagado de macieira cv. Marubakaido. Os resultados do primeiro estudo mostraram que os isolados apresentaram variação na produção de AIA. A promoção do crescimento vegetal variou nas diferentes espécies de Adesmia. No segundo estudo os resultados revelaram que os isolados testados apresentaram características fenotípicas dos gêneros Mesorhizobium e Rhizobium. Esses resultados demonstraram que os isolados EEL47310, EEL45910, EEL37910 e EEL45810 induziram uma maior produção de massa seca da parte aérea de A. latifolia. Tais isolados foram caracterizados geneticamente como Mesorhizobium sp. No terceiro estudo observou-se que o isolado EEL16010B induziu o maior crescimento no enraizamento in vitro do porta-enxerto micropropagado de macieira cv. Marubakaido. Os rizóbios produtores de AIA isolados de A. latifolia podem promover o crescimento em plântulas outras espécies do mesmo gênero. Foi testado a viabilidade do uso da utilização de caldo contendo AIA produzido pelo rizóbio EEL16010B em substituição ao AIA sintético no enraizamento in vitro do porta enxerto de macieira cv. Marubakaido. / The legume A. latifolia found in the fields of Rio Grande do Sul and Santa Catarina has a high nutritional quality and can be used in breeding programs of native grasslands. This species presents a symbiosis with rhizobia soil. These rhizobia may be used in promoting the growth of A. latifolia and other species. The objectives of this study were selected and rhizobia isolated from A. latifolia promotion for plant growth Adesmia species and rootstock cv Marubakaido micropropagated apple (Malus prunifolia Bork). To achieve these objectives were conducted three studies. The first study evaluated the promotion of growth by rhizobacteria species Adesmia nodules producing IAA. The second study characterized and evaluated the efficiency of symbiotic rhizobia from A. latifolia. The third study examined the use of rhizobia of A. latifolia for in vitro rooting and acclimatization of micropropagated rootstock of apple cv. Marubakaido. The results of the first study showed that the isolates showed variation in the production of IAA. The promotion of growth varied in different species of Adesmia. In the second study the results showed that the isolates tested showed phenotypic characteristics of the genera Mesorhizobium and Rhizobium and that these results showed that the isolates EEL47310, EEL45910, EEL37910, EEL45810 and were more effective in inducing the production of dry matter of aerial part of A. latifolia. These isolates were genetically characterized as Mesorhizobium. In the third study showed that the strain EEL16010B induced the highest growth in vitro rooting of micropropagated rootstock of apple cv. Marubakaido. The producers of IAA rhizobia isolated from A. latifolia can promote the growth of seedlings of other species of this genus. It is feasible to use broth containing IAA produced by rhizobia strain EEL16010B to replace synthetic IAA in vitro rooting of apple rootstock cv. Marubakaido

A inoculação de rizóbios em espécies leguminosas já é bastante conhecida e levou o Brasil a um avanço na produção de soja. Para garantir a qualidade desses produtos inoculantes é necessário o desenvolvimento de metodologias específicas. A detecção das estirpes constantes na embalagem no produto comercial e em solo e planta inoculada é uma forma de fiscalizar a qualidade do produto. Para tanto os objetivos deste trabalho foram detectar a presença das estirpes de bradirrizóbios recomendadas para a cultura da soja SEMIA 5079 e 5080 (Bradyrizobium japonicum) e SEMIA 587, 5019 (Bradyrizobium elkanii), que devem estar presentes nas formulações de inoculante comerciais, usando-se a técnica de REP- PCR. Detectar a presença destas estirpes em nódulos de plantas de soja inoculadas e em solo de área cultivada. Foi instalado um experimento com soja em vasos, na casa de vegetação. A soja foi inoculada com as estirpes de Bradyrhizobium e com inoculantes comerciais. Os nódulos foram utilizados para a extração de DNA. Também foi realizada extração de DNA de amostras de solo cultivado com soja e de nódulos de soja coletados do campo e de estirpes puras e produto inoculante. As extrações foram realizadas com os Kits: Wizard DNA Purification® (Promega corp., Madison, USA) e Power Soil. Após foi realizada a amplificação do DNA genômico utilizando-se a técnica de REP - PCR com o primer iniciador BOX A1 e com o primer ERIC 1 e ERIC2. O perfil de bandas da amplificação do DNA genômico da mistura de estirpes pela PCR com os oligonucleotideos iniciadores ERIC e BOX A possibilitou a diferenciação de produtos contendo misturas de estirpes diferentes. Nas condições deste trabalho, a técnica de amplificação pela PCR com BOXA1 ou ERIC do DNA de nódulos de plantas inoculadas não se mostra adequada para a identificação das estirpes de rizóbios que induziram a formação destes nódulos em plantas de soja cultivada em casa de vegetação.Não ocorreu a amplificação do DNA extraído de amostras de solo cultivado com soja pela reação em cadeia da polimerase com os oligonucleotídeos iniciadores BOX A e ERIC. A amplificação pela PCR com BOXA1 do DNA extraído de nódulos de plantas cultivadas a campo apresenta baixa similaridade com os obtidos das amostras de produtos inoculantes comerciais utilizados e não permite a identificação da mistura de estirpes que foram inoculadas nas plantas. / Inoculation of rhizobia in legume species is very well known and led Brazil to advance in soybean production. To ensure the quality of these inoculant products is necessary to develop specific methodologies. The detection of strains contained in packaging of the commercial product and in soil and plant inoculated is a way to monitor the quality of the product. Therefore the objectives of this study were to detect the presence of “bradirrizóbios” strains recommended for soybean SEMIA 5079 and 5080 (Bradyrizobium japonicum) and SEMIA 587, 5019 (Bradyrizobium elkanii) that must be present in the formulations of commercial inoculant, by using the technique of REP-PCR. To detect the presence of these strains in inoculated nodules of soybean plants into soil and cultivated area, an experiment was set up with soybeans in pots in a greenhouse. Soybeans were inoculated with the strains of Bradyrhizobium and commercial inoculants. The nodules were used for DNA extraction. Additionally it was carried out DNA extraction from soil cultivated with soybeans and soybean nodules collected from the field and pure strains and inoculant product. The extractions were performed with the following kits: Wizard DNA Purification® (Promega corp., Madison, USA) and Power Soil. Afterwards was performed the amplification of the genomic DNA using the technique of REP - PCR primer with the starting primer A1 and with the primer ERIC1 and ERIC2. The profile of the bands’ amplification of the genomic DNA from the mixture of strains by PCR with primer ERIC and BOX A allowed to differentiate products containing mixtures of different strains. In this study conditions, the technique of PCR amplification with the BOXA 1 or ERIC of DNA from nodules of inoculated plants, is inadequate for the identification of strains of rhizobia, which induced the formation of nodules on soybean plants grown in greenhouse. It was not observed amplification of DNA extracted from soil samples cultivated with soybeans by PCR with primer BOX A and ERIC. Amplification by PCR with BOXA 1 of the DNA extracted from plants’ nodules grown in a field has low similarity with the product samples obtained from commercial inoculants used and it does not allow the identification of the mixture of strains that were inoculated in the plants.

A leguminosa Adesmia latifolia encontrada nos campos do Rio Grande do Sul e Santa Catarina apresenta alta qualidade forrageira e pode ser utilizada em programas de melhoramento de campos nativos. Essa espécie apresenta simbiose com rizóbios do solo. Tais rizóbios podem ser utilizados na promoção do crescimento de A. latifolia e outras espécies vegetais. Assim, os objetivos deste trabalho foram estudar e selecionar rizóbios nativos, isolados de A. latifolia, para a promoção de crescimento de plantas de espécies de Adesmia e do porta-enxerto micropropagado de macieira cv Marubakaido (Malus prunifolia Bork). Para atingir estes objetivos foram realizados três estudos. O primeiro estudo avaliou a promoção de crescimento em espécies de Adesmia por rizóbios produtores de ácido indolacético. O segundo estudo caracterizou e avaliou a eficiência simbiótica dos rizóbios de A. latifolia. O terceiro avaliou a utilização dos rizóbios de A. latifolia para o enraizamento in vitro e a aclimatização do porta-enxerto micropropagado de macieira cv. Marubakaido. Os resultados do primeiro estudo mostraram que os isolados apresentaram variação na produção de AIA. A promoção do crescimento vegetal variou nas diferentes espécies de Adesmia. No segundo estudo os resultados revelaram que os isolados testados apresentaram características fenotípicas dos gêneros Mesorhizobium e Rhizobium. Esses resultados demonstraram que os isolados EEL47310, EEL45910, EEL37910 e EEL45810 induziram uma maior produção de massa seca da parte aérea de A. latifolia. Tais isolados foram caracterizados geneticamente como Mesorhizobium sp. No terceiro estudo observou-se que o isolado EEL16010B induziu o maior crescimento no enraizamento in vitro do porta-enxerto micropropagado de macieira cv. Marubakaido. Os rizóbios produtores de AIA isolados de A. latifolia podem promover o crescimento em plântulas outras espécies do mesmo gênero. Foi testado a viabilidade do uso da utilização de caldo contendo AIA produzido pelo rizóbio EEL16010B em substituição ao AIA sintético no enraizamento in vitro do porta enxerto de macieira cv. Marubakaido. / The legume A. latifolia found in the fields of Rio Grande do Sul and Santa Catarina has a high nutritional quality and can be used in breeding programs of native grasslands. This species presents a symbiosis with rhizobia soil. These rhizobia may be used in promoting the growth of A. latifolia and other species. The objectives of this study were selected and rhizobia isolated from A. latifolia promotion for plant growth Adesmia species and rootstock cv Marubakaido micropropagated apple (Malus prunifolia Bork). To achieve these objectives were conducted three studies. The first study evaluated the promotion of growth by rhizobacteria species Adesmia nodules producing IAA. The second study characterized and evaluated the efficiency of symbiotic rhizobia from A. latifolia. The third study examined the use of rhizobia of A. latifolia for in vitro rooting and acclimatization of micropropagated rootstock of apple cv. Marubakaido. The results of the first study showed that the isolates showed variation in the production of IAA. The promotion of growth varied in different species of Adesmia. In the second study the results showed that the isolates tested showed phenotypic characteristics of the genera Mesorhizobium and Rhizobium and that these results showed that the isolates EEL47310, EEL45910, EEL37910, EEL45810 and were more effective in inducing the production of dry matter of aerial part of A. latifolia. These isolates were genetically characterized as Mesorhizobium. In the third study showed that the strain EEL16010B induced the highest growth in vitro rooting of micropropagated rootstock of apple cv. Marubakaido. The producers of IAA rhizobia isolated from A. latifolia can promote the growth of seedlings of other species of this genus. It is feasible to use broth containing IAA produced by rhizobia strain EEL16010B to replace synthetic IAA in vitro rooting of apple rootstock cv. Marubakaido

A inoculação de rizóbios em espécies leguminosas já é bastante conhecida e levou o Brasil a um avanço na produção de soja. Para garantir a qualidade desses produtos inoculantes é necessário o desenvolvimento de metodologias específicas. A detecção das estirpes constantes na embalagem no produto comercial e em solo e planta inoculada é uma forma de fiscalizar a qualidade do produto. Para tanto os objetivos deste trabalho foram detectar a presença das estirpes de bradirrizóbios recomendadas para a cultura da soja SEMIA 5079 e 5080 (Bradyrizobium japonicum) e SEMIA 587, 5019 (Bradyrizobium elkanii), que devem estar presentes nas formulações de inoculante comerciais, usando-se a técnica de REP- PCR. Detectar a presença destas estirpes em nódulos de plantas de soja inoculadas e em solo de área cultivada. Foi instalado um experimento com soja em vasos, na casa de vegetação. A soja foi inoculada com as estirpes de Bradyrhizobium e com inoculantes comerciais. Os nódulos foram utilizados para a extração de DNA. Também foi realizada extração de DNA de amostras de solo cultivado com soja e de nódulos de soja coletados do campo e de estirpes puras e produto inoculante. As extrações foram realizadas com os Kits: Wizard DNA Purification® (Promega corp., Madison, USA) e Power Soil. Após foi realizada a amplificação do DNA genômico utilizando-se a técnica de REP - PCR com o primer iniciador BOX A1 e com o primer ERIC 1 e ERIC2. O perfil de bandas da amplificação do DNA genômico da mistura de estirpes pela PCR com os oligonucleotideos iniciadores ERIC e BOX A possibilitou a diferenciação de produtos contendo misturas de estirpes diferentes. Nas condições deste trabalho, a técnica de amplificação pela PCR com BOXA1 ou ERIC do DNA de nódulos de plantas inoculadas não se mostra adequada para a identificação das estirpes de rizóbios que induziram a formação destes nódulos em plantas de soja cultivada em casa de vegetação.Não ocorreu a amplificação do DNA extraído de amostras de solo cultivado com soja pela reação em cadeia da polimerase com os oligonucleotídeos iniciadores BOX A e ERIC. A amplificação pela PCR com BOXA1 do DNA extraído de nódulos de plantas cultivadas a campo apresenta baixa similaridade com os obtidos das amostras de produtos inoculantes comerciais utilizados e não permite a identificação da mistura de estirpes que foram inoculadas nas plantas. / Inoculation of rhizobia in legume species is very well known and led Brazil to advance in soybean production. To ensure the quality of these inoculant products is necessary to develop specific methodologies. The detection of strains contained in packaging of the commercial product and in soil and plant inoculated is a way to monitor the quality of the product. Therefore the objectives of this study were to detect the presence of “bradirrizóbios” strains recommended for soybean SEMIA 5079 and 5080 (Bradyrizobium japonicum) and SEMIA 587, 5019 (Bradyrizobium elkanii) that must be present in the formulations of commercial inoculant, by using the technique of REP-PCR. To detect the presence of these strains in inoculated nodules of soybean plants into soil and cultivated area, an experiment was set up with soybeans in pots in a greenhouse. Soybeans were inoculated with the strains of Bradyrhizobium and commercial inoculants. The nodules were used for DNA extraction. Additionally it was carried out DNA extraction from soil cultivated with soybeans and soybean nodules collected from the field and pure strains and inoculant product. The extractions were performed with the following kits: Wizard DNA Purification® (Promega corp., Madison, USA) and Power Soil. Afterwards was performed the amplification of the genomic DNA using the technique of REP - PCR primer with the starting primer A1 and with the primer ERIC1 and ERIC2. The profile of the bands’ amplification of the genomic DNA from the mixture of strains by PCR with primer ERIC and BOX A allowed to differentiate products containing mixtures of different strains. In this study conditions, the technique of PCR amplification with the BOXA 1 or ERIC of DNA from nodules of inoculated plants, is inadequate for the identification of strains of rhizobia, which induced the formation of nodules on soybean plants grown in greenhouse. It was not observed amplification of DNA extracted from soil samples cultivated with soybeans by PCR with primer BOX A and ERIC. Amplification by PCR with BOXA 1 of the DNA extracted from plants’ nodules grown in a field has low similarity with the product samples obtained from commercial inoculants used and it does not allow the identification of the mixture of strains that were inoculated in the plants.

This study reports on the early effects of the Trifolium hybridum-Rhizobium symbiosis on Pinus contorta Doug, ex Loud (lodgepole pine), soil, and selected native plant species. Four rates of seeding (0, 10, 20, and 30 kg/ha) using inoculated Trifolium hybridum (alsike clover) seed were applied to three different site preparation treatments (broadcast burn, windrow burn, and mechanical scraping) using a split-plot design. Alsike clover and the Rhizobium inoculant were found to have excellent establishment and infectivity, and the symbiosis was assessed to be fixing nitrogen effectively. No effect of site preparation treatments was observed on establishment of the symbiosis, and clover-seeded plots averaged 76% cover by the end of the third growing season. The symbiosis had no significant (p < 0.05) effects on lodgepole pine total or incremental height or survival during the first three growing seasons, nor was there any observed effect on lodgepole pine foliar total nitrogen (N) concentration and ઠ¹⁵N values at the end of the second growing season. Small, but significant (p < 0.05) decreases were observed for lodgepole pine total and incremental diameter in the second and third growing seasons, as well as needle mass in the second growing season. The growth decreases were probably attributable to the effect of shading by the clover cover. However, lodgepole pine seedlings overtopped the clover by the end of the third growing season and shade effects are likely to decrease as tree seedlings continue to grow. After one growing season, the symbiosis significantly (p < 0.05) increased mineralizable N in the forest floor and mineral (0-15 cm) soil layers. However, no significant changes in total N were detectable. The changes in mineralizable N were likely a measure of increased microbial biomass attributable to greater amounts of rhizosphere soil in clover-seeded plots compared to controls. Available phosphorus (P) in the forest floor significantly (p < 0.05) decreased as rate of seeding increased after one growing season. The decrease of forest floor available P may be attributed to greater assimilation of P in clover-seeded plots for plant and microbial growth, as well as the additional requirements for P in the supply of biological energy needed for active N₂ fixation. All native plant species had low cover values which made interpretation of results difficult. However, percent cover of Calamagrostis canadensis, Rosa acicularis, and Spiraea betulifolia were significantly less in clover-seeded plots compared to controls at the end of the second growing season. Replacement of some herb and low-growing shrub species by legume-Rhizobium symbiosis may be desirable if the net result is an increase in site N without detrimental effects to tree crop species. It is recommended that the legume-Rhizobium symbiosis be established in the early regeneration of lodgepole pine plantations on similar sites that are inherently N deficient and have experienced further site N depletion through forestry practices such as slashburning. / Forestry, Faculty of / Graduate

No description available.

No description available.

Page i was not in the other copies of this thesis. -Digitization Centre / Tn5 mutagenesis was used to generate four independent mutants of Rhizobium meliloti that were unable to grow on protocatechuate (Pca-). Two of the Pca- mutations were mapped to a region of the second symbiotic megaplasmid (pRmeSU47b) previously shown to be required for growth on protocatechuate. This pca locus was shown to consist of the first five structural genes of the protocatechuate branch of the B-ketoadipate pathway, in the order pcaDCHGB. This gene order is the same as determined for Agrobacterium tumefaciens. An additional reading frame with homology to LysR-type regulators was found to be upstream of, and transcribed divergently to the pcaDCGHB operon. This is likely to fulfil the same role as the regulatory gene, pcaQ, of A. tumefaciens. A cosmid plasmid which carried these pea genes failed to complement the Pca- phenotype of a strain carrying a 300 kb megaplasmid deletion encompassing this pea locus. This implies that another pca locus, perhaps pcaIJ, is present within the deleted region of the rnegaplasmid. Two Pca- Tn5 insertions which did not map to the megaplasmid locus were isolated. One of these insertions appears to be in a catalase gene. / Thesis / Master of Science (MS)

No description available.

Genetic mapping of pDS15 revealed that this cosmid clone carries the Sinorhizobium meliloti TCA cycle genes mdh, sucCDAB, sdhAB and part of lpdA. Three genes (mdh, sucC , and sucA) were completely sequenced and submitted to GenBank. The nucleotide and amino acid sequences of the TCA cycle genes encoded on pDS15 were aligned and found to be highly homologous with other closely related rhizobial species. S. meliloti cells grown in LBmc express the mdh-sucCDAB operon as one transcript, based on RT-PCR results. Alternative sigma factor sigma54 was not found to have a role in mdh-sucCDAB expression. Despite considerable effort, we have not been able to isolate sucA mutants via random transposon Tn5tac1 mutagenesis to date. Homologous recombination between a plasmid-borne sucA::Tn5 and wild-type S. meliloti sucA failed to generate a bona fide mutant, as revealed by Southern blot analysis. Plasmid pDS15 was mutagenized with transposons Tn5, Tn5tac1, and Tn5-B20. Three Tn5-B20 insertions were mapped to mdh, sucD, and sucA respectively, and preliminary gene expression studies were done.