Le manioc (Manihot esculenta L. Crantz) est cultivé dans toute la zone intertropicale. Compte tenu de ses utilisations potentielles, le développement de la culture du manioc a considérablement augmenté au niveau mondial, mais il est encore considéré comme une culture de subsistance en particulier dans les régions pauvres où il est cultivé par les petits producteurs. La production du manioc est fortement contrainte par des facteurs biotiques et abiotiques, dont la bactériose vasculaire du manioc (CBB) et la nécrose bactérienne du manioc (CBN) qui sont deux maladies causées par les bactéries Xanthomonas axonopodis pv. les manihotis (Xam) et X. cassavae (Xc), respectivement. CBB est considérée comme la principale maladie bactérienne, notamment au Venezuela où la CBB a été signalée pour la première fois dans les années 70. Dans les années 90, des études ont été menées pour élucider la variabilité génétique de Xam dans différentes régions du pays, au moyen d’outils moléculaires, mettant en évidence un degré élevé de polymorphisme parmi les souches analysés.Le sujet de cette thèse porte sur la situation de CBB au Venezuela 20 ans après. Quelle est la diversité génétique actuelle de Xam au Venezuela? Quelle est la structure génétique des populations de Xam dans ce pays, et comment diffèrent-elles des souches de Xam recueillies dans les années 90? En outre, étant donné que Xam et Xc provoquent des symptômes semblables sur feuilles de manioc et présentent des caractéristiques physiologiques et morphologiques similaires, nous avons également visé à développer un nouvel outil de diagnostic moléculaire permettant une détection rapide et fiable de Xam et de Xc tout étant capable de les discriminer l’une vis à vis de l’autre.Pour atteindre nos objectifs, nous avons d'abord établi une PCR-duplex comme outil de détection moléculaire des xanthomonades infectant le manioc. Sur la base de l'analyse in silico des séquences du génome de 66 souches de Xam et une de Xc, nous avons pu sélectionner 6 paires d'amorces candidates spécifiques de Xam et six autres pour Xc. Nous avons pu développer un test de PCR-duplex qui a été validé en testant 53 souches de Xam et 25 souches de Xc issues de différents pays, 18 souches non cibles contrôles et cinq souches épiphytes associées au manioc. Cette technique représente un outil utile pour détecter et différencier Xam et Xc provenant de cultures in vitro mais aussi fonctionnelle à partir de tissues de plantes infectés.Deuxièmement, nous avons donc évalué la diversité des populations de Xam à l’aide de l’étude de microsatellites (ou MLVA pour « Multiple loci VNTR analysis »). Une enquête de terrain menée dans six États du Venezuela a permis d'évaluer la présence de la maladie, son statut et nous a permis d'établir une collection de souches de Xam dont l’analyse détaillée de la diversité a pu être réalisée. Au total nous avons isolé 202 souches de Xam issues de six localités situées dans quatre états. À l'aide d'un schéma 14-MVLA, nous avons analysé 12 populations et mis en évidence un indice élevé de la diversité génétique au sein de ces populations et entre elles, et principalement dans l'est du pays.Le développement de ce type de recherche est essentiel pour une gestion raisonnée des cultures dans le monde. Conjugué aux politiques agricoles existantes, il nous permettra de mieux comprendre les agents pathogènes d'importance agricole et les mécanismes impliqués dans leur établissement dans le temps et à travers l’espace. L'objectif à long terme est d'appliquer des mesures de contrôle efficaces et durables, ce qui conduira à des mesures de quarantaine plus efficaces pour prévenir la propagation de la maladie. / Cassava (Manihot esculenta L. Crantz) belongs to the group of roots and tubers and is cultivated in the tropics worldwide. This species is a nutritional alternative in many populations where no optimal crop production, general conditions nor technological support exist. Considering its potential uses, the global development of cassava crop has increased significantly but it is still considered a subsistence crop in poor regions lead by smallholder producers. Cassava is affected by biotic and abiotic factors during its life cycle, which heavily limiting its optimal performance. A variety of pests and diseases are known to affect cassava production. Among them are those caused by Xanthomonas axonopodis pv. manihotis (Xam) and Xanthomonas cassavae (Xc), causal agents of Cassava Bacterial Blight (CBB) and Cassava Bacterial Necrosis (CBN) diseases, respectively. CBB is considered the major bacterial disease that affects cassava crop worldwide which is also the case in Venezuela where it was first reported in the 70s. Since the 90s, studies were conducted to elucidate Xam genetic variability in different regions in the country, by means of different molecular tools available at that time. A high degree of polymorphism among the isolates was reported, whether collected from the same or different fields. The Xam population was distributed into eight clusters and no correlation was observed between genetic diversity and geographic origin.Our questions deal with the situation of CBB in Venezuela 20 years later : what is the current genetic diversity of Xam populations in Venezuela? what is the genetic structure of Xam populations and how do they differ with respect to Xam strains collected in 90s. Moreover, because Xam and Xc cause similar symptoms on cassava leaves and display similar physiological and morphological characteristics, we also aimed at developing a new molecular diagnostic tool allowing for fast and reliable detection of Xam and able to discriminate with Xc.To achieve our goals, we first established a duplex-PCR as a molecular detection tool of cassava-infecting xanthomonads. Based on in silico analysis of the genome sequences of 66 Xam and 1 Xc strains, we were able to select 6 Xam and 6 Xc primers pairs candidates, of which one set of primers for each was selected for further studies. We were able to develop a duplex-PCR assay that was validated upon testing 53 Xam strains and 25 Xc strains from different countries, 18 non-target strains, and 5 epiphytic strains associated to cassava, proving this technique a useful tool to detect and differentiate Xam and Xc from in vitro cultures and in planta.Secondly we assessed the diversity of Xam populations through a variable number of tandem repeat analysis (MLVA). A field survey conducted in six states in Venezuela enabled to evaluate the occurence of the disease, its status and allowed us to establish a strain collection for detailed diversity analysis. We isolated 202 Xam strains from six localities, localized in four states. Using a MVLA14-scheme, we analyzed 12 populations highlighting a high index of genetic diversity among and within populations, mainly in the east of the country.The development of this type of research is essential in the management of crops in the world and coupled with the existing agricultural policies, it will allow us to have a deeper understanding of pathogens of agricultural importance and the mechanisms involved in their establishment over time and across regions. The long-term objective of this is to apply control measures that are effective in time, thus establishing more stringent quarantine measures to prevent the spread of the disease.

Made available in DSpace on 2017-07-10T17:37:39Z (GMT). No. of bitstreams: 1 Roberto_Luis_Portz.pdf: 1085899 bytes, checksum: 3efb6c9ddc743240ff09e54b5f1b5d9a (MD5) Previous issue date: 2003-02-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The bacterial blight of cassava (Xanthomonas axonopodis pv. manihotis) is the most important disease in the culture of the cassava and its occurrence is generalized in all the places where it is cultivated. The intensity of the disease is related, mainly, with the resistance and age of the plant and with the soil and weather conditions. With the objective of obtaining information about the genetic variability of these bacteria in the West region of Paraná, a research was accomplished, throughout sampling of stems with similar symptoms to the caused by the disease, at Entre Rios do Oeste, Marechal Cândido Rondon, Mercedes, Missal, Nova Santa Rosa and Pato Bragado. Stems of cassava varieties destined for industry and varieties for human consume were sampled. The isolates obtained were characterized to "in vitro" amylase activity, eletrophoretic analysis of isoenzymes α and β-esterase and aggressiveness. From 61 collected materials, it was possible to obtain 19 isolated of X. axonopodis pv. manihotis. Materials of human consume varieties showed larger incidence than those for industry. Stems from Pato Bragado, Entre Rios do Oeste and Mercedes showed incidences of 10, 27 and 10%, respectively, values smaller than those of Marechal Cândido Rondon (50%) and Nova Santa Rosa (58%). The isolates could be contained in three, six and 12 different groups to amylolytic activity, aggressiveness and esterase isoenzimes, respectively. There was not relationship between the amylase activity and aggressiveness of the isolates. Based on the origin, isolates of Marechal Cândido Rondon were more aggressive than those of other sampled areas. The clustering for esterase allowed to verify that isolates from Entre Rios do Oeste, Nova Santa Rosa and Mercedes showed high similarity degree. These results indicate there to be differentiation among the isolates of the bacteria presents in the studied regions / A bacteriose ou murcha bacteriana (Xanthomonas axonopodis pv. manihotis) é a doença de maior importância econômica na cultura da mandioca e sua ocorrência está generalizada em todos os locais onde esta é cultivada. A intensidade da doença está relacionada, principalmente, com a resistência e idade da planta e com as condições edafoclimáticas. Com o objetivo de obter informações sobre a variabilidade genética desta bactéria em municípios produtores da região Oeste do Paraná, foi realizado um levantamento, através de coletas de ramas com sintomas semelhantes aos causados pela doença, em Entre Rios do Oeste, Marechal Cândido Rondon, Mercedes, Missal, Nova Santa Rosa e Pato Bragado. Foram coletadas ramas de variedades de mandioca destinadas para indústria e para mesa. Os isolados obtidos foram caracterizados quanto à atividade in vitro de amilase, análise eletroforética de isoenzimas de α e β-esterase e quanto à agressividade. A partir de 61 materiais vegetais coletados, foi possível obter 19 isolados de X. axonopodis pv. manihotis, sendo que, materiais provenientes de variedades de mesa apresentaram maior incidência em relação àquelas para indústria. Manivas provenientes de Pato Bragado, Entre Rios do Oeste e Mercedes apresentaram incidências de 10, 27 e 10%, respectivamente, valores inferiores aos de Marechal Cândido Rondon (50%) e de Nova Santa Rosa (58%). Os isolados puderam ser agrupados em três, seis e 12 grupos distintos com relação à capacidade amilolítica, agressividade e isoenzimas de esterase, respectivamente. Não houve relação entre a atividade de amilase e agressividade dos isolados. Baseado na procedência, isolados de Marechal Cândido Rondon foram mais agressivos que os provenientes das outras regiões amostradas. O agrupamento com base em esterase permitiu verificar que isolados provenientes de Entre Rios do Oeste, Nova Santa Rosa e Mercedes apresentam, entre si, alto grau de similaridade. Estes resultados indicam haver diferenciação entre os isolados da bactéria presentes nos municípios amostrados

Submitted by Helena Bejio (helena.bejio@unioeste.br) on 2017-12-04T20:57:20Z No. of bitstreams: 2 Tatiane_M_Portz_2017_ vai enviar outra(1).pdf: 3595859 bytes, checksum: eb00999666bc96e412d548793214ad1c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-12-04T20:57:20Z (GMT). No. of bitstreams: 2 Tatiane_M_Portz_2017_ vai enviar outra(1).pdf: 3595859 bytes, checksum: eb00999666bc96e412d548793214ad1c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The bacterial blight of cassava, caused by the bacterium Xanthomonas axonopodis pv. manihotis, is considered the most important disease and constitutes a serious economic problem for cassava in Brazil, since there are no efficient commercial products to manage the disease. Considering the benefits observed for agriculture through the application of homeopathy, this can become a low-cost strategy for cassava producers, because it is an environmentally correct and not residual. So, this work aims to evaluate the action of homeopathic drugs on the bacterium, the severity of the disease and on the development of cassava plants. The treatments included two homeopathic drugs (Sulphur and Silicea terra), a drug homeopathic based on Propolis, the isotheraphy in the form of nosode of X. axonopodis pv. manihotis (XAM) and hydro-alcoholic solution (30% ethyl alcohol). The dynamization undertaken were 6CH, 12CH, 18CH, 24CH and 30CH. The estimate of the antimicrobial activity in vitro was by antibiogram, using the technique "pour plate", and the bacterial growth count by the “spread plate” technique. For in vivo evaluations, cassava branches of the variety Caiuá (Olho Junto), were used in two trials. The first test was carried out with the inoculation of bacteria prior to the application of the treatments, in order to obtain curative action, and the second test was carried out the inoculation after the application of the treatments, aiming at preventive action. The results were submitted to analysis of variance by the F test and comparison of means by the Scott-knott test, both with levels of 5% of significance. In the in vitro assays, the homeopathic drugs did not present inhibition by the antibiogram, however, for bacterial growth and colony count, Sulphur treatments 12CH and 18CH, Propolis 6CH, 12CH, 18CH and 30CH, isotherapic XAM 6CH and 18CH, in addition to all the dynamizations of hydroalcohol solution caused inhibition. For the in vivo assay, the treatments interfered in the physiological activities of the plants, being the preventive application more efficient than the curative mode, in the management of the bacterial blight of cassava. The treatments Silicea terra 12CH and Sulphur 24CH, for the curative and preventive trials, respectively, favored the development of the pathogen, causing death of the plants. The treatments with Alcohol 30CH and Propolis 6CH were efficient in reducing the severity of the disease in both trials. In general, preventive homeopathic treatment tends to reestablish plant homeostasis more efficiently than curative treatment, as well as to accelerate sprout budding and increase the height of cassava plants. / A murcha bacteriana, causada pela bactéria Xanthomonas axonopodis pv. manihotis é considerada a doença mais importante e que constitui sério problema econômico para a cultura da mandioca no Brasil, uma vez que não há produtos comerciais eficientes para manejo da doença. Tendo em vista os benefícios observados para a agricultura pela aplicação da homeopatia, esta pode se tornar uma estratégia de baixo custo para os mandiocultores, pois é uma ferramenta ambientalmente correta e não residual. Assim, este trabalho objetiva avaliar a ação de preparados homeopáticos sobre a bactéria, a severidade da doença e sobre o desenvolvimento das plantas de mandioca. Os tratamentos compreenderam dois medicamentos homeopáticos (Sulphur e Silicea terra), um preparado homeopático a base de própolis, o isoterápico na forma de nosódio de X. axonopodis pv. manihotis (XAM) e solução hidro alcóolica (álcool etílico 30%) dinamizada. As dinamizações empreendidas foram 6CH, 12CH, 18CH, 24CH e 30CH. A avaliação da atividade antimicrobiana in vitro foi por antibiograma, utilizando a técnica "pour plate", e a contagem do crescimento bacteriano pela técnica "spread plate". Para as avaliações in vivo, foram utilizadas ramas de mandioca da cultivar Caiuá (Olho Junto), em dois ensaios. O primeiro ensaio foi realizado com a inoculação de bactéria anterior à aplicação dos tratamentos, a fim de obter-se ação curativa e, o segundo ensaio procedeu-se a inoculação posterior à aplicação dos tratamentos, visando ação preventiva. Os resultados foram submetidos à análise de variância pelo teste F e comparação de médias pelo teste Scott- knott, ambos com níveis de 5% de significância. Nos ensaios in vitro, os preparados homeopáticos não apresentaram inibição pelo antibiograma, no entanto, para crescimento bacteriano e contagem de colônias, os tratamentos Sulphur 12CH e 18CH, Propolis 6CH, 12CH, 18CH e 30CH, isoterápico XAM 6CH e 18CH, além de todas as dinamizações de solução hidroalcóolica 30%, causaram inibição. Para o ensaio in vivo, os tratamentos interferiram nas atividades fisiológicas das plantas, sendo a aplicação de maneira preventiva mais eficiente que o modo curativo no manejo da murcha bacteriana na mandioca. Os tratamentos Silicea terra 12CH e Sulphur 24CH, para os ensaios curativo e preventivo, respectivamente, favoreceram o desenvolvimento do patógeno, causando morte das plantas. Os tratamentos com álcool 30CH e Propolis 6CH foram eficientes na redução da severidade da doença em ambos os ensaios. De forma geral, o tratamento homeopático preventivo tende a reestabelecer a homeostasia vegetal mais eficientemente que o tratamento curativo, além de acelerar a brotação das manivas e aumentar a altura das plantas de mandioca.