Dentre os mecanismos relacionados à interação bactéria-planta, a biossíntese bacteriana de ácido indol acético (AIA) exerce um papel fundamental na promoção do crescimento vegetal, uma vez que é capaz de influenciar inúmeros processos fisiológicos nas plantas. Diferentes vias metabólicas são utilizadas pelas bactérias para a biossíntese do AIA, sendo a via do ácido indol-3-pirúvico (IPyA) a mais comumente descrita. Nesta via encontra-se o gene indol-3-piruvato descarboxilase (ipdC) com vital função na produção de AIA utilizando como precursor o aminoácido L-triptofano. Nesse contexto, estudos moleculares acerca das vias metabólicas e dos genes envolvidos nesse processo são preponderantes para o entendimento da inter-relação das vias regulatórias com a síntese do fitormônio. A rizobactéria Bacillus sp. (RZ2MS9) vem apresentando satisfatória atividade na promoção de crescimento vegetal. O sequenciamento do seu genoma apontou a presença de uma vasta gama de genes relacionados à promoção do crescimento, com destaque para genes codificadores de auxinas. Assim, o estudo teve por objetivo comprovar a função do gene ipdC na biossíntese do AIA pela via dpendente do L-triptofano através do nocaute sítio dirigido do gene ipdC na Rizobactéria Promotora do Crescimento em Plantas (RPCP) Bacillus sp. (RZ2MS9). Para tanto, foi realizado o nocaute sítio dirigido por meio da técnica de CRISPR-Cas9. O nocaute do gene ipdC foi eficiente, gerando mutantes disruptivos para o referido gene. A biossíntese do AIA pela linhagem ΔipdC apresentou reduções nas concentrações do fitormônio, de acordo com o tempo de crescimento, sendo 87,96% em 24 horas, 88,25% em 48 horas e 58,27% em 72 horas do crescimento em comparação à linhagem selvagem (WT). Além disso, a biossíntese do AIA na ausência do aminoácido L-triptofano também foi avaliada, não sendo constatada síntese do fitormônio em nenhum dos tempos crescimento, tanto na linhagem selvagem, quanto na linhagem ΔipdC. O presente estudo foi pioneiro no nocaute do gene ipdC em uma linhagem de Bacillus utilizando a técnica de CRISPR-Cas9. Os resultados obtidos contribuem para um melhor entendimento da influência do gene ipdC e da via IPyA na biossíntese do AIA pela linhagem RZ2MS9 e futuramente sera comprovado seu papel na promoção de crescimento vegetal. / Among the mechanisms related to the bacterium-plant interaction, the bacterial biosynthesis of indole acetic acid (AIA) plays a fundamental role in the promotion of plant growth, since it is capable of influencing innumerable physiological processes in plants. Different metabolic pathways are used by bacteria for the biosynthesis of IAA, with the indole-3-pyruvic acid (IPyA) pathway being the most commonly described. In this pathway, the indole-3-pyruvate decarboxylase (ipdC) gene has a vital role in the production of IAA using the amino acid L-tryptophan as a precursor. In this context, molecular studies about the metabolic pathways and the genes involved in this process are preponderant for the understanding of the interrelationship of the regulatory pathways with the phytormonium synthesis. The rhizobacterium Bacillus sp. (RZ2MS9) has been showing satisfactory activity in promoting plant growth. The sequencing of its genome pointed to the presence of a wide range of genes related to growth promotion, especially genes encoding auxins. Thus, the objective of the present study was to verify the function of the ipdC gene in the IAA biosynthesis L-tryptophan dependent through the knockout of the ipdC in the plant growth-promoting rhizobateria (PGPR) Bacillus sp. (RZ2MS9). Therefore, the knockout was realized using the CRISPR-Cas9. The knockout of the ipdC gene was efficient, generating disruptive mutants for the said gene. IAA biosynthesis by the ΔipdC strain showed reductions in phytormonium concentrations, according to the growth time, being 87.96% in 24 hours, 88.25% in 48 hours and 58.27% in 72 hours of growth compared to the Wild Type (WT). In addition, the biosynthesis of IAA in the absence of the amino acid L-tryptophan was also evaluated, with no phytormonium synthesis being observed at any growth time, both in the wild type and ΔipdC strain. The present study pioneered the knockout of the ipdC gene in a Bacillus strain using the CRISPR-Cas9. The results obtained contribute to a better understanding of the influence of the ipdC gene and the IPyA pathway in the IAA biosynthesis through the RZ2MS9 strain, and its role in plant growth promoting will be demonstrated in the future.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-03-06T18:10:51Z No. of bitstreams: 1 ANDRESSA_VON_WURMB_HELRIGUEL_LEITE_DIS.pdf: 1584575 bytes, checksum: 5f9a44ec42089e160df54cc84eaadb9a (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-03-07T14:04:49Z (GMT) No. of bitstreams: 1 ANDRESSA_VON_WURMB_HELRIGUEL_LEITE_DIS.pdf: 1584575 bytes, checksum: 5f9a44ec42089e160df54cc84eaadb9a (MD5) / Made available in DSpace on 2018-03-07T14:11:58Z (GMT). No. of bitstreams: 1 ANDRESSA_VON_WURMB_HELRIGUEL_LEITE_DIS.pdf: 1584575 bytes, checksum: 5f9a44ec42089e160df54cc84eaadb9a (MD5) Previous issue date: 2017-08-25 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Tomato (Solanum lycopersicum) cultures are frequently attacked by many pathogens, leading to yield losses. Among the pathogens, the fungus Alternaria solani (?Early blight? disease) can cause significant yield reductions and decreases the fruit quality. Due to the high susceptibility of tomato to fungal pathogens, the agricultural practices include the intensive use of agrochemicals, which may result in fruits with high levels of residual pesticides and higher production costs. The promotion of plant defense system represents an additional tool for disease control and plant growth management. Some rhizobacteria have been shown the ability to affect plant development and modulate plant defense against pathogens. Among those bacteria, the genus Streptomyces is able to synthesize molecules that directly interfere with growth, through auxin and ACC deaminase production, and indirectly, through antibiotics and siderophores synthesis. The aim of this work was to select Streptomyces (CLV04, CLV05 and CLV07) with the ability to promote seed germination and plant growth, as well as improve tomato plant resistance against A. solani. Results showed that CLV07 delayed the initial seed germination, but it did not impair the final germination process. However, CLV04, CLV05 and CLV07 reduced the early root and shoot growth. All these rhizobacteria produced high auxin levels. CLV07 was efficient for inducing plant resistance against A. solani. Furthermore, all CLVs showed antagonism by competition, decreasing the A. solani growth. Taking together all results, Streptomyces tested were deleterious for seed and initial plant growth, but CLV07 showed a potential to be used for inducing plant resistance. However, the inoculation of these rhizobacteria on seeds is not recommended. / O tomateiro (Solanum lycopersicum) ? constantemente atacado por diversos pat?genos, levando a perdas de produtividade. Dentre os principais pat?genos, destaca-se o fungo Alternaria solani que causa a doen?a ?pinta-preta? (?Early blight?), capaz de reduzir a produ??o e/ou levar ? forma??o de frutos de baixa qualidade. Devido ? grande susceptibilidade do tomateiro a pat?genos, as pr?ticas agr?colas incluem o uso intensivo de agroqu?micos que resultam, frequentemente, em tomates com altos n?veis residuais de pesticidas. A promo??o do crescimento e da defesa vegetal utilizando microrganismos representa uma ferramenta para o manejo do tomateiro. Dentre as diversas rizobact?rias com capacidade de modular o crescimento e a defesa vegetal, o g?nero Streptomyces vem se destacando. Estas bact?rias s?o capazes de sintetizar mol?culas que interferem tanto diretamente no crescimento, como a auxina e a ACC desaminase, quanto indiretamente, atrav?s da produ??o de antibi?ticos e sider?foros. Pretendeu-se, neste trabalho, selecionar isolados de Streptomyces (CLV04, CLV05 e CLV07) com capacidade de promover a germina??o e o crescimento de plantas de tomate, al?m de aumentar a resist?ncia vegetal contra A. solani. Os resultados demonstram que o CLV07 causou retardo na germina??o, mas n?o reduziu a taxa final de germina??o. Apesar das rizobact?rias CLV04, CLV05 e CLV07 produzirem elevados n?veis de auxina, elas causaram a redu??o do crescimento tanto da raiz quanto da parte a?rea de pl?ntulas de tomate cultivadas in vitro. O CLV07 foi eficiente na promo??o da defesa de plantas de tomate contra A. solani. Todas as rizobact?rias apresentaram antagonismo por competi??o, reduzindo o crescimento do mic?lio do fungo A. solani. Considerando todos os resultados, as diferentes bact?rias Streptomyces foram prejudiciais ? germina??o e ao crescimento inicial de pl?ntulas de tomate. Desta forma, n?o ? recomend?vel a utiliza??o destas bact?rias em sementes e pl?ntulas jovens. Contudo, o CLV07 apresenta potencial para ser utilizado para a indu??o de resist?ncia de plantas de tomate contra o pat?geno A. solani.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-03-15T13:05:33Z No. of bitstreams: 1 PATRICIA_FERNANDA_DASILVA_VILCHES_DIS.pdf: 1829625 bytes, checksum: d4b53df7fed69d5202d5bafec96aaa13 (MD5) / Approved for entry into archive by Tatiana Lopes (tatiana.lopes@pucrs.br) on 2018-03-23T12:17:26Z (GMT) No. of bitstreams: 1 PATRICIA_FERNANDA_DASILVA_VILCHES_DIS.pdf: 1829625 bytes, checksum: d4b53df7fed69d5202d5bafec96aaa13 (MD5) / Made available in DSpace on 2018-03-23T12:22:01Z (GMT). No. of bitstreams: 1 PATRICIA_FERNANDA_DASILVA_VILCHES_DIS.pdf: 1829625 bytes, checksum: d4b53df7fed69d5202d5bafec96aaa13 (MD5) Previous issue date: 2017-03-30 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Potatoes (Solanum tuberosum L.) are the third most consumed crop in the world, after rice and wheat. Among the diseases affecting potato, the blackleg and tuber soft rot, caused by phytobacteria Pectobacterium spp., lead to significant losses in the yield crop. Several studies have been exploring the use of plant defense inducers as a strategy to control plant diseases. The use of plant growth-promoting rhizobacteria (PGPR) in agriculture can lead to plant growth and enhancement of plant defense through the promotion of induced systemic resistance (ISR). However, the mechanisms involved in promoting ISR are still poorly understood. This study aimed to screen rhizobacteria of the genus Streptomyces with capacity to promote plant growth and induce Solanum tuberosum innate immunity. To achieve these objectives, we evaluated: i) the ability of Streptomyces isolates to produce auxin (3-indoleacetic acid), ACC deaminase (1-aminocyclopropane-1-carboxylic acid deaminase) and siderophores; ii) their capacity to promote the growth of potato plants; iii) the induction of resistance in potato plants challenged with Pectobacterium carotovorum subsp. brasiliensis and iv) the expression of genes related to defense pathways in S. tuberosum, promoted by Streptomyces and P. carotovorum. Results indicated that the CLV163 isolate presents PGPR features, such as high auxin and siderophores production, and promoted plant defense against P. carotovorum. Although CLV145 showed the highest auxin and siderophores production, it reduced the shoot dry mass and was inefficient in promoting plant defense. Moreover, the ability of Streptomyces in producing ACC deaminase was not critical for promoting plant growth. The CLV163 isolate induced a priming state in potato plants that has occurred through the activation of the AS and ET pathways, and its interaction with S. tuberosum plants did not impair the plant growth. / A batata (Solanum tuberosum L.) ? a terceira cultura agr?cola mais consumida no mundo, ficando atr?s somente do arroz e do trigo. Dentre as diversas doen?as que afetam esta cultura, a canela-preta e a podrid?o-mole, causadas pela fitobact?ria Pectobacterium spp., levam a importantes perdas na produ??o. Neste contexto, v?rios trabalhos v?m explorando o uso de indutores da defesa vegetal como estrat?gia para o controle de doen?as. O uso de rizobact?rias promotoras do crescimento vegetal (PGPR) na agricultura pode promover tanto o crescimento quanto o aumento da defesa das plantas atrav?s da indu??o da resist?ncia sist?mica induzida (ISR). Contudo, ainda s?o pouco conhecidos os mecanismos envolvidos na promo??o da ISR. Este estudo visou selecionar rizobact?rias do g?nero Streptomyces com caracter?sticas de promotoras de crescimento e indutoras da imunidade inata de Solanum tuberosum. Para tanto, foram avaliadas: i) a capacidade de isolados de Streptomyces produzirem auxina (?cido 3- indolac?tico), ACC desaminase (?cido 1-aminociclopropano-1-carbox?lico desaminase) e sider?foros; ii) a capacidade destas rizobact?rias em promover o crescimento das plantas; iii) a promo??o da resist?ncia em plantas de batata desafiadas com Pectobacterium carotovorum subsp. brasiliensis e iv) a express?o de genes relacionados a vias de sinaliza??o de respostas de defesa em S. tuberosum, promovida por Streptomyces sp e a fitobact?ria P. carotovorum. Os resultados indicam que o isolado CLV163 apresenta caracter?sticas de PGPR, sendo capaz de produzir auxina e sider?foros, al?m de promover o aumento da resist?ncia das plantas contra P. carotovorum. Contudo, a rizobact?ria CLV145, com a maior produ??o de auxina e sider?foros, causou a diminui??o da mat?ria seca de parte a?rea e n?o promoveu a defesa das plantas. A capacidade dos Streptomyces em produzirem ACC desaminase n?o foi determinante para a promo??o de crescimento vegetal. O isolado CLV163 induziu um estado de priming nas plantas de S. tuberosum L. atrav?s da ativa??o das vias do AS e ET e a sua intera??o com as plantas de batata n?o comprometeu o crescimento vegetal.

Dentre os mecanismos relacionados à interação bactéria-planta, a biossíntese bacteriana de ácido indol acético (AIA) exerce um papel fundamental na promoção do crescimento vegetal, uma vez que é capaz de influenciar inúmeros processos fisiológicos nas plantas. Diferentes vias metabólicas são utilizadas pelas bactérias para a biossíntese do AIA, sendo a via do ácido indol-3-pirúvico (IPyA) a mais comumente descrita. Nesta via encontra-se o gene indol-3-piruvato descarboxilase (ipdC) com vital função na produção de AIA utilizando como precursor o aminoácido L-triptofano. Nesse contexto, estudos moleculares acerca das vias metabólicas e dos genes envolvidos nesse processo são preponderantes para o entendimento da inter-relação das vias regulatórias com a síntese do fitormônio. A rizobactéria Bacillus sp. (RZ2MS9) vem apresentando satisfatória atividade na promoção de crescimento vegetal. O sequenciamento do seu genoma apontou a presença de uma vasta gama de genes relacionados à promoção do crescimento, com destaque para genes codificadores de auxinas. Assim, o estudo teve por objetivo comprovar a função do gene ipdC na biossíntese do AIA pela via dpendente do L-triptofano através do nocaute sítio dirigido do gene ipdC na Rizobactéria Promotora do Crescimento em Plantas (RPCP) Bacillus sp. (RZ2MS9). Para tanto, foi realizado o nocaute sítio dirigido por meio da técnica de CRISPR-Cas9. O nocaute do gene ipdC foi eficiente, gerando mutantes disruptivos para o referido gene. A biossíntese do AIA pela linhagem ΔipdC apresentou reduções nas concentrações do fitormônio, de acordo com o tempo de crescimento, sendo 87,96% em 24 horas, 88,25% em 48 horas e 58,27% em 72 horas do crescimento em comparação à linhagem selvagem (WT). Além disso, a biossíntese do AIA na ausência do aminoácido L-triptofano também foi avaliada, não sendo constatada síntese do fitormônio em nenhum dos tempos crescimento, tanto na linhagem selvagem, quanto na linhagem ΔipdC. O presente estudo foi pioneiro no nocaute do gene ipdC em uma linhagem de Bacillus utilizando a técnica de CRISPR-Cas9. Os resultados obtidos contribuem para um melhor entendimento da influência do gene ipdC e da via IPyA na biossíntese do AIA pela linhagem RZ2MS9 e futuramente sera comprovado seu papel na promoção de crescimento vegetal. / Among the mechanisms related to the bacterium-plant interaction, the bacterial biosynthesis of indole acetic acid (AIA) plays a fundamental role in the promotion of plant growth, since it is capable of influencing innumerable physiological processes in plants. Different metabolic pathways are used by bacteria for the biosynthesis of IAA, with the indole-3-pyruvic acid (IPyA) pathway being the most commonly described. In this pathway, the indole-3-pyruvate decarboxylase (ipdC) gene has a vital role in the production of IAA using the amino acid L-tryptophan as a precursor. In this context, molecular studies about the metabolic pathways and the genes involved in this process are preponderant for the understanding of the interrelationship of the regulatory pathways with the phytormonium synthesis. The rhizobacterium Bacillus sp. (RZ2MS9) has been showing satisfactory activity in promoting plant growth. The sequencing of its genome pointed to the presence of a wide range of genes related to growth promotion, especially genes encoding auxins. Thus, the objective of the present study was to verify the function of the ipdC gene in the IAA biosynthesis L-tryptophan dependent through the knockout of the ipdC in the plant growth-promoting rhizobateria (PGPR) Bacillus sp. (RZ2MS9). Therefore, the knockout was realized using the CRISPR-Cas9. The knockout of the ipdC gene was efficient, generating disruptive mutants for the said gene. IAA biosynthesis by the ΔipdC strain showed reductions in phytormonium concentrations, according to the growth time, being 87.96% in 24 hours, 88.25% in 48 hours and 58.27% in 72 hours of growth compared to the Wild Type (WT). In addition, the biosynthesis of IAA in the absence of the amino acid L-tryptophan was also evaluated, with no phytormonium synthesis being observed at any growth time, both in the wild type and ΔipdC strain. The present study pioneered the knockout of the ipdC gene in a Bacillus strain using the CRISPR-Cas9. The results obtained contribute to a better understanding of the influence of the ipdC gene and the IPyA pathway in the IAA biosynthesis through the RZ2MS9 strain, and its role in plant growth promoting will be demonstrated in the future.

Plant growth-promoting rhizobacteria (PGPR) are free-living, soil-borne bacteria that colonize the rhizosphere and, when applied to crops, enhance the growth of plants. Plant growth-promoting rhizobacteria may enhance plant growth either by direct or indirect mechanisms. The direct mechanisms of action include nitrogen fixation,production of phytohormones and lowering of ethylene concentrations. The objective of this study was to determine whether Pseudomonas putida strain 6-8 isolated from the rhizosphere of legume crops grown in Saskatchewan fields was able to promote the growth of canola cv. Smart and lentil cv. Milestone plants by direct mechanisms. Initial studies determined the effect of strain 6-8 and other known phytohormoneproducing PGPR strains on the growth of canola and lentil plants both in gnotobiotic and growth chamber conditions. Variations in the results were observed, as there were significant differences among trials. Strain 6-8 enhanced the growth of canola cv. Smart in growth pouches but not in pots in growth chamber studies. In the case of lentil cv.Milestone, strain 6-8 had no significant effect in growth pouches, but it significantly increased root dry weight, shoot dry weight and root surface area in pots in growth chamber studies. A similar effect was observed with wild-type strains GR12-2 and G20- 18. Strain GR12-2 was consistent in promoting the growth of lentil cv. Milestone both in growth pouches and in pots in growth chambers when compared to other strains and the control. The ability of the PGPR strains to produce auxin and cytokinin phytohomones in pure culture and in the canola rhizosphere was tested using the enzyme linked immunosorbent assay (ELISA). All the PGPR strains produced indole compounds and the concentration of the indoles produced increased with increasing concentrations of the precursor tryptophan. There were no significant differences among PGPR strains in production of indole-3-acetic acid (IAA) when assayed using ELISA. The concentrations of IAA secreted by PGPR strains were extremely low (0.19 µg/ml 9.80 µg/ml). Strain 6-8 produced the cytokinins, isopentenyl adenosine (IPA), zeatin riboside (ZR) and dihydroxyzeatin riboside (DHZR) in pure culture. Indole-3-acetic acid was detected in supernatants obtained from canola growth pouches inoculated with PGPR strains, but there were no significant differences in the concentrations of IAA secreted among PGPR strains. Significantly higher concentrations of IPA and ZR were observed in the rhizosphere of canola inoculated with strain 6-8 than in the non-inoculated control. Strain 6-8 produced siderophores, solubilized inorganic phosphate and used 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, as sole nitrogen source. These traits are considered to be alternative mechanisms for direct plant growth promotion. A qualitative and quantitative study of root colonization by strain 6-8 was conducted by tagging the strain with green fluorescent protein in conjunction with confocal laser scanning microscopy and by conventional plating. The populations of strain 6-8 were higher on canola roots than on lentil roots by conventional plating. Similar results were also observed in confocal laser scanning microscopy (CLSM) studies after 5, 7 and 9 days for canola and 3, 6 and 9 days for lentil. Pseudomonas putida strain 6-8 produced cytokinins and also possessed other direct growth promoting characteristics. The ability of strain 6-8 to promote the growth of canola cv. Smart in growth pouches and lentil cv. Milestone in growth chamber studies may be related to these direct growth promoting characteristics. Strain 6-8 may have potential for development as a plant growth-promoting rhizobacterial inoculant.

Plant growth-promoting rhizobacteria (PGPR) are free-living, soil-borne bacteria that colonize the rhizosphere and, when applied to crops, enhance the growth of plants. Plant growth-promoting rhizobacteria may enhance plant growth either by direct or indirect mechanisms. The direct mechanisms of action include nitrogen fixation,production of phytohormones and lowering of ethylene concentrations. The objective of this study was to determine whether Pseudomonas putida strain 6-8 isolated from the rhizosphere of legume crops grown in Saskatchewan fields was able to promote the growth of canola cv. Smart and lentil cv. Milestone plants by direct mechanisms. Initial studies determined the effect of strain 6-8 and other known phytohormoneproducing PGPR strains on the growth of canola and lentil plants both in gnotobiotic and growth chamber conditions. Variations in the results were observed, as there were significant differences among trials. Strain 6-8 enhanced the growth of canola cv. Smart in growth pouches but not in pots in growth chamber studies. In the case of lentil cv.Milestone, strain 6-8 had no significant effect in growth pouches, but it significantly increased root dry weight, shoot dry weight and root surface area in pots in growth chamber studies. A similar effect was observed with wild-type strains GR12-2 and G20- 18. Strain GR12-2 was consistent in promoting the growth of lentil cv. Milestone both in growth pouches and in pots in growth chambers when compared to other strains and the control. The ability of the PGPR strains to produce auxin and cytokinin phytohomones in pure culture and in the canola rhizosphere was tested using the enzyme linked immunosorbent assay (ELISA). All the PGPR strains produced indole compounds and the concentration of the indoles produced increased with increasing concentrations of the precursor tryptophan. There were no significant differences among PGPR strains in production of indole-3-acetic acid (IAA) when assayed using ELISA. The concentrations of IAA secreted by PGPR strains were extremely low (0.19 µg/ml 9.80 µg/ml). Strain 6-8 produced the cytokinins, isopentenyl adenosine (IPA), zeatin riboside (ZR) and dihydroxyzeatin riboside (DHZR) in pure culture. Indole-3-acetic acid was detected in supernatants obtained from canola growth pouches inoculated with PGPR strains, but there were no significant differences in the concentrations of IAA secreted among PGPR strains. Significantly higher concentrations of IPA and ZR were observed in the rhizosphere of canola inoculated with strain 6-8 than in the non-inoculated control. Strain 6-8 produced siderophores, solubilized inorganic phosphate and used 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, as sole nitrogen source. These traits are considered to be alternative mechanisms for direct plant growth promotion. A qualitative and quantitative study of root colonization by strain 6-8 was conducted by tagging the strain with green fluorescent protein in conjunction with confocal laser scanning microscopy and by conventional plating. The populations of strain 6-8 were higher on canola roots than on lentil roots by conventional plating. Similar results were also observed in confocal laser scanning microscopy (CLSM) studies after 5, 7 and 9 days for canola and 3, 6 and 9 days for lentil. Pseudomonas putida strain 6-8 produced cytokinins and also possessed other direct growth promoting characteristics. The ability of strain 6-8 to promote the growth of canola cv. Smart in growth pouches and lentil cv. Milestone in growth chamber studies may be related to these direct growth promoting characteristics. Strain 6-8 may have potential for development as a plant growth-promoting rhizobacterial inoculant.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-05-04T11:52:18Z No. of bitstreams: 1 texto completo.pdf: 497165 bytes, checksum: bc5281a82ca2c8d2550a885d7e7d6211 (MD5) / Made available in DSpace on 2017-05-04T11:52:18Z (GMT). No. of bitstreams: 1 texto completo.pdf: 497165 bytes, checksum: bc5281a82ca2c8d2550a885d7e7d6211 (MD5) Previous issue date: 2005-04-26 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Experimentos realizados por Silva (2002) objetivando o isolamento e seleção de rizobactérias indutoras de resistência sistêmica e agentes de biocontrele a múltiplos patógenos resultaram na obtenção de um isolado promissor, selecionado de rizosfera de plantas de tomateiro em cultivo comercial e identificado por análise de ácidos graxos como Bacillus cereus. Ensaios em condições de laboratório, casa de vegetação e campo foram realizados neste trabalho visando uma caracterização mais completa do isolado para uma melhor compreensão dos mecanismos de biocontrele envolvidos na interação rizobactéria-fitopatógenos. Testes bioquímicos e morfológicos confirmaram a identificação de espécie para o isolado e demonstraram sua versatilidade fisiológica e nutricional. Ainda, em ensaios laboratoriais, verificou-se a capacidade de utilização de certos antibióticos em estudos de dinâmica populacional de B. cereus e sua compatibilidade a pesticidas utilizados na cultura do tomateiro e a adjuvantes utilizados em formulações comerciais. Porém, estudos visando a avaliação da sobrevivência de células de B. cereus em uma formulação proposta veiculando o isolado não se mostraram eficientes na manutenção da viabilidade celular. Ensaios de antibiose direta contra diversos fitopatógenos demonstraram que o isolado não inibiu o crescimento da maioria dos isolamentos patogênicos testados. Em condições controladas, o isolado não promoveu o crescimento de plantas de tomateiro, e também não foi capaz de controlar Meloidogyne incognita. Porém foi capaz de aumentar a produtividade na cultura do tomateiro sob cultivo protegido e comprovar a capacidade de indução de resistência em plantas de tomateiro, quando desafiadas com Pseudomonas syringae pv. tomato. Em condições de campo, B. cereus não conseguiu controlar a epidemia de Phythophtora infestans, mesmo quando diferentes formas de dispensa da rizobactéria foram testadas, e não foi capaz de promover o crescimento de plantas de tomateiro. Em experimento realizado em infectario observou- se uma menor suscetibilidade a murcha bacteriana (Ralstonia solanacearum), permitindo que plantas de tomateiro advindas de sementes microbiolizadas com B. cereus apresentassem uma menor incidência da doença por um período de 30 dias, quando a severidade da doença igualou- se nos dois tratamentos, demonstrando que o isolado não foi efetivo em controlar a epidemia. Estudos Visando a manipulação e otimização de fatores relacionados as habilidades fisiológicas e bioquímicas do isolado fazem-se necessários para o conhecimento e exploraoção máxima de suas potencialidades, como futuros trabalhos de formulação, possibilitando a obtenção de um produto passível de utilização na produção de culturas de alto valor comercial, minimizando o uso de agrotóxicos e favorecendo a prática de uma agricultura ecologicamente correta. / Experiments carried out by Silva (2002) to isolate and select rhizobacteria inducer of systemic resistance and biological control agents against multiple pathogens resulted in the obtention of a promising isolate. It was selected in the rhizosphere of tomato plants in commercial cultivation and identified by analysis of fatty acids as Bacillus cereus. In this work, to get to a more complete characterization of the isolate, essays in laboratory, greenhouse and field conditions were carried out, for a better understanding of the biological control mechanisms involved in the rhizobacteria- phytopathogen interaction. Biochemical and morphological tests confirmed the identification of the species in the isolate, as well as demonstrated its physiologic diversity and nutritional versatility. Moreover, in laboratorial essays, the viability of using determined antibiotics to carry out studies of populational dynamics of the isolate was verified, as well as the compatibility of B. cereus to pesticides used on the tomato cultivation and adjuvants used in commercial formulations. But studies to evaluate the survival of B. cereus in a formulation containing the isolate did not prove to be efficient to keep the viability of the cells. Essays of direct antibiosis against various phytopathogens showed that the isolate was not efficient to inhibit the growth of the majority of pathogenic isolates tested. Under controlled conditions, the isolate did not promote the growth of tomato plants, and also did not have the ability to control Meloidogyne incognita. But it showed the ability to increase the productivity of the crop in sheltered cultivation, and to prove the induction of resistance in tomato plants, when they were challenged with Pseudomonas syringae pv. tomato. Under field conditions, B. cereus was unable to control an epidemy of Phythophtora infestans, even when various application forms of the rhizobacteria were tested, and it also did not promote the growth of tomato plants. In an eXperiment carried out in an “infectario” (infested microplot), a less susceptibility to bacterial wilt (Ralstonia solanacearum) was observed, allowing the tomato plants to resist with less incidence of the disease for a period of 30 days in plants grown from microbiolized seeds, although B. cereus was not effective to control the epidemy. Studies aiming at handling and optimizing factors related to the physiological and biochemical abilities of the isolate are needed for the understanding and maXimum exploitation of its potentialities. For the biological control of diseases in the tomato cultivation, besides further formulation works, there is the probability of obtaining a product that could be used in the production of crops with high commercial value and quality, and that can keep the effectivity of action of this microorganism in the biological control as well as to minimize the use of pesticides encourage the practice of an agriculture ecologically correct. / Tese importada do Alexandria

Studies on plant growth-promoting rhizobacteria (PGPR) and fungi (PGPF) as biostimulants have shown significant positive effects on plant health, fruit yield, or pest management. However, very few published studies to date have been specific regarding their effects on strawberries (Fragaria × ananassa), particularly on soilborne disease prevalence in organically grown strawberries. Empirical data on the results of using these products in commercial growing applications under various conditions would be highly valuable, especially for organic growers who have limited synthetic chemical pesticides, herbicides and fertilizers registered for use. The objective of this study is to evaluate the efficacy of biostimulant supplementation on strawberries for improving fruit yield, fruit quality, and plant health in both high-tunnel, open-sided ‘hoophouse’ and field conditions. This study consisted of two research projects. The first project investigated the effects of commercially available PGPR-based biostimulant products on strawberry plant health. The three products contained differing proprietary combinations of PGPR, primarily from the Bacillus and Lactobacillus genera. Plants were grown in two different soil types: sandy and clay, in order to investigate the effects of biostimulant supplementation in different soil conditions. In fall of 2018, 160 ‘Monterey’ strawberry plants were grown in an outdoor hoophouse in 3-gallon pots. Plants were either treated monthly with a single bacterial biostimulant product (EM-1, Accomplish LM, or Armory), or left untreated as a control. Plants were grouped into 20 blocks, each block comprised of 8 plants (each of the four treatments replicated in both soil types). Fruit yield (g), fruit sugar content (Brix), and leaf SPAD absorbance levels were measured weekly from January 27 to June 26, 2019. The treatments tested had no significant effects on fruit yield, leaf SPAD absorbance or Brix; soil type, however, did significantly impact fruit yield, with higher yields in sandy soil. The second project was a field trial beginning in spring of 2020, in collaboration with Rutiz Farms in Arroyo Grande, CA, involving a total of 480 ‘Chandler’ strawberry plants. The farm is organically managed and has a history of soilborne diseases, including Verticillium dahliae. These plants were either treated monthly with one of three microbial biostimulant products: a product containing a proprietary strain of Trichoderma harzianum biocontrol fungus (TrichoSym), and two of the same PGPR-based products used the previous year (Accomplish LM and Armory); or left untreated as a control. The experiment was laid out in a randomized complete block design with four blocks, with each block consisting of 4 plots for each of the 4 treatments; each plot contained 30 plants. Fruit yield (g) per plot was measured weekly throughout the 2020 growing season and phenotypic disease incidence was measured biweekly. Soil samples were taken at three different points throughout the season, cultured on selective media, and analyzed to obtain estimates of V. dahliae colony-forming units (CFU) per gram soil. The treatments tested had no significant effect on fruit yield, phenotypic disease incidence, or V. dahliae CFU/g soil. The results are inconclusive as to whether this lack of effect is due to viability of the products themselves, ineffective application techniques resulting in lack of rhizosphere colonization, or some combination of these. Further research is needed to determine whether or not supplementation with microbial biostimulants can produce reliable, beneficial results in strawberries.

La rhizosphère est le siège de multiples interactions biotiques, contrôlées au moins en partie par l’échange de molécules signal entre les racines des plantes et les microorganismes qui leur sont associés. Dans le contexte des symbioses associatives entre les PGPR (Plant-Growth Promoting Rhizobacteria) et les plantes, le rôle et l’importance de ces molécules restent encore largement méconnus. Ainsi, l’influence de différents signaux rhizosphériques sur l’expression génique et les propriétés phytobénéfiques des PGPR a été étudiée par, (i) la caractérisation chimique d’une nouvelle molécule phytobénéfique d’Azospirillum et de son impact sur l’architecture du système racinaire, (ii) une approche ciblée portant sur l’analyse de l’expression in situ du gène phytobénéfique acdS de PGPR, et (iii) une approche globale permettant d’identifier la réponse du transcriptome d’Azospirillum à un métabolite secondaire bactérien. Ces travaux ont permis d’identifier des signaux rhizosphériques impliqués dans le contrôle de la colonisation racinaire et/ou des propriétés de phytostimulation des PGPR sur leur plante hôte / In rhizosphere, numerous biotic interactions are controlled by molecular signals exchanged between plant roots and microorganisms. In the case of the associative symbiosis between PGPR (Plant-Growth Promoting Rhizobacteria) and plants, the role and importance of these molecules are largely unknown. The impact of several rhizospheric signals on PGPR gene expression and plant beneficial properties was thus studied by performing (i) the chemical characterization of a new Azospirillum plant-stimulatory molecule whose positive impact on root development was revealed, (ii) an approach targeting the in situ expression of the acdS plant beneficial gene in PGPR, and (iii) a global approach to identify the Azospirillum transcriptomic response to a bacterial secondary metabolite. This works allowed the identification of rhizospheric signals controlling PGPR root colonization and/or plant growth promotion

As rizobactérias promotoras de crescimento (PGPR) são microrganismos que ocorrem naturalmente no solo, são conhecidas por proporcionar melhorias no desenvolvimento das plantas atuando de diversas formas. Bacillus subtilis (cepa GB03) é uma PGPR disponível para comercialização como fungicida biológico concentrado, utilizada no tratamento de sementes de diversas culturas como algodão, soja, amendoim, trigo e cevada. Destaca-se pela capacidade de promover o crescimento de plantas por meio da emissão de voláteis. Vários estudos com Arabidopsis thaliana já comprovaram que B. subtilis (GB03) é capaz de auxiliar no desenvolvimento da planta por meio da promoção de crescimento e pela supressão de patógenos habitantes de solo. No entanto, o seu papel na proteção de plantas contra a herbivoria de insetos ainda não é bem caracterizado. Deste modo, buscou-se avaliar os efeitos da interação de B. subtilis (GB03) com plantas de rúcula (Eruca sativa) e Plutella xylostella (traça-das-crucíferas). Este inseto pertence à ordem Lepidoptera, considerado praga de maior importância no cultivo de Brassicaceae ao redor do mundo. Devido à sua alta prolificidade e capacidade de adaptação e ao seu curto ciclo de vida, tornou-se uma das pragas mais resistentes e de difícil controle da agricultura. Atualmente, os custos em escala mundial com o controle da praga anualmente chegam em torno de US $ 4 a 5 bilhões. Foram utilizados dois tratamentos, plantas inoculadas com B. subtilis (GB03) e controle (plantas não inoculadas). Avaliou-se o crescimento de plantas de rúcula e o peso seco de parte aérea. Para avaliar o desempenho e dano de P. xylostella em ambos tratamentos, previamente foram pesados grupos de quinze lagartas e submetidas a alimentação de plantas de rúcula durante 24 horas. Posteriormente, as lagartas foram retiradas e pesadas novamente e a área foliar consumida foi calculada por meio do software editor de imagens ImageJ®. A preferência de oviposição do inseto foi testada por meio de olfatometria, composta apenas de pistas olfativas e em arenas contendo tanto pistas olfativas quanto visuais. A emissão de voláteis foi caracterizada quantitativamente e qualitativamente por cromatografia gasosa e analisada por espectrometria de massas. A inoculação com GB03 em plantas de rúcula promoveu melhor crescimento das plantas em relação ao tratamento controle, ao mesmo tempo em que diminuiu os danos pelo consumo alimentar do inseto na planta. P. xylostella não apresentou distinção entre os odores das plantas nos testes de olfatometria. Entretanto, observou-se menor número de ovos em plantas com GB03 nos bioensaios de arena. Não foram constatadas diferenças significativas na emissão total de voláteis entre os tratamentos com e sem GB03, no entanto, foram encontradas concentrações diferentes dos compostos (Z)-3-hexenol e 2-ethyl-1-decanol. Outros testes devem ser realizados com a finalidade de estabelecer o papel desempenhado por GB03 na indução de defesas de plantas contra insetos. / The plant growth promoting rhizobateria (PGPR) are microorganisms that naturaly live in the soil, known by improving the plants\' development in many ways. Bacillus subtilis (strain GB03) is a comercial available PGPR, sold as a concentrated biological fungicide, applied in seed treatment of different cultures as cotton, soybean, peanut, wheat and barley. Moreover, it stands out by its capacity of plant growth promoting via volatiles emission. Several studies with Arabidopsis thaliana showed that B. subtilis (GB03) can help the plant development via growth promotion and by soil pathogens supression. However, its role in plant protection against insect herbivory has not been characterized yet. Thus, it aimed to evaluate the interaction effects among B. subtilis (GB03), arugula plants (Eruca sativa) and Plutella xylostella (Diamondback moth). This insect belongs to the order Lepidoptera and have been considered the main pest in Brassicaceae fields around the world. Due to its high prolificacy and plasticity in field survival, and its short life cycle, it has become one of the most resistant and hard control pest in agriculture. Currently, the annualy world costs with this pest control is about US $ 4-5 bilions. Here, it was used two treatments, innoculated plants with B. subtilis (GB03) and control (non-innoculated plants). The arugula plants growth and dry mass of shoots were evaluated. To analyze the performance and damage by P. xylostella in both treatments, it was previously weighed groups with fifteen caterpillars and submitted to feeding on arugula plants during 24 hours. After that, the caterpillars were removed and weighed again and the consumed leaf area was calculated by the image editor software ImageJ®. The insect oviposition preference was tested by olfactometry, with only olfactory cues and in arenas containing both, olfactory and visual cues. Volatiles emission was quantitatively and qualitatively characterized by gas chromatography and analyzed by mass spectrometry. GB03 innoculation in arugula plants promoted a better growth when compared to control, and, at the same time, there was an increasing in the plant damage by insect food consumption. P. xylostella did not show distinction between odors of the plants in olfactometry tests. Although, it was observed less number of eggs in plants with GB03 in arena bioassays. It was not found significant differences in total volatile emission between the treatments with and without GB03, even though different concentrations of (Z)-3-hexenol and 2-ethyl-1-decanol were observed. Other tests must be performed in order to estabilish the role played by GB03 in plant induction defense against insects.