The potential of increasing disease problems through the use of recycled irrigation water in horticultural operations is a serious concern, yet basic research on waterborne plant pathogens in Virginia is lacking. In this work seasonal fluctuations and locations of Pythiaceae in a recycled water irrigation system at a container nursery were determined. <i>Pythium</i> spp. were recovered more frequently and in greater numbers than <i>Phytophthora</i> spp. Species of <i>Phytophthora</i> recovered in filtering assays were identified as <i>P. capsici, P. citricola, P. citrophthora, P. cryptogea, P. drechsleri,</i> and <i>P. nicotianae. P. cryptogea</i> and <i>P. drechsleri</i> were the only <i>Phytophthora</i> spp. recovered from baits placed on the surface of the irrigation reservoir, whereas a greater diversity of species was recovered from baits placed at depths. Hymexazol-amended medium was found to have limitations in recovery of <i>Phytophthora</i> spp. In pathogenicity tests, <i>P. cactorum, P. capsici, P. citrophthora,</i> and <i>P. nicotianae</i> caused significant mortality of <i>Salvia officinalis</i> and <i>P. cactorum</i> showed limited pathogenicity on <i>Gerbera jamesonii</i>. Asymptomatic (aboveground) plants were found to harbor inoculum long after <i>Phytophthora</i>-inoculation. Fresh weight analyses of roots and shoots of asymptomatic plants demonstrated that <i>Phytophthora</i> inoculation may either reduce or stimulate plant shoot growth, but little effect is apparent on roots. Irrigation with naturally infested irrigation water reduced plant growth. This research provides data for prioritizing development of detection technology and management practices for plant pathogens in irrigation water. The results may also lead to improvements in conventional water assay protocols for plant pathogens. / Master of Science

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Um dos principais fatores limitantes da produção do melão é a ocorrência de doenças. Entre os patógenos mais importantes estão as bactérias, que causam perdas significativas na produção. Causada pela bactéria Acidovorax avenae subsp. citrulli (Aac), a mancha-aquosa-do-melão, também conhecida como mancha-bacteriana-dofruto, é uma doença grave, que tem preocupado produtores do nordeste, sendo que todos os tipos de melão apresentam suscetibilidade ao patógeno. A principal fonte de inóculo para esta bactéria é a semente infectada. Este trabalho teve como objetivo o desenvolvimento de um meio semi-seletivo para detecção e identificação de Acidovorax avenae subsp. citrulli em sementes de melão, para testes de rotina em laboratórios de patologia de sementes. Por meio de testes de fungitoxicidade, antibiogramas qualitativos e quantitativos e testes bioquímicos foi desenvolvido o seguinte meio semiseletivo para a detecção de Aac em sementes de melão: dextrose (5 g/L), NaCl (5 g/L), peptona (5 g/L), KH2PO4 (2 g/L), MgSO4.7H2O (0,2 g/L), vermelho de fenol (0,012 g/L), uréia (25 g/L), agar (17 g/L), benomil (100 mg/L), nistatina (200 mg/L) e amoxicilina (15 mg/L). / One of the main limiting factors of the melon production is the occurrence of diseases. The bacteria are among the most important pathogens causing significant losses in the production. Caused by the bacterium Acidovorax avenae subsp. citrulli (Aac), the bacterial-fruit-blotch is a serious disease that affects all types of melon and has worried northeast producers. The main source of inoculum for this bacterium is the infected seed. This work had as objective the development of a semi-selective medium to detect and identify Acidovorax avenae subsp. citrulli in melon seeds, for routine tests in seed pathology laboratories. By means of fungitoxicity tests, qualitative and quantitative antibiograms and biochemical tests the following semi-selective medium to detect Aac in melon seeds was developed: dextrose (5 g/L), NaCl (5 g/L), peptone (5 g/L), KH2PO4 (2 g/L), MgSO4.7H2O (0,2 g/L), phenol red (0,012 g/L), urea (25 g/L), agar (17 g/L), benomil (100 mg/L), nistatina (200 mg/L) and amoxicilin (15 mg/L).

Um dos principais fatores limitantes da produção do melão é a ocorrência de doenças. Entre os patógenos mais importantes estão as bactérias, que causam perdas significativas na produção. Causada pela bactéria Acidovorax avenae subsp. citrulli (Aac), a mancha-aquosa-do-melão, também conhecida como mancha-bacteriana-dofruto, é uma doença grave, que tem preocupado produtores do nordeste, sendo que todos os tipos de melão apresentam suscetibilidade ao patógeno. A principal fonte de inóculo para esta bactéria é a semente infectada. Este trabalho teve como objetivo o desenvolvimento de um meio semi-seletivo para detecção e identificação de Acidovorax avenae subsp. citrulli em sementes de melão, para testes de rotina em laboratórios de patologia de sementes. Por meio de testes de fungitoxicidade, antibiogramas qualitativos e quantitativos e testes bioquímicos foi desenvolvido o seguinte meio semiseletivo para a detecção de Aac em sementes de melão: dextrose (5 g/L), NaCl (5 g/L), peptona (5 g/L), KH2PO4 (2 g/L), MgSO4.7H2O (0,2 g/L), vermelho de fenol (0,012 g/L), uréia (25 g/L), agar (17 g/L), benomil (100 mg/L), nistatina (200 mg/L) e amoxicilina (15 mg/L). / One of the main limiting factors of the melon production is the occurrence of diseases. The bacteria are among the most important pathogens causing significant losses in the production. Caused by the bacterium Acidovorax avenae subsp. citrulli (Aac), the bacterial-fruit-blotch is a serious disease that affects all types of melon and has worried northeast producers. The main source of inoculum for this bacterium is the infected seed. This work had as objective the development of a semi-selective medium to detect and identify Acidovorax avenae subsp. citrulli in melon seeds, for routine tests in seed pathology laboratories. By means of fungitoxicity tests, qualitative and quantitative antibiograms and biochemical tests the following semi-selective medium to detect Aac in melon seeds was developed: dextrose (5 g/L), NaCl (5 g/L), peptone (5 g/L), KH2PO4 (2 g/L), MgSO4.7H2O (0,2 g/L), phenol red (0,012 g/L), urea (25 g/L), agar (17 g/L), benomil (100 mg/L), nistatina (200 mg/L) and amoxicilin (15 mg/L).

Achromobacter xylosoxidans est un bacille à Gram négatif non fermentaire émergent dans la mucoviscidose. A Dijon, la prévalence de patients colonisés est élevée au Centre de Ressources et de Compétences de la Mucoviscidose par rapport à la moyenne nationale, et augmente également chez des patients hospitalisés atteints d’autres pathologies. Les réservoirs et le mode de contamination des patients sont inconnus.Les objectifs de ce travail étaient de décrire la situation épidémiologique dans notre CRCM et de mettre en évidence d’éventuelles sources de contamination pour les malades en Bourgogne.Dans une première étude rétrospective, toutes les souches d’A. xylosoxidans isolées chez les patients du CRCM depuis le début de leur suivi jusqu’en 2010 ont été analysées. Sur 120 patients, 21 ont présenté au moins une culture positive. Le génotypage par électrophorèse en champ pulsé n’a montré que de rares cas de transmissions croisées. Les résistances acquises aux antibiotiques sont fréquentes : ciprofloxacine (dès primocolonisation), ceftazidime et carbapénèmes (colonisations chroniques). Notre protocole de détection d’A. xylosoxidans dans l’environnement a permis d’isoler 50 souches (lavabos, douches, rivières, lacs) : 33 à l’hôpital, 9 à domicile et 8 dans la nature. Ces isolats partagent des caractéristiques avec les souches cliniques : 6 génotypes communs et résistance constante à la ciprofloxacine. Le risque de contamination des patients à domicile ou à l’hôpital est donc réel.D’autres études sont en cours afin de mieux comprendre l’émergence de cette bactérie, ses mécanismes de résistance acquise aux antibiotiques et les facteurs favorisant sa sélection dans l’environnement. / Achromobacter xylosoxidans is an aerobic nonfermentative Gram-Negative rod considered as an important emerging pathogen among cystic fibrosis (CF) patients worldwide. In Dijon (Burgundy), the prevalence of colonised patients is high among CF patients and increasing among non-CF hospitalised patients. The natural habitat of this organism as well as the possible sources of patient contamination remain unknown.The aims of this study were to report the first epidemiological data about A. xylosoxidans in a French CF centre and to identify potential reservoirs of this organism in Burgundy. In a retrospective study, all the isolates recovered from the patients affiliated with our centre in 2010 since their first visit were analysed. Out of 120 patients, 21 (17.5%) had at least one positive culture with A. xylosoxidans. Genotyping analysis by Pulsed Field Gel Electrophoresis revealed that cross-Contamination was very rare. Acquired resistance was frequent to ciprofloxacin (since primocolonisation), to ceftazidime and to carbapenems (in persistent colonisations). Thanks to our protocol designed to detect A. xylosoxidans in environment, a total of 50 strains were isolated in hospital (33 isolates), domestic (9 isolates) and outdoor (8 isolates) samples, mainly in handwashing sinks, showers, and water. These environmental isolates shared characteristics with clinical ones: 6 genotypes in common and a constant resistance to ciprofloxacin. These results reveal potential sources of contamination for the patients at home or in hospital. Further studies are needed to help understanding the emergence of this bacterium and the mechanisms involved in acquired antibiotic resistance.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2018-03-02T13:04:32Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Carlos Rafael.pdf: 2751688 bytes, checksum: 31c547702ea9189c4ded0e1d56077de5 (MD5) / Made available in DSpace on 2018-03-02T13:04:32Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Carlos Rafael.pdf: 2751688 bytes, checksum: 31c547702ea9189c4ded0e1d56077de5 (MD5) Previous issue date: 2017-08-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O conhecimento da dinâmica espacial de doenças de plantas, aliado a quantificação de inóculo e monitoramento das condições meteorológicas é de grande importância para a adoção de estratégias de manejos adequados e com menor impacto ambiental. Sendo assim, os objetivos deste trabalho de pesquisa foram caracterizar a distribuição, variabilidade espacial e possíveis relações entre atributos referentes ao mofo branco e atributos de plantas de soja além de avaliar técnicas de identificação e quantificação de ascósporos de Sclerotinia sclerotiorum durante o florescimento da soja. Foram realizadas amostragens através de uma malha georreferenciada em uma área de 12 hectares (ha) no município de Mauá-da-Serra-PR nas safras 2013/14 e 2014/15 e uma área de quatro hectares no município de Ponta Grossa-PR na safra 2015/16. Foram utilizados quadrats com área útil de 7,2 m² para avaliação das seguintes variáveis: escleródios presentes no solo, estande da cultura, incidência, índice de doença, rendimento, escleródios produzidos na colheita, além da deposição do bioaerossol, partes de folha e flores da soja como uso de Meio Semi-seletivo a Sclerotinia sclerotiorum (MSS) e Reação em cadeia da polimerase quantitativa (qPCR), em 36 pontos de amostragem na área de Ponta Grossa-PR, em três datas durante o florescimento da soja. Os dados foram analisados com uso de estatística descritiva, ajuste de semivariogramas e matriz de correlação de Pearson. As condições meteorológicas observadas nos três experimentos foram adequadas para o desenvolvimento da cultura da soja e do mofo branco na soja. As variáveis referentes a cultura da soja apresentaram coeficientes de variação baixos ou moderados e as variáveis referentes ao mofo branco apresentaram coeficiente de variação muito elevados. Os ajustes dos semivariogramas apresentaram diferenças nos três experimentos, com efeito pepita puro, ajuste linear, esférico e exponencial. A variável escleródios do solo apresentou efeito pepita puro nos dois experimentos em Mauá-da-Serra. Foram encontradas correlações significativas positivas entre a incidência do mofo branco e a produção de escleródios na colheita, além de correlação negativa com o rendimento nos três experimentos. A identificação de inóculo de mofo branco com incubação em MSS mostrou-se a técnica com maior sensibilidade, mas demanda de até 15 dias para a confirmação de S. sclerotiorum, tanto para bioaerossol, como flores e partes de folhas de soja. Foi possível otimizar o protocolo de extração de DNA e ciclo de reação para qPCR, além de gerar uma curva padrão com DNA oriundo de ascósporos de S. sclerotiorum, com ajuste ao modelo linear (R²=0,99) e eficiência de 92,2%. O uso de qPCR mostrou-se promissor para partes de folhas, sendo possível resultados em um dia de trabalho. / The knowledge of the spatial dynamics of plant diseases, allied with the quantification of inoculum and monitoring of the meteorological conditions, is important for the adoption of adequate management strategies and with less environmental impact. The aim of this work was to characterize the distribution, spatial variability and possible relations between white mold attributes and soybean plants attributes, as well as to evaluate techniques for identification and quantification of ascospores of Sclerotinia sclerotiorum during soybean flowering. Samplings were carried out through a georeferenced grid on a field of 12 hectares (ha) in the municipality of Mauá-da-Serra-PR in the 2013/14 and 2014/15 crop seasons and, on a field of four hectares in the municipality of Ponta Grossa-PR in the 2015/16 crop season. Quadrats with a useful area of 7.2 m² were used to evaluate the following variables: sclerotia in the soil, crop stand, incidence, severity, yield, sclerotia produced at harvest, as well as bioaerosol deposition, soybean leaf parts and flowers on semi-selective medium to Sclerotinia sclerotiorum (MSS) and quantitative polymerase chain reaction (qPCR), at 36 sampling points in the Ponta Grossa- PR field, at three dates during soybean flowering. Data were analyzed using descriptive statistics, semivariogram adjustment and Pearson correlation matrix. The meteorological conditions observed in the three experiments were adequate for the development of soybean crop and white mold disease. The variables related to soybean plants, showed low or moderate coefficients of variation and the variables related to white mold showed a high coefficient of variation. The semivariograms adjustments showed differences in the three experiments, with pure nugget effect, linear, spherical and exponential adjustments. The variable sclerotia of soil showed pure nugget effect on the two experiments in Mauá-da-Serra. Significant positive correlations were found between the incidence of white mold and the production of sclerotia at harvest, in addition to negative correlation to yield in the three experiments. The identification of white mold inoculum with incubation on MSS was shown to be the most sensitive technique, but takes up to 15 days for the confirmation of S. sclerotiorum pathogen for bioaerossol, flowers and parts of soybean leaves. It was possible to optimize the protocol of DNA extraction and reaction cycle for qPCR, besides generating a standard curve with DNA from ascospores of S. sclerotiorum, adjusted to the linear model (R² =0, 99) and efficiency of 92.2%. The use of qPCR showed promise results for leaf parts, being possible achieve concluding results in one working day.