Diplodia pinea causes Diplodia tip blight on more than 30 different pine species. During the past 10 years, Diplodia tip blight has emerged as a serious problem in landscape and Christmas tree farms in this region. Surveys of diseased and symptomless Austrian pines revealed that latent infections of symptomless shoots by D. pinea were common. Latent infections may account for the recently observed rapid decline of mildly diseased pines in our region. To investigate the colonization habits of D. pinea within its host, molecular cytology was attempted and traditional histology was performed on naturally infected, diseased and asymptomatic Austrian pine tissues. I devoted much effort to developing a transformation system for D. pinea. Ultimately I did not succeed in this goal, but I was able to develop a highly efficient protocol for Agrobacterium tumefaciens-mediated transformation of another pathogenic fungus, Colletotrichum graminicola, in the process. The work that I did should help in future efforts to transform D. pinea, something that will be essential if it is to become a tractable system for the study of fungal latency. Traditional histological methods were more successful, and provided important information about the nature of latent infections. Very sparse epiphytic and subcuticular fungal growth was observed in healthy shoots, however, no fungal tissues were present within the shoots. In diseased and latently infected shoots, crevices created between the needle bundles and the shoots were filled with fungal material, and hyphae were observed colonizing the needle sheaths. Hyphae were also observed breaching the shoot epidermal layer in these crevices and colonizing the underlying periderm. D. pinea colonization was extensive in all tissues of diseased shoots early in symptom development. In contrast, localized pockets of degradation were observed in the periderm and adjacent cortical cells located around areas of needle attachment in asymptomatic, latently infected shoots. The mechanism that operates to prevent expansion of these infected pockets in the latently infected shoots is still unclear. Obvious signs of pine defense mechanisms were only observed in 2 shoots. My observations were consistent with the idea that colonization progresses into the vascular tissues, and that this results in symptom development. Vascular colonization may occur more readily if the host is stressed. My research lays the groundwork for future efforts to understand the nature of the transformation from latent to pathogenic infection.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-02-06T12:12:53Z No. of bitstreams: 2 Tese - Vanessa Duarte Dias - 2016.pdf: 2283005 bytes, checksum: 467160bb33424613a588c7251fabc187 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-02-06T12:13:29Z (GMT) No. of bitstreams: 2 Tese - Vanessa Duarte Dias - 2016.pdf: 2283005 bytes, checksum: 467160bb33424613a588c7251fabc187 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-02-06T12:13:30Z (GMT). No. of bitstreams: 2 Tese - Vanessa Duarte Dias - 2016.pdf: 2283005 bytes, checksum: 467160bb33424613a588c7251fabc187 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-06-30 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The sugarcane production areas are increasing in Brazil due to increased ethanol consumption by flex fuel cars. The planted area is growing, but productivity has been declining in recent years, and factors such as incidence of diseases in the crop may be contributing to this situation. Among the diseases, bacteria such as scald of the leaves and ratoon stunting disease are of great importance to the crop because they can reduce productivity by up to 30%. In addiction and the symptoms are not always displayed in the field, thus requiring advanced techniques to detect such bacterial diseases For Leifsonia xyli subsp. xyli, which causes rickets in the ratoon cane sugar control measure most commonly used besides varietal resistance is thermally treating the billets that will serve seedlings. Thus, the purpose of the first study was to perform the heat water treatment of billets with the addition of kasugamycin antibiotic dosage 300mL/100L H2O in order to try to reduce bacterial escape from the standard treatments, as also in other time and temperature combinations proposed: T1 = 52°C/30 '; T2 = 52°C/1hr; T3 = 50°C/1hr; T4 = 50°C/2hrs; T5 = 52°C/30 '+ antibiotic; T6 = 52°C/1hr + antibiotic; T7 = 50°C/1hr + antibiotic; T8 = 50°C/2hrs + antibiotic; T9 = antibiotic and T10 = control. Moreover, among techniques for diagnosis of such diseases, the most used by laboratories are the serological tests which have the advantage of quantitatively detecting the presence of bacteria on the stems but, however, has the disadvantage of detecting only when the bacterial population is relatively high. PCR technique that is one of the techniques considered most sensitive, has not been used in practice, as this high sensitivity has not been used, the protocols do not detect bacterial diseases in the case of latent infection, where bacterial title is relatively low. So the other work aimed at improving the Xanthomonas albilineans detection developing a LAMP protocol, and compared to other techniques of detection and isolation in semiselective medium, PCR and nested PCR for both symptomatic samples and for asymptomatic and the latter where population in general pathogen is low, we improved Nested one protocol to detect the leaf scald in latent infections by comparing the vascular fluid extraction techniques combined with four different DNA extraction protocols. / O cultivo de cana-de-açúcar está em expansão no Brasil, devido principalmente ao crescente consumo de etanol por carros bicombustíveis. A área plantada está em crescimento, mas a produtividade nos últimos anos vem decrescendo, e fatores como incidência de doenças podem estar contribuindo para tal situação. Dentre as doenças, as bacterianas, como o raquitismo das soqueiras e a escaldadura das folhas, possuem grande importância para a cultura, pois podem reduzir a produtividade em até 30%. Nem sempre os sintomas são visualizados em campo, necessitando assim de técnicas avançadas de detecção ou controle. Para a bacteria Leifsonia xyli subsp. xyli (Lxx), que causa o raquitismo das soqueiras em cana-de-açúcar a medida de controle mais utilizada além da resistência varietal é tratar termicamente os toletes que servirão de mudas. Assim o objetivo do primeiro trabalho foi realizar o tratamento térmico dos toletes com a adição de antibiótico casugamicina à dosagem de 300mL/100L de H2O com o intuito de tentar reduzir o escape bacteriano dentre os tratamentos padrões, como também em outras combinações de tempo e temperatura propostos: T1= 52ºC/30’; T2= 52ºC/1hr; T3= 50ºC/1hr; T4= 50ºC/2hrs; T5= 52ºC/30’ + antibiótico; T6= 52ºC/1hr + antibiótico; T7= 50ºC/1hr + antibiótico; T8= 50ºC/2hrs + antibiótico; T9= antibiótico e T10= testemunha. Além disso, dentre as técnicas para diagnose de tais doenças, as mais utilizadas por laboratórios são os testes sorológicos, que tem como vantagem detectar quantitativamente a presença das bactérias nos colmos mas, no entanto, possui a desvantagem de detectar somente quando a população bacteriana está relativamente alta. Já a técnica de PCR que é uma das tecnicas consideradas mais sensíveis, não vem sendo utilizada na prática, pois esta alta sensibilidade não vem ocorrendo. Os protocolos não detectam as bacterioses no caso de infecção latente, onde o título bacteriano é relativamente baixo. Portanto, os demais trabalhos visaram aprimorar a detecção Xanthomonas albilineans desenvolvendo um protocolo LAMP, e comparando com outras tecnicas de detecção como isolamento em meio semi-seletivo, PCR e Nested-PCR, tanto para amostras sintomáticas quanto para assintomáticas. Para estas últimas onde a população do patógeno de maneira geral é baixa, aprimoramos um protocolo Nested para detectar escaldadura das folhas em infecções latentes, comparando técnicas de extração do fluído vascular combinado a quatro diferentes protocolos de extração de DNA.

Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2016-09-21T16:14:51Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_daiana_medeiros.pdf: 661783 bytes, checksum: b730e6b69207fcfbd6fb978846974286 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2016-09-21T16:46:53Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_daiana_medeiros.pdf: 661783 bytes, checksum: b730e6b69207fcfbd6fb978846974286 (MD5) / Made available in DSpace on 2016-09-21T16:46:53Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_daiana_medeiros.pdf: 661783 bytes, checksum: b730e6b69207fcfbd6fb978846974286 (MD5) Previous issue date: 2014-02-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Infecções pelo herpesvírus bovino tipo 1 (BoHV-1) têm sido amplamente detectadas em rebanhos bovinos brasileiros, e são responsáveis por grandes prejuízos econômicos na bovinocultura. Entre as doenças causadas por esse vírus estão a Rinotraqueíte infecciosa bovina (IBR), Vulvovaginite Pustular Infeciosa (IPV) e Balanopostite Pustular Infecciosa (IBP). O impacto econômico dessa enfermidade é devido principalmente a falhas reprodutivas, como retorno ao estro e abortamentos. A espécie bovina é hospedeira natural do BoHV-1, no entanto, estudos sorológicos realizados em diversos países têm sugerido que búfalos (Bubalus bubalis) podem ser suscetíveis ao herpesvírus bovino tipo 1 (BoHV-1), e outros alfaherpesvirus geneticamente relacionados. Apesar de os bovinos se infectarem naturalmente pelo BoHV-1, a distribuição dessa enfermidade na espécie bubalina não tem sido estudada no Brasil, e as informações são limitadas e suportadas apenas por inquéritos sorológicos. Dessa forma, o objetivo desse trabalho foi detectar a presença de DNA viral de BoHV-1 em 202 gânglios trigêmeos de bubalinos coletados em um abatedouro localizado no município de Pelotas através de uma Semi-Nested PCR (SN-PCR) para detecção parcial do gene da glicoproteína D (gD) do envelope do BoHV-1. Além disso, 242 amostras de soro foram testadas pela técnica de soroneutralização (SN) para a detecção de anticorpos neutralizantes frente a diferentes amostras de herpesvírus de ruminantes: BoHV-1 (cepa Los Angeles), BoHV-5 (cepa Riopel - RP), e BuHV (cepa B6). O DNA de BoHV-1 foi detectado em 30,1% dos animais, e os resultados da SN mostraram que 27,6% dos animais apresentaram anticorpos neutralizantes contra, pelo menos, um dos vírus testados. Os resultados demonstram que o BoHV-1 circula em rebanhos bubalinos na região sul do Brasil, e apesar das reatividade cruzada, anticorpos anti-BoHV-5 e anti-BuHV-B6 também foram identificados na população estudada. Este é o primeiro estudo que utiliza testes moleculares para a detecção de infecções latentes por BoHV-1 na espécie bubalina no Brasil. / Infections caused by the bovine herpesvirus type 1 (BoHV-1) have been widely detected in Brazilian cattle, and they are responsible for major economic losses to the cattle industry. Among the diseases caused by this virus there are infectious rhinotracheitis (IBR), Infectious Pustular vulvovaginitis (IPV) and balanoposthitis Infectious Pustular (IBP). The economic impact of this disease is mainly due to reproductive failures, such as return to estrus and abortions. The bovine is a natural host of BoHV-1, however, serologic studies in several countries have suggested that buffaloes (Bubalus bubalis) may be susceptible to bovine herpesvirus type 1 (BoHV-1), and other genetically related alphaherpesvirus. Despite the cattle become infected naturally by BoHV-1, the distribution of this disease in the buffalo species has not been studied in Brazil, and the information is limited and only supported by serological surveys. Thus, the aim of this study was to detect the presence of viral DNA of BHV - 1 in trigeminal ganglia of 202 buffaloes collected in a slaughterhouse located in the city of Pelotas through a semi -nested PCR (SN - PCR) for detection of partial gene glycoprotein D of BoHV-1. In addition, 242 serum samples were obtained and tested by the serum neutralization (SN ) technique for the detection of neutralizing antibodies against different samples of herpesvirus: BHV -1 ( Los Angeles strain ) , BHV -5 (strain Riopel - RP ) and BuHV (strain B6 ). The BHV -1 DNA was detected in 30.1 % of the animals and the results of SN showed that 27.68 % of the animals had neutralizing antibodies against at least one of the tested viruses. This is the first study that uses molecular tests for the detection of latent BoHV - 1 in buffalo species in Brazil.