Tobacco mosaic virus (TMV) is an economically important pathogen that has been studied for over one hundred years. Seedlings, seed coats, and nutrient solution were assayed for the presence of the virus and seed treatments were tested on seeds. Double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) and biological local lesion assay data were collected. Seed coats from seed collected from TMV infected plants were always positive for TMV regardless of chemical treatment. Seed from infected source plants have lower germination than seed from healthy plants. Trisodium phosphate and hydrochloric acid treatments reduced virus infection of seedlings when grown under controlled conditions. Virus particles were serologically and biologically detected in both the leaves and roots of seedlings mechanically inoculated with TMV. Nutrient solution collected from 28 day old seedlings, 12 days post inoculation, tested positive for biologically active TMV by ELISA and infectivity assay. Infected water in float bed production could facilitate viral movement to all seedlings sharing nutrient solution. Seed transmission of TMV was shown to occur at a rate of 0.2%. This is in contrast to other research attempting to demonstrate seed transmission where visual symptoms on seedlings have been used to assess seed transmission. / Master of Science

Tobacco mosaic virus (TMV) is an extensively studied RNA virus that reduces quality and yield in commercially grown tobacco (Nicotiana tabacum L.). The virus is transmitted mechanically, although infections have been associated with contaminated seeds with the seed coat being the source of virus. Thus, TMV transmission is said to be seedborne (as opposed to true seed transmission where the embryo is infected). The objective of this study was to identify TMV concentrations in the three components of an individual tobacco seed: seed coat (SC), endosperm (ED), and embryo (EM). Six hundred seed from TMV infected K 326 flue-cured cultivar tobacco plants were carefully dissected into the three components. Total RNA was extracted from each sample and synthesized into cDNA for analysis. A quantitative real-time PCR (RT-qPCR) assay was developed to quantify viral titers in each component, while endpoint PCR confirmed RT- qPCR results and established a threshold viral cycle (Ct) value. Endpoint PCR results revealed viral accumulation in all three components of a tobacco seed. The highest concentration of TMV was in the SC, followed by ED and EM. A similar viral concentration gradient was observed in each individual tobacco seed from all three experimental plants. This is the first detection of TMV in tobacco embryos and suggests the virus can be seed transmitted. / Master of Science / Tobacco mosaic virus (TMV) is a widely studied plant virus that affects tobacco, tomato, pepper, and many other crops throughout the world. The virus is easily transmitted through contaminated tools or machinery, workers’ hands or clothing, or when an infected leaf comes into contact with an uninfected leaf. For years, TMV transmission was said to be seedborne, not seed transmitted, meaning that seedling infection comes from the infected external seed coat of the seed. Seed transmission of the virus has yet to be proven because of the difficulty to fully separate tobacco seed tissues. The objective of this study was to identify TMV concentrations in the three components of an individual tobacco seed: seed coat (SC), endosperm (ED), and embryo (EM). Six hundred seed from TMV infected tobacco plants were carefully dissected into the three components. Total RNA was extracted from each sample, and synthesized into cDNA for analysis. A quantitative real-time polymerase chain reaction (RT-qPCR) assay was developed to quantify viral concentrations in each component. Endpoint PCR was used to confirm the quantitative results of RT-qPCR. Results revealed TMV accumulation in all three components of a tobacco seed, the highest concentration detected in the SC, followed by ED and EM; this pattern was observed from each plant. This is the first report of TMV being detected in embryos of tobacco seed which suggests that TMV can be seed transmitted.

The Tobacco mosaic virus (TMV) is a positive sense single stranded RNA virus and is found across the world. TMV can impact the overall yield and quality of the crop resulting in an economic loss. Plants that are infected with TMV show a variety of symptoms such as mosaic pattern, mottling, necrotic lesions and stunted growth. Historically, TMV has caused controversy on whether this economically significant virus is seedborne or seed transmitted. The objective of this study is to track TMV infection from infected seeds to seedlings to determine the percentage of seed transmission. This experiment used three pods from three different TMV infected cultivar K 326 flue-cured tobacco plants. Seeds from each pod were germinated in a growth chamber for approximately ten days. Samples were separated into seed coat, root and leaves after germination. Total RNA was extracted from each part and synthesized into cDNA for analysis. A quantitative real-time PCR (RT-qPCR) assay was used to determine TMV concentration of each sample. Endpoint RT-PCR was used to determine a conservative threshold value from the RT-qPCR results. These results demonstrated that TMV influenced percent germination with a range from 94% to 50%. Seed coats had a significantly higher virus titer concentration (P < 0.05) when compared to the roots and leaves. Statistical analysis revealed highly significant (P < 0.0001) differences among pods for virus titer and there is a highly significant plant by pod interaction (P < 0.0001). Endpoint RT-PCR confirmed TMV infection in leaves, roots and seed coats. Percent infection in leaves ranged from 2% to 24% and percent infection for roots ranged from 8% to 40%. Results demonstrate that TMV is seed transmitted in flue-cured tobacco. / Master of Science / The Tobacco mosaic virus (TMV) is an RNA virus that occurs globally in areas where tobacco is grown. TMV is a tobamovirus and infects over 350 plant species. TMV can reduce the yield and quality of the crop which will result in an economic loss for the grower. Plants that are infected with TMV show a variety of symptoms such as mosaic pattern, necrotic lesions, and stunted growth, and there are no effective ways to eradicate the virus. There has been controversy on whether to categorize TMV as a seedborne virus or a seed-transmitted virus because the location of the virus within a seed is unknown. This study examined seeds from three pods grown on three different TMV-infected flue-cured tobacco plants of cultivar K 326 to track TMV infection from infected seeds to seedlings. Seeds from each pod were germinated in a growth chamber for ten days and samples were separated into leaves, root and seed coat. Each sample had total RNA extracted and synthesized into cDNA for analysis. A quantitative real-time PCR (RT-qPCR) assay was used to determine TMV concentration of each sample since this technology can detect small amounts of virus. Endpoint RT-PCR was used to conservatively determine an infection threshold value from the RT-qPCR results. Percent germination of TMV infected seeds ranged from 94% to 50%. Seed coats had a significantly higher virus titer (P < 0.05) when compared to the roots and leaves in each pod. Statistical analysis showed (P < 0.0001) differences among pods for virus titer and there is a highly significant plant by pod interaction (P < 0.0001). Endpoint RT-PCR confirmed TMV infection in leaves, roots and seed coats. Percent infection in iv leaves ranged from 2% to 24% and percent infection for roots ranged from 8% to 40%. Therefore, results show that TMV is seed-transmitted in flue-cured tobacco.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-10T15:33:36Z No. of bitstreams: 1 Francisco Conrado Queiroz Carvalho.pdf: 693930 bytes, checksum: 0e5f734618ab807f4c62e74984f62954 (MD5) / Made available in DSpace on 2017-03-10T15:33:36Z (GMT). No. of bitstreams: 1 Francisco Conrado Queiroz Carvalho.pdf: 693930 bytes, checksum: 0e5f734618ab807f4c62e74984f62954 (MD5) Previous issue date: 2012-02-29 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Bacterial fruit blotch caused by Acidovorax citrulli occurs in different parts of watermelon plant, at different stages of development but symptoms are more conspicuous and easy to diagnose in fruit. BFB has caused significant economic losses to melon production in Brazil and is a major threat to the watermelon fields. This justifies the research for resistance sources to be used in breeding programs aiming to obtain varieties resistant to BFB, since they do not yet exist. To select genotypes with potential use in the management of fruit blotch, the resistance level of watermelon genotypes belonging to the Cucurbits Germplasm Active Bank for the Brazilian Northeast (Banco Ativo de Germoplasma de Cucurbitáceas para o Nordeste Brasileiro - BAG) of Embrapa Semiárido was evaluated at different plant developmental stages: seeds (74 genotypes), seedlings and plants before flowering (29 genotypes) as well as plants during flowering and fruiting (7 genotypes). The genotypes were evaluated for the incidence or severity of the disease, which was estimated with the aid of descriptive scales. Additionally, A. citrulli transmission was determined in seeds derived from symptomatic and asymptomatic fruits. No watermelon genotype was immune to fruit blotch, and the majority showed variations in resistance responses. However, the genotypes BGCIA 979, BGCIA 34 and Sugar Baby showed high levels of resistance at most stages of plant development, thereby suggesting that these genotypes possess fruit blotch resistance genes that could be used in breeding programs. Seeds from symptomatic and asymptomatic fruits of the seven tested genotypes showed transmission rates of A. citrulli up to 35.3% and 8.7%, respectively. These results confirm that asymptomatic fruits can harbor contaminated seeds that are responsible for the transmission of the bacteria. / A mancha aquosa, causada pela bactéria Acidovorax citrulli, ocorre em distintos órgãos de melancia, em diferentes estádios de desenvolvimento, sendo os sintomas mais comuns e de fácil diagnose nos frutos. Essa doença é responsável por elevadas perdas econômicas na cultura do meloeiro no Brasil e uma grande ameaça para a melancia. Isto justifica a busca de fontes de resistência a serem utilizadas em programas de melhoramento visando à obtenção de variedades dessa olerícola com resistência a doença. Com o objetivo de selecionar genótipos com potencial de utilização no manejo da mancha aquosa, avaliou-se o nível de resistência de 74 genótipos de melancia pertencentes ao Banco Ativo de Germoplasma de Cucurbitáceas para o Nordeste Brasileiro (BAG) da Embrapa Semiárido, em diferentes estádios de desenvolvimento da planta: sementes (74 genótipos), plântulas e plantas antes da floração (29 genótipos), plantas durante a floração e frutificação (7 genótipos). Os genótipos foram avaliados quanto a incidência ou severidade da doença, esta estimada com auxílio de escalas descritivas. Adicionalmente, foi determinada a transmissão de A. citrulli em sementes oriundas de frutos sintomáticos e assintomáticos. Nenhum genótipo de melancia foi imune à mancha aquosa, e a maioria apresentou variação nas reações de resistência. Porém, os genótipos BGCIA 979, BGCIA 34 e ‘Sugar Baby’ mostraram altos níveis de resistência na maioria dos ensaios realizados, indicando possuírem genes para resistência à mancha aquosa que poderão ser utilizados em programas de melhoramento. Sementes de frutos sintomáticos e assintomáticos dos sete genótipos apresentaram transmissão de A. citrulli de até 35,3% e 8,7% respectivamente, confirmando que frutos assintomáticos podem abrigar sementes contaminadas responsáveis pela transmissão da bactéria.

No description available.

La transmission aux semences est l’un des moyens les plus efficaces de survie et de dispersion pour les champignons phytopathogènes. Les semences ainsi contaminées sont altérées dans leur germination et leur viabilité. De ce fait, nous avons cherché à identifier des mécanismes moléculaires qui pourraient être impliqués dans cette capacité de transmission en utilisant le pathosystème modèle Alternaria brassicicola - Arabidopsis thaliana. Pour cela, nous avons analysé la réponse d’A. brassicicola soumis à différentes contraintes in vitro et in vivo :l’exposition à des métabolites de défenses de la famille des Brassicacées (brassinine,camalexine et isothiocyanate) et à des perturbations de la balance hydrique (dessiccation,sorbitol and PEG) ainsi que lors de la colonisation de la semence à partir des siliques. Nous avons montré que la cible probable des phytoalexines indoliques était la mitochondrie avec notamment une altération de la respiration et du potentiel membranaire mitochondrial après une courte exposition. Nos analyses ont aussi révélé que plusieurs protéines de type hydrophilines-like ou en lien avec formation des eisosomes, semblaient être impliquées dans la réponse au stress hydrique. Nous avons également montré que l’expression de la majorité des gènes codant ces protéines était dépendante d’au moins une des trois protéines kinases,AbSch9, AbNik1 and AbHog1. Enfin, nos analyses in planta ont permis d’identifier un mécanisme inattendu, impliquant le remodelage de la chromatine comme élément potentiel,de la régulation de l’expression génique du champignon lors de l’infection. / Seed transmission is one of the most effective means of survival and dispersal for plant pathogenic fungi. The contaminated seeds are altered in their germination and viability. As a result, we have sought to identify molecular mechanisms that could be involved in this transmission capacity using the Alternaria brassicicola - Arabidopsis thaliana pathosystem model. To do this, we analyzed the response of A. brassicicola subjected to different stresses in vitro and in vivo: exposure to defence metabolites of the Brassicaceae family (brassininin, camalexin and isothiocyanate) and to perturbations of the water balance (desiccation, sorbitol and PEG) as well as during seed colonization from silicics. We have shown that the likely target of indolic phytoalexins is mitochondria, including impaired respiration and mitochondrial membrane potential after short exposure. Our analyses also revealed that several hydrophiline-like proteins or proteins related to eisosome formation appeared to be involved in the response to water stress. We have also shown that the expression of the majority of genes encoding these proteins is dependent on at least one of the three protein kinases, AbSch9, AbNik1 and AbHog1. Finally, our in planta analyses identified an unexpected mechanism, involving the remodelling of chromatin as a potential element in regulating the gene expression of the fungus during infection.

Groundnut or peanut (Arachis hypogaea L.) is an important legume cultivated in several developing countries in the tropics and subtropics. It plays a significant role as a food crop in regions with alarming population growth rates. The disease “peanut clump”, which is caused by viruses in the genus Pecluvirus, has been reported from India and from several countries of West Africa. In India, the causal agent is the Indian peanut clump virus (IPCV), which is transmitted by a soil-borne root parasite, Polymyxa graminis. The virus is also transmitted by infected seed and so far no economical method of control has been found. Therefore efforts have been concentrated on understanding the epidemiology of peanut clump disease with the aim of devising cultural methods of control. The work addressed in this thesis describes how investigation in various aspects of clump disease epidemiology, including identification of alternative hosts of the virus and the vector, and of factors that contribute to survival and spread of inoculum, has led to formulation of simple cultural practices that could reduce disease incidence.

Groundnut or peanut (Arachis hypogaea L.) is an important legume cultivated in several developing countries in the tropics and subtropics. It plays a significant role as a food crop in regions with alarming population growth rates. The disease “peanut clump”, which is caused by viruses in the genus Pecluvirus, has been reported from India and from several countries of West Africa. In India, the causal agent is the Indian peanut clump virus (IPCV), which is transmitted by a soil-borne root parasite, Polymyxa graminis. The virus is also transmitted by infected seed and so far no economical method of control has been found. Therefore efforts have been concentrated on understanding the epidemiology of peanut clump disease with the aim of devising cultural methods of control. The work addressed in this thesis describes how investigation in various aspects of clump disease epidemiology, including identification of alternative hosts of the virus and the vector, and of factors that contribute to survival and spread of inoculum, has led to formulation of simple cultural practices that could reduce disease incidence.