Thèse doctorat : Microbiologie : Villeurbanne, INSA : 2007. / Titre provenant de l'écran-titre. Bibliogr. p. 181-199.

Les bactéries pectinolytiques appartenant aux genres Pectobacterium et Dickeya spp. sont des agents pathogènes chez Solanum tuberosum. Ces bactéries sont responsables de la maladie de la jambe noire et de la pourriture molle lors de la culture et du stockage des tubercules. Ce travail de thèse est divisé en deux axes: 1) Etude dela diversité d'une population du pathogène D. solani par approche de génomique comparée afin de mieux comprendre la structure génomique de cette espèce émergente. 2) L'assemblage du génome et la caractérisation génomique des facteurs de virulence chez Pectobacterium wasabiae RNS 08421A.L'analyse des génomes de 20 isolats de D. solani issus d’environnements différents, par une approche de génomique comparative associée à des analyses fonctionnelles, a révélé une forte homogénéité génétique au sein de la majorité des souches (16/20). De plus, cette analyse a permis de caractériser un nouveau sous-groupe au sein de l'espèce D. solani, représenté par la souche 0512 (1/20). En revanche, d’autres isolats (3/20) montrent des variations de quelques centaines à quelques milliers de SNPs/InDels qui sont regroupés dans des îlots génomiques. Leur analyse phylogénétique révèle qu’ils proviennent d’autres pathogènes par transferts horizontaux. Par ailleurs, l’analyse des fonctions affectées par les SNPs/InDels a permis de prédire, puis de vérifier sur pomme de terre, qu’un des isolats était faiblement virulent.La deuxième partie de mon travail porte sur l'assemblage, la caractérisation et l'analyse du génome de la souche RNS 08.42.1A de P. wasabiae, qui a été isolée en France. La génomique comparative avec 3 autres souches de P. wasabiae d'origines géographiques différentes, a révélé à la fois une forte similitude au niveau de la séquence génomique (ANI> 99%) et une synténie conservée des gènes de virulence. En outre, notre analyse a mis en évidence une nette distinction entre ces quatre souches de P. wasabiae (isolées de S. tuberosum) et la souche type japonaise P. wasabiae CFBP 3304T (isolée du raifort). Dans P. wasabiae RNS 08.42.1A, les gènes de synthèse et de perception du quorum sensing, expI/expR, présentent une plus forte homologie avec leurs orthologues chez P. atrosepticum et P. carotovurm (90%) qu'avec leurs homologues chez P. wasabiae (70%). Ceci suggère une acquisition de ces gènes par transfert horizontal au sein d'une population de pathogènes infectant la même plante hôte. / The pectolytic bacteria Pectobacterium and Dickeya species cause important diseases on Solanum tuberosum and other arable and horticultural crops. These bacteria are responsible for blackleg in the field and tuber soft rots in storage and in transit as well as in the field worldwide. The main objectives of this thesis are: 1) To study the diversity of a D. solani population using comparative genomics approaches in order to understand the genomic structure and evolution of this emerging species. 2) Characterization and genomic analysis of virulence factors in Pectobacterium wasabiae RNS 08421A.Using comparative genomics approach combined with functional assays, the analysis of the genomes of 20 isolates of D. solani from different environments, revealed a strong genetic homogeneity within the majority of strains (16/20). Moreover, this analysis allowed to characterize a new subgroup within D. solani species, represented by the 0512 strain (1/20). In contrast, some strains (3/20) showed variations from hundreds to a few thousand of SNPs/Indels which are grouped in genomic islands (GIs). Phylogenetic analysis of these GIs shows that they were acquired from other pathogens by horizontal transfer. Furthermore, the analysis of the functions affected by SNPs/Indels allowed to predict, then check on potatoes, that one of these strain was less virulent.The second part of my work involves assembly, characterization and analysis of the genome of the strain P. wasabiae RNS 08.42.1A, which was isolated in France. Comparative genomics with three other P. wasabiae strains from different geographical origins, revealed a strong similarity in the genome sequence (ANI> 99%) and conserved synteny of virulence genes. In addition, our analysis showed a clear distinction between the strains of P. wasabiae isolated from S. tuberosum and the type strain P. wasabiae CFBP 3304T (isolated from horseradish). In P. wasabiae RNS 08.42.1A, the complex system for synthesis and perception of quorum sensing signal expI/expR, exhibit higher homology with their orthologs in P. atrosepticum and P. carotovurm (90%) than their homologs in P. wasabiae (70%). This suggests acquisition of these genes by horizontal gene transfer within a population of pathogens infecting the same host plant.

A multitude of microorganisms possess the ability to metabolise molecular hydrogen (H2). The major enzyme family involved in hydrogen metabolism are Hydrogenases. These enzymes catalyse the reversible conversion of molecular hydrogen to protons and electrons (H2 ↔ 2H+ + 2e-). These enzymes have the potential to be utilised for biotechnological applications such as hydrogen fuel cells, but they also represent promising drug targets for inhibition of bacterial energy metabolism both within the gastrointestinal tract and after infection. Therefore, further understanding and discoveries made in the hydrogenase field warrants progression into applied medical and biotechnological research areas. Hydrogenases are also interesting due to their phylogeny and physiology in a large number of microbial species. These enzymes are categorised by their active site architecture. One well studied, ancient group is termed the [NiFe]-hydrogenases, which all harbour a complex NiFe(CN-)2CO active site in the 'large' catalytic subunit and usually have three iron-sulfur clusters within a 'small' electron transferring partner subunit. [NiFe]-hydrogenases have undergone massive diversification, with four major phylogenetic subgroups arising. The major part of this Thesis concerns work on a Group 4 [NiFe]-hydrogenase that functions in partnership with a formate dehydrogenase as a formate hydrogenlyase (FHL). This FHL complex generates H2 and CO2 from the disproportionation of formate (CHOO- + H+ ↔ H2 + CO2). In this Thesis, genetic and biochemical characterisation of Pectobacterium atrosepticum SCRI1043, a potato pathogen, led to the identification of a novel FHL complex. The [NiFe]-hydrogenase in this organism is similar to that of Escherichia coli Hydrogenase-4, with an extended membrane domain similar to that of respiratory Complex I. Importantly, the P. atrosepticum formate dehydrogenase is selenium-free, while previously characterised FHL complexes have selenocysteine-containing formate dehydrogenases. Using genetic and biochemical approaches it was shown that the [NiFe]-hydrogenase and a formate dehydrogenase were vital for H2 production by P. atrosepticum. Using plant infection assays it was also shown that the gene encoding the formate dehydrogenase was important for full infective ability of P. atrosepticum in potato plants and tubers. The latter part of this Thesis focuses on developing genetic tools to study this novel FHL from P. atrosepticum as well as Hydrogenase-1 and -2 from E. coli.

Pectobacterium carotovorum ssp. carotovorum (Pcc) is implicated in the destruction of ornamental plants in greenhouse recirculating systems. PCR-based detection and quantification of Pcc requires expensive instrumentation and knowledgeable users. This thesis describes the production of polyclonal antibodies and a single-domain antibody fragment (VHH) against Pcc lipopolysaccharide (LPS), and the development of user- friendly diagnostic assays for detection and quantification of the pathogen. Polyclonal ELISAs against heat-killed (HK) Pcc (limit of detection (LOD) = 81 CFU/ml; limit of quantitation (LOQ) = 216 CFU/ml) and Pcc LPS (LOD = 23 ng/ml; LOQ = 76 ng/ml) were developed. A preliminary user-friendly dipstick assay was also developed (≥ 105 CFU/ml). A phage display library was constructed (6.0 x 105 clones/ml), yielding one unique anti-Pcc LPS VHH. Using the Pcc LPS-specific VHH to produce affordable, user- friendly diagnostic assays is feasible since antibody fragments can be produced on a large scale through expression in Escherichia coli or Piccia pastoris. / Flowers Canada, CANADA-ONTARIO RESEARCH AND DEVELOPMENT (CORD) PROGRAM, Canada Research Chairs (CRC) Program, NSERC/NRC

Les bactéries Pectobacterium sont classées parmi les agents pathogènes les plus importants économiquement pour la culture de la pomme de terre. Au cours des dernières années, une augmentation des maladies dues à ces bactéries a pu être observée. Le but de ce travail de thèse était d’analyser certains aspects de la diversité liés aux Pectobacterium sp à savoir : (i) la diversité génétique liée aux régulateurs du pouvoir pathogène de la bactérie (ii) la diversité de virulence et d’agressivité des souches de Pectobacterium vis-à-vis de leurs hôtes et (iii). Le rôle et la diversité des effets induits par le lipopolysaccharides (LPS), composants de la surface bactérienne de bactéries phytopathogène ou non phytopathogène. Ce travail de thèse souligne également le rôle potentiel que pourrait jouer ces molécules LPS dans le biocontrôledes Pectobacterium sp. Différentes expérimentations cellulaires et moléculaires allant de l’identification de la bactérie à la compréhension des voies de signalisation ont été utilisées. Les résultats obtenus nous ont permis de montré, en premier lieu que, le séquençage du gène pmrA, gène connu pour être impliqué dans la régulation du pouvoir pathogène desPectobacterium, est un outil moléculaire complémentaire d’identification de sous espèces de Pcc et pourrait être aussi un moyen efficace d’évaluation de la diversité génétique intraspécifique. Dans un second temps, nous avons montré que les cultures cellulaires d’Arabidopsis thaliana pourraient être un modèle végétal d’évaluation de l’agressivité des Pectobacterium. Ceci a été obtenu par quantification des cinétiques de trois paramètres associés à la pathogénie de ces bactéries à savoir : l’activité des pectate-lyases, déterminant majeur du pouvoir pathogène des Pcc, la fuite des électrolytes, considérée comme un marqueur précoce de la mort cellulaire, et la mort cellulaire des cultures elle-même. Enfin nous avons également montré que les effets induits par les LPS chez les cellules d’Arabidopsis thaliana sont dépendant du type bactérien. En effet Les résultats obtenus nous ont permis de mettre en évidence trois types de réponses différentes aux LPS en fonction de leur origine: les réponses identiques (régulation des flux d’ions), des réponses communes mais présentant des intensités et de cinétiques différentes (production de ROS, induction de gènes de défense) et des réponses spécifiques (induction d’une PCD, alcalinisation du milieu). Ces résultats indiquent que différentes voies de signalisation pourraient être activées chez Arabidopsis thaliana. Ils nous ont permis également de mieux comprendre l’implication des LPS dans le biocontrôle contre les Pectobacterium sp. / Pectobacterium are classified among the most economically important pathogens of culture of potato. Recently, an increase in diseases caused by these bacteria was observed. The work presented in this thesis has allowed highlighting some aspects on diversity associated with Pectobacterium sp namely: (i) genetic diversity related with the pathogenicity of thebacterium (ii) the diversity in virulence and aggressiveness of Pectobacterium strains on its hosts and (iii) the role and the diversity of effects induced by lipopolysaccharide (LPS), bacterial surface components of phytopathogenic and non- phytopathogenic bacteria. First, we have demonstrated that sequencing the pmrA gene, known to be involved in the regulation of pathogenicity of Pectobacterium, is an additional molecular tool for identification of Pectobacterium subspecies. Moreover, pmrA gene could be an effective tool for evaluation of genetic diversity within species. In a second time, we have showed that cell cultures of Arabidopsis thaliana, could be used as an alternative system to evaluate rapidly and efficiently the virulence of different Pectobacterium strains. This was achieved by quantification of different parameters associated to the pathogenesis of these bacteria namely, the activity of pectate lyases, major determinant of the pathogenicity of Pcc, the electrolyte leakage, considered an early marker of cell death and the cell death itself. Finally, our data further suggest the effects induced by LPS from different origin on Arabidopsis thaliana cells, could be different. Indeed, three different types of responses to LPS have been shown: the identical responses (regulation of ion flux), the common responses but having different intensities and kinetics (ROS production, induction defense genes) and specific responses (induction of PCD, alkalinization of the medium). These results indicate that different signaling pathways could be involved in Arabidopsis thaliana in response to LPS and allowed us to highlight the potential role of LPS in the biocontrol of Pectobacterium sp.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-09-19T14:09:02Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Doutorado_Carolina_Barbosa_Malafaia_2016_PPGCB_UFPE.pdf: 5843362 bytes, checksum: c164be8fbab90a37d6db77986071cad9 (MD5) / Made available in DSpace on 2016-09-19T14:09:02Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Doutorado_Carolina_Barbosa_Malafaia_2016_PPGCB_UFPE.pdf: 5843362 bytes, checksum: c164be8fbab90a37d6db77986071cad9 (MD5) Previous issue date: 2016-01-25 / CAPES / A formação de biofilme é uma característica importante para as bactérias, por ser uma formação natural e altamente influenciada pelo ambiente circundante, confere aos microrganismos alta tolerância às adversidades e torna-se importante na virulência para patógenos. Sendo assim, apresentamos nesta tese uma investigação da adesão bacteriana e desenvolvimento de biofilme das fitobactérias Ralstonia solanacearum (Rsol) e Acidovorax citrulli (Acc), agronomicamente importantes, sobre superfícies hidrofóbicas, foi investigado também o emprego de extratos vegetais de plantas oriundas da Caatinga, na inibição da adesão bacteriana e sua capacidade bactericida contra R. solanacearum, e foi determinado o método mais eficiente na preparação amostras proteicas para R. solanacearum, A. citrulli e Pectobacterium carotovorum subsp. carotovorum (Pcc) para aplicação em estudos futuros de investigação molecular da formação de biofilmes fitopatogênicos. A formação de biofilme por diferentes isolados bacterianos após 24h de incubação em diferentes meios de cultura foi quantificado pelo método de cristal violeta e suas estruturas observadas por microscopia eletrônica de varredura e microscopia confocal. Foram avaliados também 22 extratos aquosos de 16 plantas coletadas na Caatinga quanto a capacidade de inibição da formação de biofilme de Rsol. Quanto à eficiência na obtenção de proteínas, foram testados os métodos de Trizol, Fenol, Centrifugação e Lise e avaliados através de eletroforese uni e bidimensional. Quanto a formação de biofilme os resultados obtidos indicam que, nas condições testadas, isolados de Rsol se mostrou diferente entre os isolados tanto quantitativa quando morfologicamente onde os isolados B5-5 CGH26 CGH8 e SCN 21 foram moderados ou fortes produtores de biofilme. Já os isolados de Acc não foram bons produtores de biofilme, apresentando apenas os isolados Acc1.43 e Acc 1.73 como fortes formadores de biofilme com quantidade e morfologia semelhantes. No screening de atividade antibiofilme, dentre os extratos testados apenas ramos de Harpochilus neesianus e folhas de Myroxylon peruiferum apresentaram atividade antibiofilme superior a 83% e 50%, respectivamente, e Jacaranda rugosa apresentou atividade antimicrobiana contra todos os isolados de Rsol testados. Quanto à extração de proteínas de alta qualidade o método de Lise foi o mais eficiente para Rsol e Pcc, apresentando respectivamente 369 ± 4 e 212 ± 3 diferentes spots de proteínas, contudo para Acc o método de centrifugação foi o mais indicado com 224 ± 8 spots. De acordo com os resultados deste estudo conclui-se que a formação de biofilme pode ser quantitativa e estruturalmente distinta entre isolados da mesma espécie. O screening das propriedades antimicrobianas das plantas fornece base de dados para o desenvolvimento de novos agentes antibacterianos naturais contra fitopatógenos seguros para o meio ambiente e para o desenvolvimento de estudos moleculares da formação de biofilme faz-se necessária uma prévia determinação de métodos para obtenção das macromoléculas a serem analisadas, sendo assim a seleção de métodos de extração é um ponto crucial para obtenção de amostras de qualidade para analises confiáveis. / Biofilm formation is an important feature for bacteria due to its naturally occurring and highly influence by the surrounding environment, giving to the microorganisms high tolerance to adversity and becoming essential in virulence for pathogens. Thus, we present in this thesis an investigation about bacterial adhesion and biofilm development of the phytobacteria Ralstonia solanacearum (Rsol) and Acidovorax citrulli (Acc), agronomically important, on hydrophobic surfaces; it was also investigated the use of plant extracts from the Caatinga region through the inhibition of the bacterial adhesion and its bactericidal activity against R. solanacearum. The most efficient method to prepare protein samples for R. solanacearum, A. citrulli and Pectobacterium carotovorum subsp. carotovorum (Pcc) was determined to be applied in future studies of molecular investigation of the formation of pathogenic biofilms. The biofilm formation by different bacterial strains after 24 h incubation in distinct culture media was quantified by the crystal violet method and its structures were observed by scanning electron microscopy and confocal microscopy. There were also evaluated 22 aqueous extracts from 16 plants collected in the Caatinga as its potential of inhibition of Rsol biofilm formation. In what concerns the efficiency in obtaining proteins, Trizol, Phenol, centrifugation, and Lyse were the methods evaluated by one- and two-dimensional electrophoresis. The results for biofilm formation demonstrate that, under the tested conditions, Rsol strains were different, both quantitatively and morphologically, and the strains namely B5-5, CGH26, CGH8, and SCN 21 were moderate or strong biofilm producers. Regarding the results for Acc strains, it is possible to note that they were not good biofilm producers, unless the strains Acc1.43 and Acc1.73 that were considered strong biofilm producers with similar quantity and morphology patterns. In relation to the screening of antibiofilm activity, only branches of Harpochilus neesianus and leaves of Myroxylon peruiferum presented antibiofilm activity with values higher than 83% and 50%, respectively, and Jacaranda rugosa showed activity antimicrobial against all the tested Rsol strains. The extraction of high quality proteins was performed most efficiently by the Lysis method for Rsol and Pcc, respectively with 369 ± 4 and 212 ± 3 different spots of proteins, however the centrifuge method was better for Acc with 224 ± 8 spots. According to the results of this study it is possible to conclude that biofilm formation can be quantitatively and structurally distinct from strains of the same species. The screening of the antimicrobial properties of the plants provides data as a basis for the development of new natural antibacterial agents against safe phytopathogens for the environment; in addition, for the development of molecular studies about the biofilm formation it is necessary a preliminary determination of the methods suitable for obtaining the macromolecules to be analyzed, so the selection of extraction methods is a crucial point for obtaining quality samples for reliable analysis.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-01T14:56:39Z No. of bitstreams: 1 DIS_MAILA_PACHECO_DIAS_COMPLETO.pdf: 1237790 bytes, checksum: 5f0ce43f31e6bb7c9d9d5d922daa8668 (MD5) / Made available in DSpace on 2017-06-01T14:56:39Z (GMT). No. of bitstreams: 1 DIS_MAILA_PACHECO_DIAS_COMPLETO.pdf: 1237790 bytes, checksum: 5f0ce43f31e6bb7c9d9d5d922daa8668 (MD5) Previous issue date: 2016-03-29 / Solanum lycopersicum L., tomato, is an herbaceous plant belonging to the Solanaceae family. Its fruits are consumed worldwide, reaching the world production of 160 million tons per year. In Brazil, it is the second vegetable in economic importance. However, the tomato is attacked by numerous diseases that lead to loss of production and /or poor quality of the fruit, such as the hollow stem, the disease caused by Pectobacterium spp. Due to the large number of diseases caused by plant pathogens, the tomato is a culture in which a significant amount of agrochemicals is used. Therefore, this species is among the vegetables with the greatest amount of residual pesticides. Under these circumstances, it is essential to develop sustainable plant defense techniques in order to reduce the use of agrochemicals. Then, changes in plant metabolism related to defense must be understood so that new strategies and new products can be developed. Disease control using soil microorganisms has been considered as an alternative, since the rhizobacteria, in addition to promoting plant growth, may induce resistance as the result of activation of the natural plant defenses. These, calls plant growth promoting rhizobacteria (PGPR), has been explored for their biofertilizers, biopesticides and phyto-stimulating abilities. The aims of this study were to characterize biochemically the Streptomyces spp. isolates, to determine the antagonism against Pectobacterium carotovorum subsp. brasiliensis (Pcb), to determine the ability of Streptomyces spp. on promoting growth of tomato plants and to evaluate the modulation of the defense-related metabolism of tomato plants when treated with Streptomyces spp. The possible influence of Streptomyces spp. on reducing soft rot disease in tomato plants was also evaluated. Biochemical characterization was evaluated through the ability of Streptomyces spp. on producing siderophores, solubilizing phosphate, and activity of amylase and lipase, as well as volatile organic compounds (VOC) production. Antagonism of Streptomyces spp. against Pcb was determined by dual-culture method and I-plate for VOC effect analysis. Plant growth promotion was evaluated through VOC emission and by direct interaction with Streptomyces spp. isolates (PM1, PM3, PM4, PM5, PM6 e PM9). Enzymes related to plant defense were colorimetric analyzed in plants treated with isolates of Streptomyces spp. Evaluation of soft rot disease was performed on plants treated with Streptomyces spp. and challenged with Pcb through the area under the disease progression curve (AUDPC) and plant mortality. Isolates of Streptomyces spp. displayed characteristics of PGPR and 32 volatile compounds were identified from the different isolates. PM3 was the isolate showing efficient antagonism against Pcb. Most of the isolates promoted increase of root and shoot length of tomato plants by VOC although PM5 was efficient on promoting growth by direct interaction with Streptomyces spp. Treatment with Streptomyces spp. modulated the activity of defense-related enzymes and decrease incidence of soft rot disease. / O tomateiro (Solanum lycopersicum L.) ? uma planta herb?cea pertencente ? fam?lia Solanaceae. Seus frutos s?o consumidos mundialmente, chegando ? produ??o mundial de 160 milh?es de toneladas por ano. No Brasil, ? a segunda hortali?a em import?ncia econ?mica. Contudo, o tomateiro ? alvo de in?meras doen?as que levam ? perda de produ??o e/ou m? qualidade dos frutos, como por exemplo, a doen?a Talo oco causada por Pectobacterium spp. Devido ao elevado n?mero de doen?as causadas por fitopat?genos, o tomateiro ? uma cultura onde se utiliza uma quantidade expressiva de agroqu?micos, estando entre as hortali?as que apresentam maior quantidade de agrot?xicos residuais. Por isso, torna-se imprescind?vel o desenvolvimento de t?cnicas sustent?veis de defesa para o vegetal, a fim de reduzir o uso destes compostos. Para este fim, ? fundamental compreender as altera??es no metabolismo vegetal relacionado ? defesa, para que novas estrat?gias e novos produtos agr?colas possam ser desenvolvidos. O controle de doen?as utilizando microrganismos de solo tem sido considerado uma alternativa, uma vez que as rizobact?rias, al?m de promoverem o crescimento vegetal, podem induzir ? resist?ncia como consequ?ncia da ativa??o da defesa vegetal. Estas, chamadas rizobact?rias promotoras de crescimento vegetal (PGPR), v?m sendo exploradas quanto ? capacidade biofertilizante, fito-estimuladora e biopesticida. Os objetivos deste estudo foram caracterizar seis isolados de Streptomyces spp. como PGPR, determinar o antagonismo contra Pectobacterium carotovorum subsp. brasiliensis (Pcb), determinar a capacidade de isolados de Streptomyces spp. na promo??o do crescimento de plantas de tomate e avaliar a modula??o do metabolismo relacionado ? defesa das plantas de tomate quando tratadas com Streptomyces spp. A poss?vel influ?ncia de Streptomyces spp. na redu??o da doen?a Talo oco em plantas de tomate tamb?m foi avaliada. A caracteriza??o bioqu?mica de isolados de Streptomyces spp. foi realizada por meio da capacidade de produzir sider?foros, solubilizar fosfato, e da atividade de amilase e lipase, bem como a produ??o de compostos org?nicos vol?teis. O antagonismo de Streptomyces spp. contra Pcb foi determinado pelo m?todo de dupla cultura e placa com barreira para an?lise do efeito de compostos org?nicos vol?teis (VOC). A promo??o do crescimento das plantas foi avaliada por meio de emiss?o de VOC e pela intera??o direta com os isolados de Streptomyces spp. (PM1, PM3, PM4, PM5, PM6 e PM9). Enzimas relacionadas ? resposta de defesa foram analisadas colorimetricamente em plantas tratadas com isolados de Streptomyces spp. A avalia??o da doen?a Talo oco foi realizada em plantas tratadas com Streptomyces spp. e desafiadas com Pcb atrav?s da ?rea sob a curva de progresso da doen?a e da mortalidade das plantas em 24 dias. Os isolados de Streptomyces spp. mostraram caracter?sticas de PGPR e 32 compostos vol?teis foram identificados como produtos dos diferentes isolados. PM3 foi o isolado mais eficiente quanto ao antagonismo contra Pcb. A maioria dos isolados promoveu o aumento do comprimento de raiz e da parte a?rea do tomateiro por VOC, embora PM5 tenha sido tamb?m eficiente na promo??o do crescimento atrav?s da intera??o direta com Streptomyces spp. O tratamento com Streptomyces spp. modulou a atividade de enzimas relacionadas ? defesa e diminuiu a incid?ncia da doen?a Talo oco.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-17T15:01:49Z No. of bitstreams: 1 Katia Cilene da Silva Felix.pdf: 2202729 bytes, checksum: 8bf95b820172f7d6b51dfd5e4ca39314 (MD5) / Made available in DSpace on 2017-03-17T15:01:49Z (GMT). No. of bitstreams: 1 Katia Cilene da Silva Felix.pdf: 2202729 bytes, checksum: 8bf95b820172f7d6b51dfd5e4ca39314 (MD5) Previous issue date: 2012-12-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The soft rot caused by Pectobacterium carotovorum subsp. carotovorum is the main bacterial disease of Chinese cabbage and lettuce in Brazil and the world. Chemical control of this disease has not been efficient, being necessary to search for new alternatives of control. Given the growing importance of soft rot as a limiting factor for the production of these vegetables in the regions of the Agreste and Mata of Pernambuco state, Brazil and the difficulty of effective control measures, the present study aimed to: (i) determine the effect Calcium in the control of soft rot in Chinese cabbage, (ii) select lettuce cultivars as promising sources of resistance to soft rot. Were tested two sources of calcium in different concentrations, calcium nitrate [Ca(NO3)2] a 0.00; 0.15 and 0.3 g L-1 and calcium chloride [CaCl2] a 0.00; 1 and 5 g L-1 applied leaf spraying and soil drench. When applied with both methods, Ca(NO3)2 was effective in controlling soft rot, as it reduced the disease severity by up to 48.5% when sprayed onto the leaves (0.15 g L-1). A significant increase in the leaf calcium content was observed only in the plants that were sprayed with higher doses of Ca(NO3)2 and CaCl2. In all of the calcium treatments, light microscopy analyses revealed an increased number of chloroplasts and improved structuring of the palisade parenchyma, while transmission electron microscopy analyses revealed an increased cell wall thickness that was especially evident for the 0.15 g L-1 Ca(NO3)2 treatment applied by leaf spraying and soil drenching. Of the 41 genotypes tested, 14 were moderately resistant when inoculated with Pcc-C, with severity scores ranging from 3.5 to 4.0; 27 genotypes were susceptible. Eleven of these genotypes, four susceptible and seven moderately resistant were selected to test their resistance stability against three pathogen isolates with different degrees of virulence (Pcc-36, Pcc-A1.1 and Pcc-23). Most of the genotypes evaluated (77%) exhibited the same reaction observed in the selection assays only against isolate Pcc-36: Veneza Roxa was susceptible, while Alface Grega, Mimosa Salad Bowl Roxa, Livia, Livinia, Salad Bowl, Vitória de Santo Antão and Saia Veia were moderately resistant. Vitória de Santo Antão was the only genotype that was also moderately resistant to isolates Pcc-A1.1 and Pcc-23. This genotype can be used as a promising source of stable and durable soft rot resistance. / A podridão mole causada por Pectobacterium carotovorum subsp. carotovorum é a principal fitobacteriose das culturas da couve-chinesa e alface no Brasil e mundo. O controle químico dessa doença não tem sido eficiente, sendo necessária a busca por novas alternativas de controle. Tendo em vista a crescente importância da podridão mole como fator limitante para a produção destas hortaliças nas mesorregiões do Agreste e Mata do estado de Pernambuco, Brasil e a dificuldade de medidas efetivas de controle, o presente trabalho teve como objetivos: (i) verificar o efeito do cálcio no controle da podridão mole em couve-chinesa; (ii) selecionar cultivares de alface como fontes de resistência à podridão mole. Foram testadas duas fontes de cálcio em diferentes concentrações, nitrato de cálcio (Ca(NO3)2) a 0,00; 0,15 e 0,3 g L-1) e cloreto de cálcio (CaCl2) a 0,00; 1 e 5 g L-1, aplicadas via pulverização foliar e rega do solo. Ca(NO3)2, nas duas formas de aplicação foi eficiente no controle da podridão mole, reduzindo a severidade da doença em até 48,5% via aplicação foliar (0,15 g L-1). Verificou-se aumento significativo do conteúdo de cálcio foliar apenas nas plantas pulverizadas com as maiores doses de Ca(NO3)2 e CaCl2. Em todos os tratamentos com cálcio, a microscopia de luz revelou aumento do número de cloroplastos e melhor estruturação do parênquima paliçádico, enquanto a microscopia eletrônica de transmissão evidenciou aumento de espessura da parede celular, com destaque para o tratamento Ca(NO3)2 a 0,15 g L-1 via pulverização foliar e rega do solo. De acordo com os resultados obtidos, Ca(NO3)2 a 0,15 e 0,3 g L-1, aplicado via pulverização foliar e rega do solo, apresentou potencial para o controle da podridão mole em plantas de couve-chinesa, estando essa eficiência associada a melhor estruturação e integridade da parede celular. Dos 41 genótipos de alface avaliados quanto à reação à podridão mole, visando identificar fontes de resistência, 14 apresentaram reação como moderadamente resistentes, com severidade variando de 3,5 a 4,0, enquanto 27 genótipos foram suscetíveis. Destes genótipos, 11 foram selecionados para o ensaio de estabilidade da resistência em relação a três isolados do patógeno com diferentes níveis de virulência, sendo quatro suscetíveis e sete moderadamente resistentes. A maioria dos genótipos avaliados (77%) voltou a expressar a mesma reação do ensaio de seleção em relação ao isolado Pcc-36, ou seja, reação susceptível (Veneza Roxa) e moderadamente resistente (Alface Grega, Mimosa Salad Bowl Roxa, Livia, Livinia, Salad Bowl, Vitória de Santo Antão e Saia Veia). Apenas o genótipo Vitória de Santo Antão manteve a reação de moderadamente resistente também para os isolados Pcc-A1.1 e Pcc-23. Portanto, esse genótipo pode ser utilizado como uma fonte promissora de resistência estável e durável contra a podridão mole.

Pectobacterium carotovorum subsp. brasiliense (Pcb) is a plant pathogenic bacterium that causes blackleg and tuber soft rot disease of potato worldwide. Pectobacterium spp. are characterized by the secretion of large quantities of plant cell wall degrading enzymes. As the name indicates, Pectobacteria are pectinolytic pathogens, producing enzymes such as pectate lyase, polygalacturonase, and many others that are used to catalyse the breakdown of pectin, the main plant cell wall component. Consequently, virulence of Pectobacteria is highly reliant upon the production and secretion of macerating enzymes. Hence these bacteria are also referred to as “brute-force” pathogens. Infection and disease symptoms on plants commonly result in the development of blackleg disease, a characteristic black-like decay extending on the stems of infected potato plants. Furthermore, the infection of tubers results in the development of soft rot disease. Pcb is of particular interest in that among Pectobacterium spp. such as Pectobacterium atrosepticum (Pa), P. carotovorum subsp. carotovorum (Pcc), and P. wasabiae, Pcb strains are reported to be the most aggressive and virulent pathogens causing blackleg and soft rot disease of potato in many growing regions across the world. The fact that strains of Pcb were recently reported and isolated in South Africa has necessitated that this work be undertaken in order to characterise this newly described important pathogen of potato in regard to its phenotypic, genetic diversity, virulence and host range traits. Therefore in this work Pcb strains were subjected to multilocus phylogenetic analyses (MLSA) in order to investigate and determine whether there is any interspecies and intraspecies genetic diversity among the South African Pcb isolates. It was thus established that there is a significant genetic diversity that exists both on an interspecies and intraspecies level among Pcb isolates. As a result we sought to investigate further if the level of genetic diversity observed can be reflected in terms of the pathogen’s virulence, biochemical, phenotypic as well as host range characteristics. The results of virulence assays on potato tubers and stems indicated that Pcb strains are significantly much more virulent on potato compared to closely related Pectobacterium spp. such as Pa and Pcc. Moreover, the level of intraspecies diversity observed through phylogeny was also evident and reflected on the phenotypic, virulence and host range characteristics of the pathogen. This study also focused on investigating virulence factors employed by Pectobacterium spp. during infection. Such factors include the ability to produce and secrete of various extracellular macerating enzymes, as well as screening for the presence of virulence associated effectors and phytotoxin genes. It was of interest to observe that Pcb strains have the ability to grow and produce substrate-degrading enzymes much more rapidly compared to Pa and Pcc. This phenomenon was also observed in virulence assays where Pcb strains were noted to cause more rapid and most severe maceration symptoms on potato tubers and stems. Thus in agreement with other studies, our results suggests that Pcb is a uniquely sophisticated but diverse plant pathogen which can be considered to be one of the most aggressive causal agents of blackleg and soft rot disease of potato in South Africa. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Microbiology and Plant Pathology / Unrestricted

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