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Previous issue date: 2017-03-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The plant growth promoting rhizobacteria (PGPR) can increase agricultural productivity by promoting growth through production of plant hormones, facilitating the uptake of nutrients and chemicals on the soil, as well as inhibiting plant stress factors. Streptomyces spp. (Stm) are bacteria with great biotechnological potential, because in addition to its growth promotion and plant defense induction, they are also known as great producers of secondary metabolites, including antibiotics and phenazines. Soybean [Glycine max (L) Merrill] is one of the main legume crop grown around the world and Brazil is the second largest producer. Its production is affected by many diseases, among which bacterial pustule caused by the pathogen Xanthomonas axonopodis pv. glycines (Xag). The objective of this project was to evaluate isolates of Streptomyces spp. obtained from the rhizosphere of Fabaceae plants regarding characteristics of PGPR, as well as the modulation capacity of soybean defenses in response to the phytopathogen Xag. Eleven isolates of Streptomyces spp. were screened for PGPR traits by siderophores production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, indole-3-acetic acid (IAA) and phenazines. For a taxonomic identification and growth evaluation of soybean plants, three isolates were selected for their biochemical characteristics. The growth promoting assay was performed in greenhouse using bacterized seeds with the selected isolate and sterile distilled water was use for the control. Length, fresh and dry weight from shoot and root at 15, 30 and 45 days of cultivation were the evaluated parameters. For evaluation of the induction capacity of defense mechanisms of soybean plants, the isolate that obtained the best performance in the growth promotion test was selected. Seeds of soybeans, from sensitive cultivars and resistant to Xag, were bacterized with the selected Stm isolate and grown under greenhouse conditions. The plants were challenged with Xag 15 days after emergence. The treatments consisted of (a) plants treated with sterile distilled water (absolute control); (b) plants bacterized with Stm CLV45 (Stm45); (c) water-treated and Xag challenged plants; and (d) plants bacterized with StmCLV45 and challenged with Xag (Stm45+Xag). The enzymatic responses related to the defense pathways were evaluated biochemically, by analyzing the activity of phenylalanine ammonia lyase (PAL) and by the production of phenolic compounds at times 0, 24, 48, 72 and 144 hours post infection (hpi) of Xag. The expression of the genes related to the defense in Xag challenged soybean plants was determined by the relative expression of the genes PAL, JAZ, ERF5 and PR1 by qPCR at times 0, 12, 24 and 48 hpi. The results of the biochemical analysis indicated the isolates CLV42, CLV44 and CLV46 as the major producers of siderophores and CLV41, CLV45 and CLV46 isolates with higher ACC deaminase activity. All isolates were able to produce IAA, highlighting the isolate CLV45, which produced 398.53 ?g AIA g-1 cell. Phenazine pyocyanin (PYO) was also detected in all isolates, but the same did not occur for the 1-carboxylic acid phenazine (PCA), only produced by CLV41, CLV43 and CLV45. The isolates CLV42, CLV44 and CLV45 were selected for their PGPR characteristics for the growth promotion trial of greenhouse soybean plants and taxonomically characterized as species of the genus Streptomyces. None of the isolates evaluated in the trial caused a growth deficit in soybean plants. The CLV45 isolate significantly promoted the growth of soybean shoots in 36.63%, corroborated by the highest dry mass, 17.97%, in relation to the control group, being selected for soybean defense pathways induction. Expression of PAL gene was moderately enhanced in susceptible Stm45+Xag plants at 12 hpi, followed by increase of PAL enzyme activity from 48 to 144 hpi, although corresponding accumulation of phenolic compounds was not recorded. In the resistant cultivar, the highlighted expression of PAL in Stm45+Xag plants resulted in high activity of this enzyme. Enhanced expression of ERF5 and decrease on JAZ gene at 12 hpi in Stm45+Xag plants from both cultivars suggested that ET and JA play a concert role on induced systemic defense by Streptomyces sp. CLV45 against Xag in soybean. / As rizobact?rias promotoras de crescimento de plantas (PGPR) podem aumentar a produtividade agr?cola, atuando atrav?s da promo??o de crescimento vegetal por meio de fitorm?nios reguladores de crescimento, facilitando a capta??o de nutrientes e de compostos qu?micos no solo, bem como inibindo fatores de estresse vegetal. Bact?rias do g?nero Streptomyces spp. (Stm) apresentam grande potencial biotecnol?gico, pois al?m de promoverem o crescimento e a indu??o de defesa vegetal, tamb?m s?o conhecidas pela grande produ??o de metab?litos secund?rios, incluindo antibi?ticos e fenazinas. A soja [Glycine max (L.) Merrill] ? uma das principais leguminosas cultivadas no mundo, sendo o Brasil o segundo maior produtor. Sua produ??o ? afetada por in?meras doen?as, como a P?stula bacteriana causada pelo fitopat?geno Xanthomonas axonopodis pv. glycines (Xag). O objetivo deste trabalho foi avaliar 11 isolados de Streptomyces spp. oriundos da rizosfera de plantas de Fabaceae quanto ?s caracter?sticas de PGPR, bem como, ? capacidade de modula??o das vias de defesa de plantas de soja em resposta ? fitobact?ria patog?nica Xag. Os isolados foram avaliados quanto ?s caracter?sticas de PGPR pela produ??o de sider?foros, de ?cido indolac?tico (AIA), da enzima 1-aminociclopropano-1-?cido carbox?lico (ACC) desaminase e de fenazinas. Para a identifica??o taxon?mica e a avalia??o da promo??o de crescimento de plantas de soja foram selecionados tr?s isolados com caracter?sticas de PGPR. O ensaio de promo??o de crescimento ocorreu em casa de vegeta??o por meio da microbioliza??o das sementes pelos isolados selecionados e o controle com ?gua destilada est?ril. Os par?metros avaliados foram: comprimento, massa fresca e seca, de parte a?rea e raiz, aos 15, 30 e 45 dias de cultivo. Para a avalia??o da capacidade de indu??o dos mecanismos de defesa de plantas de soja, foi selecionado o isolado que obteve o melhor desempenho no ensaio de promo??o de crescimento. Sementes de soja, de cultivar sens?vel e resistente ? Xag, foram microbiolizadas com o isolado de Stm selecionado e cultivadas em casa de vegeta??o. As plantas obtidas foram desafiadas com Xag, 15 dias ap?s a sua emerg?ncia. Os tratamentos consistiram de (a) sementes tratadas com ?gua destilada est?ril (controle absoluto); (b) sementes microbiolizadas com StmCLV45 (Stm45); (c) sementes tratadas com ?gua destilada est?ril e plantas desafiadas com Xag (Xag); e (d) sementes microbiolizadas StmCLV45 e plantas desafiadas com Xag (Stm45+Xag). As respostas enzim?ticas relacionadas ?s vias de defesa foram avaliadas bioquimicamente, pela an?lise da atividade da fenilalanina am?nia liase (PAL) e pela produ??o dos compostos fen?licos, nos tempos 0, 24, 48, 72 e 144 horas p?s inocula??o (hpi) da Xag. A express?o dos genes relacionados ? defesa das plantas de soja desafiadas com Xag foi determinada pela express?o relativa de JAZ, ERF5, PAL e PR1 por qPCR, nos tempos 0, 12, 24 e 48 hpi. Os resultados da an?lise bioqu?mica indicaram os isolados CLV42, CLV44 e CLV46 como maiores produtores de sider?foros e os isolados CLV41, CLV45 e CLV46 com maior atividade de ACC desaminase. Todos os isolados foram capazes de produzir AIA, com destaque para o isolado CLV45, que produziu 398,53 ?g AIA g-1 de c?lulas. A fenazina piocianina (PYO) tamb?m foi detectada em todos os isolados, entretanto o mesmo n?o ocorreu para a fenazina 1-?cido carbox?lico (PCA), somente produzida por CLV41, CLV43 e CLV45. Os isolados CLV42, CLV44 e CLV45 foram selecionados por suas caracter?sticas de PGPR para o ensaio de promo??o do crescimento de plantas de soja em casa de vegeta??o e caracterizados taxonomicamente como esp?cies do g?nero Streptomyces. Nenhum dos isolados avaliados no ensaio causou d?ficit de crescimento em plantas de soja. O isolado CLV45 promoveu significativamente o crescimento de parte a?rea de plantas soja, em 36,63%, corroborado pela maior massa seca, 17,97%, em rela??o ao grupo controle, sendo selecionado para avalia??o nas vias de defesa da soja. A express?o do gene PAL foi moderadamente aumentada em plantas suscet?veis Stm45+Xag em 12 hpi, seguido por aumento da atividade da enzima PAL de 48 a 144 hpi, embora o ac?mulo correspondente de compostos fen?licos n?o tenha sido registrado. Na cultivar resistente, a express?o de PAL em plantas Stm45+Xag resultou em alta atividade desta enzima. A express?o aumentada de ERF5 e a diminui??o de express?o do gene JAZ em 12 hpi em plantas Stm45+Xag de ambas as cultivares sugeriram que etileno e ?cido jasm?nico desempenharam fun??o na defesa sist?mica induzida por Streptomyces sp. CLV45 contra Xag em plantas de soja.
| Identifer | oai:union.ndltd.org:IBICT/oai:tede2.pucrs.br:tede/7467 |
| Date | 31 March 2017 |
| Creators | Horstmann, Juliana Lopes |
| Contributors | Santar?m, Eliane Romanato |
| Publisher | Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Programa de P?s-Gradua??o em Biologia Celular e Molecular, PUCRS, Brasil, Faculdade de Bioci?ncias |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da PUC_RS, instname:Pontifícia Universidade Católica do Rio Grande do Sul, instacron:PUC_RS |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 8198246930096637360, 600, 600, 600, 600, 36528317262667714, -1634559385931244697, 2075167498588264571 |
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