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O uso de bactérias promotoras de crescimento vegetal ou agentes de biocontrole como inoculantes agrícolas é uma alternativa importante e ecologicamente correta, com grandes benefícios na agricultura para substituir, ou ao menos suplementar, a excessiva utilização de fertilizantes e pesticidas. Neste trabalho avaliamos a capacidade de biocontrole e de competência rizosférica de três bactérias com características de promoção de crescimento vegetal (Plant growth promoting - PGP): Bacillus mycoides B38V, Paenibacillus riograndensis SBR5 e Burkholderia sp. 89. As três bactérias avaliadas apresentaram grande versatilidade na utilização de substratos, o que poderia lhes garantir uma vantagem competitiva no ambiente rizosférico. Porém, inconsistências foram observadas nos ensaios em câmara de crescimento, ou seja, as características de PGP e de biocontrole observadas in vitro não se refletiram em benefícios para a planta. A linhagem 89 destacou-se pela produção de um metabólito estável com ampla atividade contra fungos fitopatogênicos. Através de abordagens genômicas e de análises multilocus, descrevemos Burkholderia sp. 89 como uma nova espécie membro do complexo Burkholderia cepacia, denominada de B. catarinensis 89T. O sequenciamento de seu genoma, seguido de uma análise pela ferramenta AntiSMASH, revelou a presença de um agrupamento gênico de peptídeo sintetases não ribossomais (NRPS) relacionadas com a biossíntese do sideróforo ornibactina e um agrupamento híbrido NRPS-policetídeo sintetase responsável pela biossíntese do glicolipopeptideo cíclico com atividade antifúngica burkholdina. Como estratégia de purificação de metabólitos secundários foi utilizada a metodologia da mineração de genoma combinada com fracionamento guiado por bioensaios seguida de análises em espectrômetro de massas. Desta forma, purificamos com sucesso duas variantes de ornibactina, D e F (761 e 789 Da, respectivamente), e detectamos a variante ornibactina B (m/z= 733) e as moléculas sinalizadoras homoserina lactonas C6-HSL, 3OH-C8-HSL e C8-HSL. Análises de espectrometria de massas demonstraram a presença de um grupo de metabólitos com massas de 1240, 1254, 1268, 1216, 1244 e 1272 Da, que, provavelmente, são novas variantes do antifúngico burkoldina. Sendo assim, B. catarinensis 89T possui potencial biotecnológico com possíveis aplicações farmacêuticas e agronômicas para o biocontrole de fungos fitopatogênicos. / The use of plant growth promotion bacteria or biocontrol agents as agricultural inoculants is an important eco-friendly alternative to substitute, or at least supplement, the excessive use of fertilizers and pesticides. In this work, we evaluated the biocontrol potential and rhizosphere competence of three bacteria that had shown plant growth promotion (PGP) abilities: Bacillus mycoides B38V, Paenibacillus riograndensis SBR5 and Burkholderia sp. 89. All three bacteria presented great versatility in their substrate utilization, which could enable them to survive in a competitive rhizosphere environment. However, inconsistencies were observed in the greenhouse experiments, whereas their interesting abilities observed in vitro did not result in benefits to the plants. Strain 89 produces a stable metabolite with a wide range of antifungal activity. Genomic comparisons and multilocus sequence analysis revealed Burkholderia sp. 89 as a new species of the Burkholderia cepacia complex and we described it as B. catarinensis 89T. We sequenced its genome and analyzed it with the AntiSMASH tool. This in silico prediction revealed the presence of a nonribosomal peptide synthetase (NRPS) cluster, which is related to the production of the siderophore ornibactin. Moreover, a hybrid NRPS- polyketide synthetase cluster for the production of the antifungal cyclic glicolipopeptide burkholdin was also found. A genome mining combined with a bioassay-guided fractionation with further mass spectrometry analysis was applied for the purification of these compounds. This approach enabled us to purify and characterize two variants of the siderophore ornibactin, D and F (761 and 789 Da, respectively). Also, we could detect the variant ornibactin B (m/z= 733) and the quorum sensing molecules homoserine lactones C6-HSL, 3OH-C8-HSL and C8-HSL in the supernatant of B. catarinensis 89T. Mass spectrometry analysis showed the presence of a group of metabolites with the masses 1240, 1254, 1268, 1216, 1244 and 1272 Da, which are probably new variants of the antifungal metabolite burkoldin. Therefore, B. catarinensis 89T has a great biotechnological potential for the production of metabolites with pharmaceutical and agricultural applications for the biocontrol of phytopathogenic fungi.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Os microrganismos sÃo essenciais para manter as funÃÃes do solo em Ãreas naturais e agrÃcolas, por causa de seu envolvimento em processos-chave na estrutura do solo, envolvendo a decomposiÃÃo de matÃria orgÃnica, a ciclagem de nutrientes e obtenÃÃo de energia, a fixaÃÃo de nitrogÃnio atmosfÃrico, a decomposiÃÃo de xenobiÃticos e o controle biolÃgico de pragas e doenÃas. Partindo do pressuposto de que ambientes de sistemas agrÃcolas tÃm mais indivÃduos r-estrategistas, enquanto Ãreas de floresta tÃm mais indivÃduos K-estrategistas, objetivou-se neste estudo avaliar a dinÃmica populacional e atividade dos microrganismos na rizosfera de fruteiras tropicais e comparar com uma Ãrea de vegetaÃÃo natural do semiÃrido cearense. Foram coletadas amostras de solo sob influÃncia de abacaxizeiro, cultivares MD2 e Imperial, de sapotÃcea, clone BRS 228, em Ãreas sem a influÃncia das fruteiras e numa faixa de vegetaÃÃo natural adjacente ao pomar, no municÃpio de Trairi, estado do CearÃ. As amostragens de solo superficial (camada de 0 a 10 cm) foram realizadas em junho, setembro e dezembro de 2010, para posterior anÃlise de atributos microbiolÃgicos e bioquÃmicos do solo. A biomassa microbiana e o quociente metabÃlico (qCO2) variaram entre as Ãreas e as Ãpocas avaliadas, enquanto o teor de carbono orgÃnico e o quociente microbiano (qMIC) variaram apenas com as Ãreas e a respiraÃÃo edÃfica mudou ao longo do ano. Houve pouca variaÃÃo na estimativa da quantidade glomalina entre as Ãreas e foi observada uma tendÃncia de reduÃÃo desta proteÃna ao longo dos perÃodos do ano. A fosfatase Ãcida e a urease apresentaram maior atividade no solo sob vegetaÃÃo natural, enquanto a enzima desidrogenase foi mais ativa em Ãreas do pomar. A densidade populacional de bactÃrias cultivÃveis foi maior no solo do pomar, enquanto a de fungos cultivÃveis foi semelhante para as Ãreas avaliadas e variando entre os perÃodos do ano. A densidade de esporos de fungos micorrÃzicos arbusculares (FMA) no solo foi influenciada negativamente pelo estabelecimento de fruteiras, em comparaÃÃo com a Ãrea de vegetaÃÃo natural. A prevalÃncia de Glomus foi comum nas diferentes Ãreas, e a abundÃncia relativa das comunidades foi maior no solo com vegetaÃÃo natural. Para as condiÃÃes do estudo pode-se inferir: os parÃmetros microbiolÃgicos sÃo sensÃveis à cobertura vegetal e podem ser usados para avaliar a qualidade do solo; os teores de carbono orgÃnico e da biomassa microbiana, a respiraÃÃo basal e os quocientes metabÃlico e microbiano do solo sÃo influenciados pelo manejo do solo no pomar e as prÃticas agrÃcolas influenciam na diversidade de FMA, selecionando provavelmente os fungos r-estrategistas. / Microorganisms are essential to keep the normal soil functioning in both natural and agricultural areas, mainly due to their implication in key processes occurring in soil structure, such as organic matter decomposition, nutrients cycling and energy captation, atmospheric nitrogen fixation, xenobiotics degradation and biological pests and diseases control. Assuming the existence of a higher number of r-strategists individuals in agricultural systems, while forest areas have more K-strategists, this study aimed to evaluate the populational dynamics and microbial activity in the rhizosphere of fruit plants, comparing them to a natural vegetation from the semiarid region of CearÃ. Soil samples under the influence of pineapple plants, cultivars MD2 and imperial, and of sapodilla plants, clone BRS 228, and also without the influence of fruit plants, but from a natural vegetation adjacent to the orchard, were collected. This collection was made in Trairi municipality, Cearà state. Superficial soil sampling (layer from 0 to 10 cm) were made in June, September and December 2010, for microbiological and biochemical analysis. Microbial biomass and metabolic quotient (qCO2) varied between the areas and periods evaluated, whereas the organic carbon content and the microbial quotient (qMIC) varied only between areas. Soil respiration varied along the year. There was a little variation in the glomalin concentration estimation between the areas and there was a tendency of reduction of this protein along the year. Acid phosphatase and urease showed greater activity in soil under natural vegetation, while the dehydrogenase enzyme was most active in orchard areas. The population density of culturable bacteria was higher in the orchard soil, while the populations of culturable fungi were similar between areas and ranged between periods of the year. The spores density of arbuscular mycorrhizal fungi (AMF) in soil was negatively influenced by the establishment of fruit plants, when compared to the natural vegetation area. The prevalence of Glomus species was common in different areas; relative abundance of those fungi communities was higher in soil under natural vegetation. Under conditions of this study we conclude: microbiological parameters are sensitive to the type of vegetation cover and can be used to evaluate the soil quality, the organic carbon and microbial biomass, basal respiration and metabolic and microbial quotients are influenced by the soil and orchard management. Agricultural practices can influence the diversity of AMF species, presumably selecting the r-strategists fungi.

In tropical soils, phosphorus (P) is adsorbed onto the oxide surface reducing the availability to plants. Thus, the solubility of the P sources and the particular plant characteristics which increases the absorption efficiency becomes important in the rhizosphere where roots and soil microorganisms interacts together. The objectives of this research were to evaluate sugarcane cultivars in relation to changes in the rhizosphere and the efficiency of P absorption and to evaluate the dynamics of P in the rhizosphere of sugarcane under the application of filter cake combined with mineral fertilizers. Four experiments were conducted in a greenhouse. The soil was a sandy clay loam Ferralsol from Piracicaba - SP, with low soil P, arranged in a randomized block design with four replications for each experiment. The experiment 1 was conducted in a 5x2 factorial arrangement with four sugarcane cultivars: RB92-579; RB85-5156; RB86-7515; RB86-6928 or no plants subjected to presence or absence of phosphate fertilizer (78.4 mg P kg-1) and soil adhering to the root was considered rhizosphere. The experiment 2 evaluated five doses of phosphate fertilizers: 0; 9.8; 19.6; 39.2 and 78.4 mg P kg-1 soil with the most promising cultivar for P absorption from experiment 1, and the rhizosphere was collected in five distances from the rhizoplane (mm): i) 0-2; ii) 2-4; iii) 4-6; iv) 6-8; v) 8-10. Experiment 3 was conducted in a 2x3 factorial: absence or presence of filter cake (5 g DM kg-1) with rock phosphate, soluble phosphate (78.4 mg soluble P kg-1 soil) or phosphate absence, and the rhizosphere soil was sampled 2 mm from the rhizoplane. Experiment 4 involved five filter cake rates: 0; 2.5; 5; 10 and 15 g DM kg-1 applied to the bulk soil or in the planting furrow. The cultivar RB96-6928 showed better adaptation characteristics under low availability of phosphorus, with good root development and P uptake. The rate 38.5 mg P kg-1 was the most suitable to promote optimal seedling development. The filter cake was efficient to increase P uptake and the interaction between filter cake and rock phosphate was efficient to increase production factors as the number of tillers per plant. The highest P uptake occurred when filter cake was applied to planting furrow at a rate of 10 g DM kg-1. / Em solos tropicais o fósforo (P) é adsorvido nas superfícies dos óxidos, reduzindo a disponibilidade às plantas. Assim, a solubilidade das fontes de P e as características particulares da planta que aumentam a eficiência de absorção tornam-se importantes na rizosfera, onde as raízes, microrganismos e solo interagem biologicamente de forma intensa. Os objetivos desta pesquisa foram avaliar cultivares de cana-de-açúcar quanto a mudanças na rizosfera e eficiência na absorção de P e avaliar a dinâmica do P na rizosfera de cana-de-açúcar sob a aplicação de torta de filtro juntamente com fertilizantes minerais. Foram conduzidos quatro experimentos em casa de vegetação. O solo utilizado foi um Latossolo Vermelho amarelo arenoso da região de Piracicaba - SP, com baixo teor de P, em delineamento em blocos ao acaso com quatro repetições para todos os experimentos. O experimento 1 foi conduzido em esquema fatorial 5x2, com 4 cultivares de cana-de-açúcar: RB92-579; RB85-5156; RB86-7515; RB86-6928 ou ausência de plantas, submetidos a presença ou ausência de adubação fosfatada (78.4 mg P kg-1), e o solo aderido a raiz foi amostrado com sendo rizosférico. O experimento 2 avaliou cinco doses de adubação fosfatada: 0; 9.8; 19.6; 39.2 e 78.4 mg P kg-1 solo com o cultivar mais promissor para absorção de P do experimento 1, sendo o solo rizosférico coletado em cinco distâncias a partir do rizoplano (mm): i) 0-2; ii) 2-4; iii) 4-6; iv) 6-8; v) 8-10. O experimento 3 foi conduzido em esquema fatorial 2x3: ausência ou presença de torta de filtro (5 g MS kg-1) com fosfato natural, fosfato solúvel (78.4 mg P solúvel kg-1 solo) ou ausência de fosfato, sendo o solo rizosférico amostrado a 2 mm do rizoplano. O experimento 4 envolveu cinco doses de torta de filtro: 0; 2.5; 5; 10 e 15 g MS kg-1 aplicados no volume total do solo ou no sulco de plantio. A cultivar RB96-6928 mostrou características de melhor adaptação sob baixa disponibilidade de P, com destacado desenvolvimento radicular e boa absorção de P. A dose 38.5 mg P kg-1 foi a mais apropriada para promover um adequado desenvolvimento das mudas. A torta de filtro foi eficiente para aumentar a absorção de P e a interação entre torta de filtro e fosfato natural foi eficiente para incrementar fatores de produção como o número de perfilhos por planta. A maior absorção de P se deu quando a torta de filtro foi aplicada no sulco de plantio na dose de 10 g MS kg-1.

Actinobactérias ocorrem em diversos ambientes e possuem grande potencial na produção de enzimas, antibióticos e fármacos. O presente trabalho teve como objetivo o isolamento, a identificação e a bioprospecção de actinobactérias da rizosfera de milho. Foram isoladas 60 linhagens de actinobactérias de plantas sadias de milho, cultivadas em diferentes regiões do Estado de São Paulo. Os isolados com maior atividade, CCMA30 e CCMA33, foram identificados por meio do seqüenciamento parcial do gene 16S rRNA como pertencentes à espécie Streptomyces griseorubiginosus, nestes isolados foi também detectada a presença de genes PKS.O fracionamento do extrato bruto da linhagem CCMA 33 revelou que a fração 7 (m/z 719-768) com atividade antifúngica, foi submetida a fragmentação (MS/MS), onde pelas buscas no dicionário de produtos naturais confirmou-se a presença de Julichromes Q 6-6, substância sintetizada por policetídeos e de mais duas Julichromes (Q 1-3 e Q 3-3). Estes resultados evidenciam a produção de biocompostos de interesse biotecnológico por actinobactérias da rizosfera. / Actinobacteria occur in different environments and have a great potential in producing enzymes, antibiotics and medical molecules. The present work aimed the isolation, identification and the bioprospection by rhizosphere actinobactérias. Sixty strains were isolated from healthy plants of maize, cultivated in different regions Socorro, Serra Negra and Ribeirão Preto. The two isolates with higher activity CCMA30 and CCMA33 were identified by the partial sequencing of the 16S rDNA gene as belonging to the species Streptomyces griseorubiginosus. In these isolates it was detected the presence of PKS genes. The fractioning of the crude extract of the strain CCMA 33 has revealed that fraction 7 (m/z 719-768) displayed antifungal activity and it was submitted to fragmentation (MS/MS), where by searches in the dictionary of natural products detected the presence of Julichromes Q6-6 and the presence of two other Julichromes (Q1-3 and Q3-3). These results highlight the production of secondary metabolites of biotechnological interest by rhizosphere actinobacteria.

Revegetation of bauxite residue is hampered by a lack of understanding of the limitations imposed on plant growth in highly saline and alkaline growth mediums. In this study, several of these growth limiting factors were investigated. The toxicity of the hydroxyl ion (OH-) was examined using a solution culture system developed to allow studies at high pH without nutritional limitations. Also using this solution culture system, the effect of the high Na and Mg concentrations of bauxite residue on the Ca nutrition of plants was investigated. As the toxicity of Al at high pH is not known, a study was conducted to examine the rhizotoxicity of aluminate (Al(OH)4-) and polycationic Al at high pH. The ability of plant roots to reduce rhizosphere pH in bauxite residue was also considered. A novel gypsum application method was assessed for its efficiency at improving the Ca status of bauxite residue. Manual adjustment, ion exchange resins and automated titration were examined for their suitability for nutrient solution pH control in alkaline conditions. For short-term studies, it was found that a solution without supply of Cu, Fe, Mn and Zn, and aerated with CO2 depleted air, greatly reduced nutrient precipitation at high pH, thus eliminating nutritional differences between treatments. Manual pH adjustment and the use of ion exchange resins as pH buffers were unsuitable methods of pH control. In contrast, pH control by automated titration had little effect on solution composition while maintaining constant pH. The solution culture system was used to examine OH- toxicity in mungbeans (Vigna radiata (L.) Wilczek cv. Emerald), with root length reduced at a bulk solution pH of 8.5 and greater. The effect of Ca activity ratio (CAR) and pH on Ca uptake by mungbeans and Rhodes grass (Chloris gayana cv. Pioneer) in Na dominated solution cultures and in soil was investigated. Changes in pH in the alkaline range were shown to have no effect on the critical CAR of 0.024 (corresponding to 90 % relative root length) for mungbeans grown in solution culture. Results from soil grown mungbeans confirmed those from solution culture, with a critical CAR of 0.025. A critical CAR of 0.034 was also established for soil grown Rhodes grass. However, using dilute nutrient solutions dominated by Mg at pH 9.0, root growth was found to be more limited than had been observed for Na solutions, with growth reduced beneath a critical CAR of 0.050. Using a CAR equation modified with plasma membrane binding constants (to incorporate the differing antagonistic effects of Mg and Na), new critical CAR values were calculated for Na (0.56) and Mg (0.44) dominated solutions. This modified CAR equation permits the calculation of CAR irrespective of the dominant salt present. Solubilities of various gypsum sources and size fractions in seawater were studied to investigate the effectiveness of gypsum addition to the residue sand pipeline, rather than as a direct field application. The dissolution rate constant varied with gypsum source (analytical grade (AR) > phosphogypsum (PG) > mined gypsum (MG)) due to reactivity and surface area differences, generally reaching saturation within 15 s (AR) to 30 min (MG > 2.0). The ability of bauxite residue to remove Ca from solution (due to cation exchange and precipitation) was also examined; the quantity of the total solution Ca adsorbed was found to be small (5 %). These low rates of solution Ca adsorption, comparatively rapid dissolution rates, and long pumping times (20 min), preclude the application of gypsum to the residue sand/seawater slurry as a method for residue amelioration. Dilute, alkaline (pH 9.5) nutrient solutions were used to investigate the effects of aluminate (Al(OH)4-) on mungbean root growth. Although root growth in Al(OH)4- solutions was slightly limited, the symptoms associated with this growth reduction were observed to be similar to those caused by the Al13 polycation at concentrations lower than that which can be detected. Also, when roots displaying these symptoms were transferred to fresh Al(OH)4- solutions, no root tip lesions were observed, and root hair growth on the lateral roots improved. Thus, Al(OH)4- is considered to be non-toxic, with the observed reduction in root growth in solutions containing Al(OH)4- due to the gradual formation of toxic Al13 in the bulk nutrient solution resulting from the acidification of the alkaline nutrient solution by the plant roots. The effect of Mn deficiency in Rhodes grass and of legume inoculation in lucerne (Medicago sativa L. cv. Hunter River), on the rhizosphere pH of plants grown in highly alkaline bauxite residue was investigated. In response to Mn deficiency in residue sand, Rhodes grass was observed to increase acidification of its rhizosphere (being up to 1.22 pH units lower than the bulk soil). Due to its ability to fix atmospheric N2 rather than relying on soil N (NO3-) reserves, inoculated lucerne (1.75 pH unit decrease) was also found to acidify its rhizosphere to a greater extent than non-inoculated lucerne (1.16 pH unit decrease).

Revegetation of bauxite residue is hampered by a lack of understanding of the limitations imposed on plant growth in highly saline and alkaline growth mediums. In this study, several of these growth limiting factors were investigated. The toxicity of the hydroxyl ion (OH-) was examined using a solution culture system developed to allow studies at high pH without nutritional limitations. Also using this solution culture system, the effect of the high Na and Mg concentrations of bauxite residue on the Ca nutrition of plants was investigated. As the toxicity of Al at high pH is not known, a study was conducted to examine the rhizotoxicity of aluminate (Al(OH)4-) and polycationic Al at high pH. The ability of plant roots to reduce rhizosphere pH in bauxite residue was also considered. A novel gypsum application method was assessed for its efficiency at improving the Ca status of bauxite residue. Manual adjustment, ion exchange resins and automated titration were examined for their suitability for nutrient solution pH control in alkaline conditions. For short-term studies, it was found that a solution without supply of Cu, Fe, Mn and Zn, and aerated with CO2 depleted air, greatly reduced nutrient precipitation at high pH, thus eliminating nutritional differences between treatments. Manual pH adjustment and the use of ion exchange resins as pH buffers were unsuitable methods of pH control. In contrast, pH control by automated titration had little effect on solution composition while maintaining constant pH. The solution culture system was used to examine OH- toxicity in mungbeans (Vigna radiata (L.) Wilczek cv. Emerald), with root length reduced at a bulk solution pH of 8.5 and greater. The effect of Ca activity ratio (CAR) and pH on Ca uptake by mungbeans and Rhodes grass (Chloris gayana cv. Pioneer) in Na dominated solution cultures and in soil was investigated. Changes in pH in the alkaline range were shown to have no effect on the critical CAR of 0.024 (corresponding to 90 % relative root length) for mungbeans grown in solution culture. Results from soil grown mungbeans confirmed those from solution culture, with a critical CAR of 0.025. A critical CAR of 0.034 was also established for soil grown Rhodes grass. However, using dilute nutrient solutions dominated by Mg at pH 9.0, root growth was found to be more limited than had been observed for Na solutions, with growth reduced beneath a critical CAR of 0.050. Using a CAR equation modified with plasma membrane binding constants (to incorporate the differing antagonistic effects of Mg and Na), new critical CAR values were calculated for Na (0.56) and Mg (0.44) dominated solutions. This modified CAR equation permits the calculation of CAR irrespective of the dominant salt present. Solubilities of various gypsum sources and size fractions in seawater were studied to investigate the effectiveness of gypsum addition to the residue sand pipeline, rather than as a direct field application. The dissolution rate constant varied with gypsum source (analytical grade (AR) > phosphogypsum (PG) > mined gypsum (MG)) due to reactivity and surface area differences, generally reaching saturation within 15 s (AR) to 30 min (MG > 2.0). The ability of bauxite residue to remove Ca from solution (due to cation exchange and precipitation) was also examined; the quantity of the total solution Ca adsorbed was found to be small (5 %). These low rates of solution Ca adsorption, comparatively rapid dissolution rates, and long pumping times (20 min), preclude the application of gypsum to the residue sand/seawater slurry as a method for residue amelioration. Dilute, alkaline (pH 9.5) nutrient solutions were used to investigate the effects of aluminate (Al(OH)4-) on mungbean root growth. Although root growth in Al(OH)4- solutions was slightly limited, the symptoms associated with this growth reduction were observed to be similar to those caused by the Al13 polycation at concentrations lower than that which can be detected. Also, when roots displaying these symptoms were transferred to fresh Al(OH)4- solutions, no root tip lesions were observed, and root hair growth on the lateral roots improved. Thus, Al(OH)4- is considered to be non-toxic, with the observed reduction in root growth in solutions containing Al(OH)4- due to the gradual formation of toxic Al13 in the bulk nutrient solution resulting from the acidification of the alkaline nutrient solution by the plant roots. The effect of Mn deficiency in Rhodes grass and of legume inoculation in lucerne (Medicago sativa L. cv. Hunter River), on the rhizosphere pH of plants grown in highly alkaline bauxite residue was investigated. In response to Mn deficiency in residue sand, Rhodes grass was observed to increase acidification of its rhizosphere (being up to 1.22 pH units lower than the bulk soil). Due to its ability to fix atmospheric N2 rather than relying on soil N (NO3-) reserves, inoculated lucerne (1.75 pH unit decrease) was also found to acidify its rhizosphere to a greater extent than non-inoculated lucerne (1.16 pH unit decrease).

Some plant-beneficial bacteria with biopesticide potential produce antimicrobial compounds that are tightly involved in multitrophic interactions occurring in the phytosphere among which antagonism toward a diverse range of phytopathogens. However, there is a crucial need for an accurate assessment of antibiotic production rate in this environment colonized by these bacteria. In this context, we conducted the present thesis work with the scope to better understand how two different Bacillus genus strains produce lipopeptides in the rhizosphere of plants. In the first part, one of the strains (B. amyloliquefaciens S499) was selected out of other isolates for its technological traits and in vitro inhibition growth activity of plant pathogenic fungi. We further demonstrated its biocontrol potential in tomato open-field experiments where plantings have been devastated by a local fungus preliminary identified as Fusarium semitectum. In a second part, we combined two mass spectrometry-based approaches (electrospray ionization and imaging) to analyze the pattern of surfactin, iturin and fengycin lipopeptide families produced in planta by strain S499. Our results show that rhizosphere conditions are conducive for surfactin synthesis but not for other types of lipopeptides and that the lipopeptide pattern can be markedly influenced by nutritional factors, biofilm formation and oxygen availability. In a last part, surfactin gene expression (srfA) level was evaluated in situ on tomato root using the reporter gene (LacZ) inserted in B. subtilis strain BGS3. Results showed effective expression of srfA and production of surfactin in biologically important level quantities upon establishment of bacterial population on roots. Our results also demonstrate that BGS3 developing in colonies, efficiently utilizes the main substrates from plant exudates to produce surfactins. The production may also be favored in bacteria growing slowly in the rhizosphere. Globally, this work contributes to better appreciate the impact of some environmental factors on the in situ biosynthesis of lipopeptides by strains of Bacillus which is probably an essential step for improving the level and reliability of their efficacy as biological agents for the control of plant diseases.

Naphthenic acids (NAs) are toxic organic acid compounds released during the caustic hot-water extraction of crude oil from oil sands in north-eastern Alberta, Canada. NAs subsequently accumulate in the large volume of oil sands process water (OSPW) produced daily by oil sands operations. The complexity of dealing with a mixture of over 200 individual NA compounds, combined with their acute aquatic toxicity and large volume of production has made them an emerging pollutant of concern for western Canada. The following thesis outlines a variety of experiments designed to determine the potential to use wetland plants to enhance the dissipation of NAs from OSPW (phytoremediation). <p>Investigations were carried out with three native emergent macrophyte species cattail (<i>Typha latifolia</i>), common reed (<i>Phragmites australis </i>subsp. <i>americanus</i>), and hard-stem bulrush (<i>Scirpus acutus</i>) to see if they enhanced the dissipation of NAs from a hydroponic system. Dissipation of NAs (at 30 mg L-1 and 60 mg L-1) was investigated with both a commercially available NA mixture as well as with a NA mixture extracted from the OSPW. Dissipation of NAs was also investigated under the different ionized forms of NAs (ionized, pH = 7.8; and non-ionized, pH = 5.0) to better elucidate the mechanisms of NA uptake and toxicity in plants. Phytotoxicity of NAs was investigated in hydroponic experiments through fresh weight gain and evapotranspiration was monitored throughout the experiment by water uptake. Commercially available NA mixture was more phytotoxic than oil sands NAs mixture. As well, NAs were found to be more phytotoxic in their non-ionized form therefore indicating that they may be taken up through an ion-trap‟ mechanism. However despite this, no significant dissipation of total NAs was observed from planted hydroponic systems. Nevertheless there was a significant change in the distribution (percent abundance) of individual NA families of certain size. These changes were related to the one- and two-ring NA compounds (Z = -2 and Z = -4). Despite not detecting any dissipation of total NAs from the systems, plants were able to reduce the toxicity of a NA system over 30 days by 45% as determined by Daphnia magna acute toxicity bioassays; a 11% greater reduction than unplanted systems.<p> Studies were also conducted investigating the microbial community inhabiting cattail roots exposed to NAs. It was observed that the rhizosphere community changed with NA exposure, with a general increase in potentially pathogenic bacteria and a decrease in bacteria previously found to be beneficial to plant growth. The observed microbial community change could be an indirect effect of the Phytotoxicity experienced by aquatic macrophytes exposed to NAs. Synchrotron-sourced, fourier transform microspectroscopy analysis of root cross sections revealed that there were significant physiological changes to those roots exposed to NAs. These changes were identified as being cell death in the plant root epidermis as well as a change in the chemistry of parenchyma cells in the root pith. It is not known if these changes are a direct effect of NAs to the plant or due to changes of the associated rhizosphere community in the roots or some combination of both these factors.

Naphthenic acids (NAs) are toxic organic acid compounds released during the caustic hot-water extraction of crude oil from oil sands in north-eastern Alberta, Canada. NAs subsequently accumulate in the large volume of oil sands process water (OSPW) produced daily by oil sands operations. The complexity of dealing with a mixture of over 200 individual NA compounds, combined with their acute aquatic toxicity and large volume of production has made them an emerging pollutant of concern for western Canada. The following thesis outlines a variety of experiments designed to determine the potential to use wetland plants to enhance the dissipation of NAs from OSPW (phytoremediation). <p>Investigations were carried out with three native emergent macrophyte species cattail (<i>Typha latifolia</i>), common reed (<i>Phragmites australis </i>subsp. <i>americanus</i>), and hard-stem bulrush (<i>Scirpus acutus</i>) to see if they enhanced the dissipation of NAs from a hydroponic system. Dissipation of NAs (at 30 mg L-1 and 60 mg L-1) was investigated with both a commercially available NA mixture as well as with a NA mixture extracted from the OSPW. Dissipation of NAs was also investigated under the different ionized forms of NAs (ionized, pH = 7.8; and non-ionized, pH = 5.0) to better elucidate the mechanisms of NA uptake and toxicity in plants. Phytotoxicity of NAs was investigated in hydroponic experiments through fresh weight gain and evapotranspiration was monitored throughout the experiment by water uptake. Commercially available NA mixture was more phytotoxic than oil sands NAs mixture. As well, NAs were found to be more phytotoxic in their non-ionized form therefore indicating that they may be taken up through an ion-trap‟ mechanism. However despite this, no significant dissipation of total NAs was observed from planted hydroponic systems. Nevertheless there was a significant change in the distribution (percent abundance) of individual NA families of certain size. These changes were related to the one- and two-ring NA compounds (Z = -2 and Z = -4). Despite not detecting any dissipation of total NAs from the systems, plants were able to reduce the toxicity of a NA system over 30 days by 45% as determined by Daphnia magna acute toxicity bioassays; a 11% greater reduction than unplanted systems.<p> Studies were also conducted investigating the microbial community inhabiting cattail roots exposed to NAs. It was observed that the rhizosphere community changed with NA exposure, with a general increase in potentially pathogenic bacteria and a decrease in bacteria previously found to be beneficial to plant growth. The observed microbial community change could be an indirect effect of the Phytotoxicity experienced by aquatic macrophytes exposed to NAs. Synchrotron-sourced, fourier transform microspectroscopy analysis of root cross sections revealed that there were significant physiological changes to those roots exposed to NAs. These changes were identified as being cell death in the plant root epidermis as well as a change in the chemistry of parenchyma cells in the root pith. It is not known if these changes are a direct effect of NAs to the plant or due to changes of the associated rhizosphere community in the roots or some combination of both these factors.