<i>Rhizobium leguminosarum</i> are well known for their ability to fix nitrogen (N). In addition, their capacity to solubilize phosphate has been receiving attention in recent years. The work presented in this thesis examined two aspects of screening and evaluating dicalcium phosphate (Pi) (CaHPO4) solubilizing <i> R. leguminosarum</i>. The objectives of this study were to: 1) identify a medium that is sensitive and effective as a screening tool for phospahte solubilizing <i>R. leguminosarum</i>; 2) determine the effect of N and carbon (C) on growth and P solubilization of <i>R. leguminosarum</i> isolates; 3) determine the relationship between the ability to solubilize CaHPO4 by <i>R. leguminosarum</i> isolates on solid medium and in liquid broth of same composition; and 4) assess and compare the ability of <i>R. leguminosarum</i> isolates to solubilize different P sources in soil under growth chamber conditions.<p>In this study, 30 <i>R. leguminosarum</i> isolates were evaluated for phosphate solubilization in broth and solid formulations of three different media, Yeast Mannitol Extract (YEM), Botanical Research Institute Phosphate Nutrient medium (MNBRI) and Pikovskaya Phosphate medium (PVK). All media contain CaHPO4 as the only phosphorus (P) source. The <i>R. leguminosarum</i> isolates were selected on the basis of their different plasmid profiles, indicative of genetically distinct isolates. <p>All 30 isolates increased the Pi concentration in solution to varying degrees in liquid cultures but performance varied from one medium to another. The highest average solution Pi concentration achieved by the 30 <i>R. leguminosarum</i> isolates was obtained from PVK cultured broth. CaHPO4 solubilization by <i>R. leguminosarum</i> isolates in liquid was associated with a decrease in pH. Among the three tested media, the lowest pH by the thirty <i>R. leguminosarum</i> isolates was obtained in PVK. Ability of the isolates to solubilize CaHPO4 on the solid media was not comparable to the performance of the isolates grown in liquid because only fewer <i>R. leguminosarum</i> isolates showed visible P solubilization on the solid media.<p>The composition and formulation of medium influence the ability of the <i>R. leguminosarum</i> isolates to solubilize CaHPO4. Effects of N and C concentrations on the growth and CaHPO4 solubilization by nine <i>R. leguminosarum</i> isolates were examined in liquid formulation. Ammonium N had a greater influence on the growth and CaHPO4 solubilization by <i>R. leguminosarum</i> isolates than C at the tested levels. The growth of isolates was inhibited by ammonium N at 0.5 g L-1 as (NH4)2SO4 meaning there were less viable cells in this N concentration than were of ammonium N at 0.1 g L-1. The ability of isolates to solubilize Pi however was not affected by ammonium N at 0.5 g L-1 as (NH4)2SO4. The media containing low N (0.1 g (NH4)2S04 L-1) both Pi solubilization and growth of <i>R. leguminosarum</i> isolates were not affected.<p><i>R. leguminosarum</i> isolates were tested for their effects on growth and P uptake of canola plants in P-deficient soils amended with different P sources. <i>R. leguminosarum</i> isolates were selected separately based on their ability to solubilize CaHPO4 from the three screening media. A quadrant model was used based on the ability of the 30 <i>R. leguminosarum</i> isolates to solubilize CaHPO4 on both solid and liquid formulations within a medium. The effect of <i>R. leguminosarum</i> on canola dry mass, tissue Pi content and total Pi uptake varied from one isolate to another, but was not different from the controls. The quadrant model failed to correlate isolates able to solubilize CaHPO4 in laboratory screening to isolates able to solubilize P in the growth chamber. Despite the influence of the medium composition and formulation, none of tested media predicted Pi solubilization ability by the <i>R. leguminosarum</i> isolates in soils under growth chamber conditions, from their Pi solubilization of laboratory screenings. <p>The work of this thesis demonstrates that phosphate solubilization is a complex process that depends on both organism and soil. Growth condition is an important factor for a <i>R. leguminosarum</i> isolate to express its ability to solubilize CaHPO4. Liquid media screenings illustrate an isolates ability to solubilize CaHPO4 under nonstressful conditions, but solid media screenings demonstrate the P solubilization result of an isolate under more stressful conditions. The lack of relationship in P solubilization ability by <i>R. leguminosarum</i> isolates, between laboratory methods to soil test, means neither liquid or solid media can provide a definitive selection process. Additional parameters should be investigated to modify the soil bioassay protocols and ultimate selection procedures. These include pH conditions, isolate colonization, growth, and survival on plants and rhizosphere.

<i>Rhizobium leguminosarum</i> are well known for their ability to fix nitrogen (N). In addition, their capacity to solubilize phosphate has been receiving attention in recent years. The work presented in this thesis examined two aspects of screening and evaluating dicalcium phosphate (Pi) (CaHPO4) solubilizing <i> R. leguminosarum</i>. The objectives of this study were to: 1) identify a medium that is sensitive and effective as a screening tool for phospahte solubilizing <i>R. leguminosarum</i>; 2) determine the effect of N and carbon (C) on growth and P solubilization of <i>R. leguminosarum</i> isolates; 3) determine the relationship between the ability to solubilize CaHPO4 by <i>R. leguminosarum</i> isolates on solid medium and in liquid broth of same composition; and 4) assess and compare the ability of <i>R. leguminosarum</i> isolates to solubilize different P sources in soil under growth chamber conditions.<p>In this study, 30 <i>R. leguminosarum</i> isolates were evaluated for phosphate solubilization in broth and solid formulations of three different media, Yeast Mannitol Extract (YEM), Botanical Research Institute Phosphate Nutrient medium (MNBRI) and Pikovskaya Phosphate medium (PVK). All media contain CaHPO4 as the only phosphorus (P) source. The <i>R. leguminosarum</i> isolates were selected on the basis of their different plasmid profiles, indicative of genetically distinct isolates. <p>All 30 isolates increased the Pi concentration in solution to varying degrees in liquid cultures but performance varied from one medium to another. The highest average solution Pi concentration achieved by the 30 <i>R. leguminosarum</i> isolates was obtained from PVK cultured broth. CaHPO4 solubilization by <i>R. leguminosarum</i> isolates in liquid was associated with a decrease in pH. Among the three tested media, the lowest pH by the thirty <i>R. leguminosarum</i> isolates was obtained in PVK. Ability of the isolates to solubilize CaHPO4 on the solid media was not comparable to the performance of the isolates grown in liquid because only fewer <i>R. leguminosarum</i> isolates showed visible P solubilization on the solid media.<p>The composition and formulation of medium influence the ability of the <i>R. leguminosarum</i> isolates to solubilize CaHPO4. Effects of N and C concentrations on the growth and CaHPO4 solubilization by nine <i>R. leguminosarum</i> isolates were examined in liquid formulation. Ammonium N had a greater influence on the growth and CaHPO4 solubilization by <i>R. leguminosarum</i> isolates than C at the tested levels. The growth of isolates was inhibited by ammonium N at 0.5 g L-1 as (NH4)2SO4 meaning there were less viable cells in this N concentration than were of ammonium N at 0.1 g L-1. The ability of isolates to solubilize Pi however was not affected by ammonium N at 0.5 g L-1 as (NH4)2SO4. The media containing low N (0.1 g (NH4)2S04 L-1) both Pi solubilization and growth of <i>R. leguminosarum</i> isolates were not affected.<p><i>R. leguminosarum</i> isolates were tested for their effects on growth and P uptake of canola plants in P-deficient soils amended with different P sources. <i>R. leguminosarum</i> isolates were selected separately based on their ability to solubilize CaHPO4 from the three screening media. A quadrant model was used based on the ability of the 30 <i>R. leguminosarum</i> isolates to solubilize CaHPO4 on both solid and liquid formulations within a medium. The effect of <i>R. leguminosarum</i> on canola dry mass, tissue Pi content and total Pi uptake varied from one isolate to another, but was not different from the controls. The quadrant model failed to correlate isolates able to solubilize CaHPO4 in laboratory screening to isolates able to solubilize P in the growth chamber. Despite the influence of the medium composition and formulation, none of tested media predicted Pi solubilization ability by the <i>R. leguminosarum</i> isolates in soils under growth chamber conditions, from their Pi solubilization of laboratory screenings. <p>The work of this thesis demonstrates that phosphate solubilization is a complex process that depends on both organism and soil. Growth condition is an important factor for a <i>R. leguminosarum</i> isolate to express its ability to solubilize CaHPO4. Liquid media screenings illustrate an isolates ability to solubilize CaHPO4 under nonstressful conditions, but solid media screenings demonstrate the P solubilization result of an isolate under more stressful conditions. The lack of relationship in P solubilization ability by <i>R. leguminosarum</i> isolates, between laboratory methods to soil test, means neither liquid or solid media can provide a definitive selection process. Additional parameters should be investigated to modify the soil bioassay protocols and ultimate selection procedures. These include pH conditions, isolate colonization, growth, and survival on plants and rhizosphere.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-10-19Bitstream added on 2014-06-13T19:03:38Z : No. of bitstreams: 1 barroso_cb_dr_jabo.pdf: 424873 bytes, checksum: c11fcf246d6ada6dcb329194e9814468 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Devido a baixa disponibilidade de P no solo e a alta capacidade do fungo Aspergillus niger F111 em solubilizar fosfatos inorgânicos, este trabalho teve por objetivo geral avaliar a possibilidade de inocular no solo esporos ou pellets imobilizados com vista a prolongar sua habilidade de solubilização e averiguar o mecanismo de solubilização de fosfatos inorgânicos de Ca, Al e Fe por este fungo. Os pellets inoculados em meio de cultura agitado proporcionaram maior solubilização dos fosfatos, principalmente o fosfato de Fe por ser de baixa solubilidade. No solo, os pellets livres e imobilizados promoveram as maiores solubilizações de fosfato de Fe e maior produção de CO2. Avaliando-se o efeito da fonte de N, as seguintes proporções foram obtidas na solubilização dos fosfatos de Ca, glicina > Al, nitrato de amônio > Fe, ácido l-glutâmico. Os açúcares que mais solubilizaram os fosfatos foram manitol, maltose e d-galactose. Dentre os metais somente o FeCl3.6H2O promoveu maior solubilização do fosfato de Fe e os metais FeSO4.7H2O e FeCl3.6H2O promoveram maiores solubilizações do fosfato de Ca. As concentrações de álcoois que mais favoreceram a solubilização do fosfato de Fe foram 3 e 4% de etanol e metanol, para o fosfato de Ca foi 3% de etanol. A combinação dos metais com o metanol, indicou que o metanol foi o principal responsável pela solubilização. Fatores como queda do pH, a maior produção de ácidos e o menor crescimento do fungo influíram neste trabalho, principalmente em relação a solubilização do fosfato de Fe. No solo, os pellets solubilizaram quantidades semelhantes de fosfato de Fe que os esporos imobilizados de A. niger, podendo ser utilizados com vantagem devido a sua facilidade de obtenção. / Considering the low P availability in the soil and the high capability of Aspergillus niger F111 in solubilizing inorganic phosphates, this work aimed to evaluate the possibility of inoculating spores or immobilized pellets in the soil to prolong the solubilization capability and study the solubilization mechanism of inorganic calcium phosphate, aluminum phosphate and iron phosphate by this fungus. Pellets inoculated in culture medium under agitation allowed higher phosphate solubilization, especially iron phosphate, which is low soluble. In the soil, free and immobilized pellets allowed the highest solubilization of iron phosphate and CO2 production. Evaluating the effect of N sources, the following proportions were obtained in the solubilization of calcium phosphates, glycine > Al, ammonium nitrate > Fe, l-glutamic acid. The sugars that most solubilized phosphates were mannitol, maltose and d-galactose. Among the metals, only FeCl3.6H2O promoted higher iron phosphate solubilization, and FeSO4.7H2O and FeCl3.6H2O promoted higher solubilization of calcium phosphate. The alcohol concentrations that most favored iron phosphate solubilization were 3 and 4% of ethanol and methanol, while the highest solubilization of calcium phosphate was reached with 3% ethanol. The combination of metals with methanol indicated this alcohol was mainly responsible for solubilization. Factors as pH decrease, higher acid production and lower A. niger growth influenced the results, especially in the solubilization of iron phosphate. In the soil, pellets and immobilized spores solubilized similar amounts of iron phosphate. Pellets are thus preferable because they are more easily obtained.

Orientador: Ely Nahas / Banca: Regina Teresa Rosim Monteiro / Banca: Jonas Contiero / Banca: Antônio Carlos Monteiro / Banca: Lúcia Maria Carareto Alves / Resumo: Devido a baixa disponibilidade de P no solo e a alta capacidade do fungo Aspergillus niger F111 em solubilizar fosfatos inorgânicos, este trabalho teve por objetivo geral avaliar a possibilidade de inocular no solo esporos ou pellets imobilizados com vista a prolongar sua habilidade de solubilização e averiguar o mecanismo de solubilização de fosfatos inorgânicos de Ca, Al e Fe por este fungo. Os pellets inoculados em meio de cultura agitado proporcionaram maior solubilização dos fosfatos, principalmente o fosfato de Fe por ser de baixa solubilidade. No solo, os pellets livres e imobilizados promoveram as maiores solubilizações de fosfato de Fe e maior produção de CO2. Avaliando-se o efeito da fonte de N, as seguintes proporções foram obtidas na solubilização dos fosfatos de Ca, glicina > Al, nitrato de amônio > Fe, ácido l-glutâmico. Os açúcares que mais solubilizaram os fosfatos foram manitol, maltose e d-galactose. Dentre os metais somente o FeCl3.6H2O promoveu maior solubilização do fosfato de Fe e os metais FeSO4.7H2O e FeCl3.6H2O promoveram maiores solubilizações do fosfato de Ca. As concentrações de álcoois que mais favoreceram a solubilização do fosfato de Fe foram 3 e 4% de etanol e metanol, para o fosfato de Ca foi 3% de etanol. A combinação dos metais com o metanol, indicou que o metanol foi o principal responsável pela solubilização. Fatores como queda do pH, a maior produção de ácidos e o menor crescimento do fungo influíram neste trabalho, principalmente em relação a solubilização do fosfato de Fe. No solo, os pellets solubilizaram quantidades semelhantes de fosfato de Fe que os esporos imobilizados de A. niger, podendo ser utilizados com vantagem devido a sua facilidade de obtenção. / Abstract: Considering the low P availability in the soil and the high capability of Aspergillus niger F111 in solubilizing inorganic phosphates, this work aimed to evaluate the possibility of inoculating spores or immobilized pellets in the soil to prolong the solubilization capability and study the solubilization mechanism of inorganic calcium phosphate, aluminum phosphate and iron phosphate by this fungus. Pellets inoculated in culture medium under agitation allowed higher phosphate solubilization, especially iron phosphate, which is low soluble. In the soil, free and immobilized pellets allowed the highest solubilization of iron phosphate and CO2 production. Evaluating the effect of N sources, the following proportions were obtained in the solubilization of calcium phosphates, glycine > Al, ammonium nitrate > Fe, l-glutamic acid. The sugars that most solubilized phosphates were mannitol, maltose and d-galactose. Among the metals, only FeCl3.6H2O promoted higher iron phosphate solubilization, and FeSO4.7H2O and FeCl3.6H2O promoted higher solubilization of calcium phosphate. The alcohol concentrations that most favored iron phosphate solubilization were 3 and 4% of ethanol and methanol, while the highest solubilization of calcium phosphate was reached with 3% ethanol. The combination of metals with methanol indicated this alcohol was mainly responsible for solubilization. Factors as pH decrease, higher acid production and lower A. niger growth influenced the results, especially in the solubilization of iron phosphate. In the soil, pellets and immobilized spores solubilized similar amounts of iron phosphate. Pellets are thus preferable because they are more easily obtained. / Doutor