Submitted by Maike Costa (maiksebas@gmail.com) on 2017-07-07T12:56:32Z No. of bitstreams: 1 arquivototal.pdf: 3524208 bytes, checksum: 1dfa6b7ebfd1711a48733025c4052ee9 (MD5) / Made available in DSpace on 2017-07-07T12:56:32Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3524208 bytes, checksum: 1dfa6b7ebfd1711a48733025c4052ee9 (MD5) Previous issue date: 2017-02-13 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The human body has a large bacterial flora, but when these bacteria, the principle of commensal character, become part of site other than it's natural, they can cause some severe diseases. Researches that are based either on the search of new drugs from plants or on the improvement of phytotherapics are in prominence and continue to play an important role nowadays. In this perspective, the aim was to carry out a phytochemical study of Solanum capsicoides All. fruits to isolate and characterize the chemical substances of this species and to use in silico studies to carry out investigations of new molecules potentially active for methicillin resistant Staphylococcus aureus (MRSA) and Escherichia coli using a database created with secondary metabolites isolated from Solanum genus. The phytochemical study of Solanum capsicoides All. fruits resulted in the isolation of three substances: carpesterol, acetylated glucose and 4-hydroxybenzaldehyde. A review of the literature led to the creation of a database with 421 different secondary metabolites isolated from the Solanum genus. Two databases from CHEMBL were selected. The first one with activity against MRSA and another against E. coli. The compounds were classified according to the pIC50 values to generate and validate the model using "Random Forest"(RF). The structure of six new target proteins against S. aureus obtained from the PDB (Protein database) were used for virtual screening of the based on the receptor structure using docking studies by the Molegro Virtual Docker, reaching to select Solanum database molecules capable of interacting in the binding sites of proteins. The RF prediction model for MRSA obtained a percent accuracy of 81%, area under the Receiver Operating Characteristic (ROC) curve of 0.885, selecting 8 molecules with an active potential above 60%. The prediction model for Escherichia coli obtained an accuracy rate of 88%, area under ROC curve of 0.932, selecting 4 molecules with potential probability above 84%. The study of the coupling of six target enzymes to S. aureus selected an average of 50 molecules from the bank of 421 isolated molecules of the genus Solanum with an ability to interact with on active site of each enzyme. In addition, it was possible to obtain 1 molecule with active potential and interaction capacity with 5 enzymes studied, 7 molecules interacting with 3 enzymes and 6 with 2 enzymes of S. aureus. The rutin, a molecule potentially active in the in silico study for S. aureus and E. coli, together with carpesterol, were tested in vitro against these bacteria. Microbiological tests have shown that carpesterol has no antimicrobial activity for the studied strains, and that the rutin has activity only for E. coli. An interaction study with strains of S. aureus ATCC 25923, a standard strain sensitive to all antibiotics, and SAM-01, a multidrug resistant strain, was designed. There was interaction only between rutin and oxacillin, one of the three antibiotics studied in the interaction, for a strain SAM- 01, reducing the resistance of this strain. / O ser humano possui uma vasta flora bacteriana que é comensal, mas quando essas bactérias, a princípio de caráter comensal, passam a fazer parte de outro sítio que não o seu natural, podem causar graves patogenias. Pesquisas que se fundamentam na busca por novos medicamentos a partir de plantas ou no melhoramento de fitoterápicos já existentes vem se destacando e continuam a desempenhar um papel importante nos dias de hoje. Nessa perspectiva objetivou-se realizar um estudo fitoquímico dos frutos de Solanum capsicoides All. para isolar e caracterizar substâncias químicas desta espécie e, utilizando estudos in silico, realizar investigações de novas moléculas potencialmente ativas para Staphylococcus aureus resistente a meticilina (MRSA) e Escherichia coli, utilizando um banco de dados criado com metabólitos secundários isolados do gênero Solanum. O estudo fitoquímico dos frutos de Solanum capsicoides All. resultou no isolamento de três substancias: carpesterol, glicose acetilada e 4-hidroxi-benzaldeído. A revisão de literatura levou à criação de um banco de dados com 421 diferentes metabólitos secundários isolados do gênero Solanum. Foram selecionados dois bancos de dados obtidos a partir do CHEMBL. O primeiro com atividade contra S. aureus multirresistente (MRSA) e o outro contra E. coli. Os compostos foram classificados de acordo com valores de pIC50 para gerar e validar o modelo utilizando “Random Forest”(RF). A estrutura de seis novas proteínas alvo contra S. aureus obtidas do Protein Data Bank (PDB) foram utilizadas para triagem virtual baseada na estrutura do receptor utilizando estudos de “docking” com o software Molegro Virtual Docker, a fim de selecionar moléculas do banco de dados de Solanum com potencial capacidade de interagir nos sítios de ligação dessas proteínas. O modelo RF de predição para S. aureus multirresistente obteve uma porcentagem de acerto de 81%, área sob a curva Receiver Operating Characteristic (ROC) de 0,885, selecionando 8 moléculas com potencial ativo superior a 60%. O modelo de predição para Escherichia coli obteve taxa de acerto de 88%, área sob curva ROC de 0,932, selecionando 4 moléculas com probabilidade de potencial ativo superior a 84%. O estudo do docking das seis enzimas alvo para S. aureus selecionou uma média de 50 moléculas do banco de 421 moléculas isoladas do gênero Solanum com a capacidade de interagir no sitio ativo de cada enzima. Analisando moléculas multitarget, foi possível obter 1 molécula com potencial ativo e capacidade de interação com 5 das 6 enzimas estudadas, 7 moléculas interagindo com 3 enzimas e 6 com 2 enzimas de S. aureus. A rutina, uma molécula potencialmente ativa no estudo in silico para S. aureus e E. coli, juntamente com o carpesterol, foram testadas in vitro contra essas bactérias. Os testes microbiológicos mostraram que o carpesterol não possui atividade antimicrobiana para as cepas estudadas, e que a rutina possui atividade apenas para a cepa de E. coli. Foi realizado ainda estudo de interação com as cepas de S. aureus ATCC 25923, uma cepa padrão sensível a todos os antibióticos, e SAM-01, uma cepa multirresistente. Houve interação apenas entre a rutina e a oxacilina, um dos três antibióticos estudados na interação, para a cepa SAM-01, diminuindo a resistência dessa cepa.

Submitted by CARLOS ALBERTO CAVALCANTE DE ALBUQUERQUE null (albuquerque.carlos@hotmail.com) on 2018-04-19T14:49:51Z No. of bitstreams: 1 Dissertação Carlos final.pdf: 12149144 bytes, checksum: e45321e644e989ee6c3556aa60433475 (MD5) / Approved for entry into archive by Maria Lucia Martins Frederico null (mlucia@fca.unesp.br) on 2018-04-19T15:43:55Z (GMT) No. of bitstreams: 1 albuquerque_cac_me_botfca.pdf: 12026676 bytes, checksum: d87594f7ed62488795381734b4628e83 (MD5) / Made available in DSpace on 2018-04-19T15:43:55Z (GMT). No. of bitstreams: 1 albuquerque_cac_me_botfca.pdf: 12026676 bytes, checksum: d87594f7ed62488795381734b4628e83 (MD5) Previous issue date: 2018-01-12 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O gênero Bacillus tem demonstrado em diversos estudos eficiência no controle de estresses bióticos e abióticos, tornando-se uma alternativa viável para o manejo desses problemas. O objetivo desse estudo foi avaliar o potencial de isolados de Bacillus spp. em induzir resistência em plantas de tomateiro a estresses bióticos (Fusarium oxysporum f. sp. lycopersici raça 3 e Oidium), abiótico (estresse salino) e em promover crescimento. Inicialmente, foi realizada uma seleção in vitro utilizando testes bioquímicos (produção de ácido indol acético e sideróforos, solubilização de fosfato, fixação assimbiótica de nitrogênio e produção de celulase e de catalase) com 154 isolados. Posteriormente, com quatro isolados selecionados nessa primeira fase, mais um produto comercial à base de Bacillus subtilis QST-713, foram avaliados o potencial desses isolados na promoção de crescimento das plantas, na indução de resistência ao estresse salino e no controle de F. oxysporum f. sp. lycopersici e Oidium em plantas de tomate. Os isolados de Bacillus estimularam o crescimento das plantas no ensaio de promoção de crescimento sem estresse salino. No segundo estudo avaliou-se a indução de resistência ao estresse salino mediada por Bacillus e foram observados efeitos dos isolados de Bacillus na altura das plantas aos 41 e 48 dias após a semeadura e para a massa do sistema radicular, também os isolados de Bacillus estimularam a produção das enzimas peroxidase, fenilalanina amônia-liase, polifenoloxidase e de L-prolina indicando que os isolados de Bacillus ativaram ao menos dois mecanismos de defesa contra o estresse com sal. O isolado AP3 foi o mais eficiente no controle de Fusarium oxysporum f. sp. lycopersici raça 3 e de Oidium do tomateiro. Diante dos resultados conclui-se que o gênero Bacillus possuí versatilidade em promover controle biológico e o desenvolvimento das plantas em situação de estresse. / The Bacillus genus has demonstrated in several studies efficiency in the control of biotic and abiotic stresses, making it a viable alternative for the management of these problems. The objective of this study was to evaluate the potential of Bacillus spp. in inducing resistance in tomato plants to biotic stresses (Fusarium oxysporum f. sp. lycopersici race 3 and Oidium), abiotic (saline stress) and in growth promoting. Initially, an in vitro selection was performed using biochemical tests (indolacetic acid and siderophore production, phosphate solubilization, nitrogen assimilation and cellulase and catalase production) with 154 isolates. Then, with four isolates selected in this first phase, plus a commercial product based on Bacillus subtilis QST713, the potential of these isolates in the promotion of plant growth, induction of resistance to saline stress and in the control of Fusarium and Oidium in tomato plants were evaluated. Bacillus isolates stimulated plant growth in the growth promotion trial without saline stress. The second study evaluated the induction of resistance to saline stress mediated by Bacillus and observed effects of Bacillus isolates at plant height at 41 and 48 days after sowing and for root system mass. Bacillus isolates also stimulated production of the enzymes peroxidase, phenylalanine ammonia-lyase, polyphenoloxidase and L-proline, indicating that Bacillus isolates activated at least two defense mechanisms against salt stress. The AP3 isolate was the most efficient in the control of Fusarium oxysporum f. sp. lycopersici race 3 and Oidium in tomato. We concluded that Bacillus has the versatility to promote biological control and the development of plants under stress.

Orientador: Wagner Bettiol / Resumo: O gênero Bacillus tem demonstrado em diversos estudos eficiência no controle de estresses bióticos e abióticos, tornando-se uma alternativa viável para o manejo desses problemas. O objetivo desse estudo foi avaliar o potencial de isolados de Bacillus spp. em induzir resistência em plantas de tomateiro a estresses bióticos (Fusarium oxysporum f. sp. lycopersici raça 3 e Oidium), abiótico (estresse salino) e em promover crescimento. Inicialmente, foi realizada uma seleção in vitro utilizando testes bioquímicos (produção de ácido indol acético e sideróforos, solubilização de fosfato, fixação assimbiótica de nitrogênio e produção de celulase e de catalase) com 154 isolados. Posteriormente, com quatro isolados selecionados nessa primeira fase, mais um produto comercial à base de Bacillus subtilis QST-713, foram avaliados o potencial desses isolados na promoção de crescimento das plantas, na indução de resistência ao estresse salino e no controle de F. oxysporum f. sp. lycopersici e Oidium em plantas de tomate. Os isolados de Bacillus estimularam o crescimento das plantas no ensaio de promoção de crescimento sem estresse salino. No segundo estudo avaliou-se a indução de resistência ao estresse salino mediada por Bacillus e foram observados efeitos dos isolados de Bacillus na altura das plantas aos 41 e 48 dias após a semeadura e para a massa do sistema radicular, também os isolados de Bacillus estimularam a produção das enzimas peroxidase, fenilalanina amônia-liase, polifenoloxidas... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Made available in DSpace on 2018-08-01T22:35:03Z (GMT). No. of bitstreams: 1 tese_8433_Rodolfo Ferreira de Mendonca.pdf: 1124384 bytes, checksum: ab37968507959f59b13bdb6b4a472d5b (MD5) Previous issue date: 2017-02-22 / O tomateiro é uma cultura de grande importância econômica e social para o Brasil, que pode ser afetada por muitos problemas fitossanitários, sendo a pinta preta uma das principais doenças. O objetivo deste trabalho foi avaliar a utilização de diferentes práticas culturais associadas ao uso de fungicida no manejo da pinta preta do tomateiro em condições de campo. Foram conduzidos dois experimentos em blocos casualizados, com parcelas sub-subdivididas na área experimental do Centro de Ciências Agrárias e Engenharias da Universidade Federal do Espírito Santo, em Alegre ES. No primeiro experimento, as parcelas foram constituídas de três sistemas de tutoramento, as subparcelas, de três intervalos entre as aplicações de fungicidas, e as sub-subparcelas, por três sistemas de remoção de folhas, com três repetições. No segundo experimento, as parcelas foram constituídas de três espaçamentos entre plantas, as subparcelas, de três intervalos entre as aplicações de fungicidas, e as sub-subparcelas, por três alturas de poda, com três repetições. Foram realizadas avaliações semanais da severidade da pinta preta, empregando-se escala diagramática própria para cálculo dos valores da Área Abaixo da Curva de Progresso da Doença (AACPD) para cada tratamento. Foram calculadas também a duração da área foliar sadia (HAD) e da absorção da área foliar sadia (HAA) e avaliada a produtividade do tomateiro em função dos tratamentos utilizados. A viabilidade econômica de cada tratamento foi calculada pela subtração entre receita e custo de cada parcela nos dois experimentos. Utilizou-se o programa computacional R para as análises estatísticas. Para o primeiro experimento, a eficácia da aplicação de fungicida ficou comprovada quando verificam-se os maiores valores de AACPD em parcelas sem aplicação. O tutoramento vertical proporcionou maior produtividade independente da remoção de inóculo e do intervalo de aplicação de fungicida. O efeito da remoção de folhas doentes reduziu o progresso da pinta preta e aumentou a produtividade do tomateiro, sendo que a remoção de folhas com valores ≥ 16% de severidade foi o sistema mais efetivo, permitindo reduzir o número de aplicações de fungicida sem comprometer a eficácia de controle. O efeito da altura de poda influenciou no progresso da pinta preta do tomateiro. Observou-se maiores produtividades com espaçamentos de 45 e 75 cm entre plantas que foram podadas na altura do 7º e 10º cachos. No espaçamento de 30 cm, o lucro foi maior quando foi feita a poda no 3º cacho. Conclui-se portanto que plantas conduzidas no tutoramento vertical, com aplicação quinzenal de fungicida e remoção de inóculo com severidade ≥ 16% no primeiro experimento e plantas conduzidas no espaçamento de 45 cm entre plantas com aplicação semanal de fungicida e poda na altura do 10º cacho no segundo experimento foram mais efetivas em reduzir o progresso da doença e aumentar HAD, HAA, produtividade e lucro. Palavras-chave: Solanum lycopersicum, Alternaria solani, controle cultural, controle químico, perdas.

Made available in DSpace on 2014-07-29T15:16:34Z (GMT). No. of bitstreams: 1 pabline marinho 1.pdf: 394787 bytes, checksum: 1f2c3791104aa80d0a7f1fcfead4fa07 (MD5) Previous issue date: 2008-02-23 / Solanum paniculatum L., popularly known as jurubeba, is a widespread plant species used in Brazilian folk medicine as a tonic, antifever agent, colagogue, bitter, and eupeptic to treat liver and gastric dysfunctions. The chemical composition of Solanum paniculatum has long been studied and many substances have been isolated from the entire plant, including alkaloids, resins, glucids and saponins. The aim of this study was to evaluate the mutagenic, antimutagenic and cytotoxic effects of jurubeba s ethanolic leaf and fruits extracts using micronucleus test in mice bone marrow. The experimental procedure was performed according Schimid (1975). Swiss mice were orally treated with three different concentrations (100, 200 or 300 mg/kg) of leaf or fruit extracts of S. paniculatum. To evaluate the antimutagenic activity were used the same doses of the extract simultaneously with a single dose of Mitomicin C (4mg/kg) i.p. Cytotoxicity was evaluated by the polychromatic and normochromatic erythrocytes ratio. Our results have indicated that the ethanolic leaf extract of S. paniculatum did not exhibit mutagenic effect in mice, but it showed antimutagenic effects in this extract towards MMC. The cytotoxicity was strongly demonstrated specially for higher doses. Meanwhile, the ethanolic fruit extract of S. paniculatum was mutagenic only in dose of 200 mg/Kg at time of 48 hours. It was showed the cytotoxic activity for higher doses. However, no antimutagenic effect was evidenced by fruit extract. / Solanum paniculatum L., vulgarmente conhecida como jurubeba, ocorre em toda a América tropical, especialmente no Cerrado. A ela são atribuídas propriedades medicinais, sendo popularmente utilizada no tratamento da icterícia, da hepatite crônica, de febres intermitentes, ressaca, indigestão e como cicatrizante, além de usos culinários. A análise fitoquímica dessa espécie detectou substâncias como: alcalóides, glicoalcalóides, saponinas e resinas. Devido à grande utilização da planta Solanum paniculatum pela população como recurso terapêutico e alimentício, o presente estudo teve como objetivo avaliar a atividade mutagênica, antimutagênica e citotóxica do extrato etanólico das folhas e frutos de S. paniculatum utilizando o Teste do Micronúcleo em medula óssea de camundongos. O procedimento experimental foi realizado de acordo com Schimid (1975). Os animais foram tratados via gavage em três diferentes concentrações (100, 200 ou 300 mg/kg) do extrato etanólico das folhas ou dos frutos de Solanum paniculatum. As mesmas dosagens juntamente com uma dose única de Mitomicina C (4 mg/Kg) i.p. foram administradas na avaliação da antimutagenicidade. Genotoxicidade e antigenotoxicidade foram avaliadas pela freqüência de eritrócitos policromáticos micronucleados, enquanto a citotoxicidade foi avaliada pela relação entre eritrócitos policromáticos e normocromáticos. Os resultados mostraram que o extrato etanólico das folhas de S. paniculatum não demonstrou ação mutagênica, no entanto, em doses mais elevadas o extrato exibiu a atividade citotóxica e antimutagênica. Quanto ao extrato etanólico dos frutos, este demonstrou atividade mutagênica na dose de 200 mg/Kg em 48 horas de administração, foi mostrada ação citotóxica em doses mais elevadas e não foi observada ação moduladora de mutagenicidade induzida por mitomicina C.

Fusarium is a soilborne fungus which can live in soil for long periods of time. It is known to cause wilt, root rot and crown rot diseases in a diverse group of crop plants. Of all the diseases caused by Fusarium the most important are the vascular wilts. Pathogens that cause wilting usually enter their host plant through young roots and then grow into and up the water-conducting vessels of the root and stem. The vessels become blocked and water supply to the leaves is limited. This results in the potato plant being weak resulting in yellowing of leaves, browning of stems and production of smaller tubers. Fusarium is diverse and widely distributed and can be isolated from agricultural soils and plant material. The study was done to determine the occurrence of this pathogen in the South African potato industry. Samples of plant material showing wilt symptoms were collected from nine potato production regions. Fungal isolations were made from tubers using a Fusarium selective medium, i.e Peptone PCNB Agar. The isolates were examined morphologically and those resembling Fusarium were further identified using molecular techniques. DNA sequence analysis of the translation elongation factor 1-α gene was done on the isolates. DNA-based techniques have increasingly become the tool of choice for understanding the genetic diversity and phylogeny of Fusarium species. The pathogenicity of the isolates from all the regions was also investigated on potato cultivar Caren. The DNA results confirmed Fusarium as the pathogen causing Fusarium wilt on potatoes. Two species of Fusarium were identified; namely F. oxysporum and F. solani. F. oxysporum was more prevalent and occurred in all regions compared to F. solani. F. oxysporum is best known for the plant pathogenic strain, which cause wilt, root rot and crown rot diseases on a wide variety of crops, often limiting crop production. It is also known to be phylogenetically diverse. In the pathogenicity test, the isolates were found to be virulent and one was highly virulent therefore confirming their ability to cause wilting of potatoes. The effect of silicon on Fusarium wilt of potatoes was investigated in this study to assess its effectiveness in the control of Fusarium wilt. An in vitro study using potassium silicate was done to determine if silicon can inhibit the growth of Fusarium at different concentrations. The results showed that at low concentrations of potassium silicate the growth of Fusarium was not inhibited, while at a high concentration, there was inhibition. Greenhouse pot trials were conducted to determine the effect of silicon soil amendments on Fusarium wilt of potatoes, tuber yield and the production of phenolics in the cell wall of potato peels. The levels of chlorogenic, caffeic and ferulic acids were also investigated. The following treatments were used: control, silicon ash (~99% Si), slag (30% Si), fly ash (50% Si) and lime (calcium carbonate) as a pH control. Treatments were divided into those inoculated with Fusarium and those without Fusarium. Results showed that for silicon treatments not inoculated with Fusarium, slag had the highest tuber yield, followed by lime, fly ash and silicon ash when compared to the control. Silicon treatments inoculated with Fusarium did not improve the yield as the control had the highest yield and the occurrence Fusarium wilt was not reduced in silicon treatments. In this regard silicon did not have an effect on Fusarium wilt because symptoms were visible in the silicon amended treatments. The results for phenolic acid content showed that ferulic acid levels were too low for analysis; for chlorogenic acid, concentrations were generally lower in the silicon treatments than in the treatments without silicon; and caffeic acid levels were higher in silicon treatments than treatments without silicon, as a result of increased production of as defence mechanism against invading pathogens. However, this is the first study on the effect of silicon on Fusarium wilt of potatoes and its influence on the production of phenolics. Further research is required to understand the role of silicon in potato pathosystems. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Microbiology and Plant Pathology / unrestricted

Pectobacterium carotovorum (Pbc), Dickeya spp., Pectobacterium atrosepticum (Pba), and a new, atypical strain, Pectobacterium carotovorum subsp. brasiliensis (Pbcb) can cause potato blackleg, soft rot and aerial stem rot. To determine the impact and extent of these pathogens on the South African potato industry, samples were collected from 72 potato fields in 10 potato production regions during the 2006/7 production seasons. During these seasons, blackleg outbreaks occurred in commercial production fields causing severe economic losses. Bacteria isolated from diseased material that were Gram-negative, facultative anaerobic with pectolytic ability were identified using a Multiplex PCR targeting the 16S-23S region. Isolates were subjected to partial sequencing of the 16S-23S rDNA and a subsequent PCR-RFLP of the 16S-23S rDNA region. Comparison of RFLP patterns of isolates to reference cultures confirmed the identity of the South African blackleg strains as Pbcb. This is the first report of Pbcb in South Africa. A total of 128 isolates were obtained from 10 production regions. Of these 77% were shown to be Pbcb, 17% Pbc and 6% unkown. Dickeya spp. and Pba were not detected in South Africa. From imported tubers 14 isolates were obtained, 13 of which were identified as Pbcb and one isolate as Pbc. Thus Pbcb was shown to be the most important causal agent of blackleg and soft rot in South Africa and poses a threat to the South African potato industry. Through a questionnaire survey the farmers` knowledge of soft rot / blackleg disease complex and the need for research on these potato diseases in South Africa was determined. A total of 41 questionnaires were collected from potato growers and analysed. Estimates of economic losses experienced by growers due to soft rot / blackleg disease complex, ranged from 1 to 70%. It appears that in South Africa disease symptoms are mainly prevalent at temperate (10 – 20°C) to warm climates with prolonged wet or humid conditions. To reduce disease incidence, low generation seed tubers need to be planted and good crop rotation systems need to be followed. Blackleg is mainly a seed-borne disease and is therefore difficult to control. This prompted the search for a possible management strategy to increase plant / tuber resistance to blackleg and soft rot and to minimise losses. The study also focussed on the effect of a calcium silicate slag soil amendment on phenolic formation in cell walls of potato peels, and subsequent tuber resistance to Pectobacterium carotovorum subsp. brasiliensis (Pbcb) . Pot trials were conducted with the following treatments: Control with and without the pathogen; slag (30% Si) with and without the pathogen; lime treatment (CaCO3) as pH control with and without the pathogen. Results from pot trials show that phenol production in potato stems and tubers increased in both lime and slag treatments, compared to the controls. This suggests that soil pH may play a role in phenol production. This could, however be due to increased silicon absorption by plants at higher pH values. Results also indicate that the best Si source to use is Calmasil, since Si and Ca combined have a synergistic effect in enhancing tuber resistance. This is, however, the first study on the effect of Si on defence responses of potatoes and further research is required to elucidate the modes-of-action of Si in the potato plant. This study has enhanced the understanding of the etiology of soft rot / blackleg diseases in South Africa and opened up new possibilities for the use of Si in commercial production to improve plant health status. However the need for future research on this disease complex has been highlighted in this thesis. / Dissertation (MSc)--University of Pretoria, 2009. / Microbiology and Plant Pathology / unrestricted

The BIRD protein JACKDAW (JKD) belong to the INDETERMINATE DOMAIN (IDD) protein family shown to regulate many developmental processes in plants. JKD encodes a Zinc Finger Protein expressed in the root ground tissue and regulates root patterning in Arabidopsis thaliana (Arabidopsis). Recent and unpublished study indicates that JKD is involved in plant defense response in Arabidopsis. Here we study the JKD function in tomato plants (Solanum lycopersicum). We analyzed the tomato JKD orthologues (Solyc09g007550 (Solyc09) and Solyc10g084180 (Solyc10)) mutant lines, which were generated by Crispr-Cas and TILLING (Targeting Induced Local Lesions in Genomes). Our data indicate that, like in Arabidopsis, Solyc09 controls root ground tissue patterning; the mutant lines show extra cell division in the inner cortex and disturbed stem cell patterning. In addition, we found that both Solyc09 and Solyc10 control the root and stem thickness and regulate tomato leaf shape. To further investigate whether Solyc09 and Solyc10 have a function in tomato when subjected to biotic stress, we evaluated the mutants response to the necrotrophic fungi Botrytis cinerea. We found that the tomato bird mutants have less infection when compared to the control. Taken together our data show that Solyc09 and Solyc10 genes play an essential role in tomato root, shoot development, and in plant immune response to the pathogenic fungi.

Squalene synthase (EC 2.5.1.2.1; SQS) is located at a branch point in the isoprenoid pathway and catalyzes the condensation of two molecules of farnesyl diphosphate to form squalene. SQS activity contributes to the formation of triterpenes and sterols, including phytosterols, brassinosteroids, cholesterol, and in potato plants, steroidal glycoalkaloids (SGAs). These compounds have diverse functions in the plant. SGAs are defense compounds that deter feeding by potato pests. The wild potato Solanum chacoense accumulates higher amounts of SGAs than cultivated potato and some of its accessions produce leptines, a rare class of SGAs that is toxic to Colorado potato beetle. Unlike most eukaryotes, higher plants have more than one gene coding for SQS. Three sqs gene homologs were isolated from S. chacoense, sqs1Sc, sqs2Sc, and sqs4Sc, that have 74 to 83% identity at the amino acid level. Some of the amino acid differences between sqs isoforms are likely to affect enzyme activity. Each of the three genes contained an intron in the 3'UTR. This feature may have a role in the nonsense-mediated decay of incomplete sqs mRNAs. A partial SQS polypeptide retaining catalytic activity but lacking the membrane anchoring domain could adversely affect a cell with the randomly distributed accumulation of squalene. The mRNA of sqs1Sc and sqs2Sc was detected in all tissues whereas sqs4Sc transcript was limited to bud tissue. The sqs2Sc transcript was less uniformly distributed in the plant than sqs1Sc and accumulated most abundantly in floral tissue. The results demonstrate that the three sqs genes have different patterns of gene expression and encode proteins with different primary structures indicating distinct roles in plant squalene metabolism. / Master of Science

Tomato serves as an important model organism for Solanaceae in both molecular and agronomic research. With whole genome sequencing in progress, there is a need to study functional genetics through mutant lines that exceed the practical limitations imposed by the popular research cultivar, Micro-Tom. This project utilized Agrobacterium transformation and the transposon tagging construct, Ac-DsATag-Bar_gosGFP, to produce activation tagged and knockout mutants in the processing tomato variety, M82. The construct contained hygromycin resistance (hyg), green fluorescent protein (GFP), and maize transposase (TPase) on the stable Ac element, along with a 35S enhancer tetramer and glufosinate herbicide resistance (BAR) on the mobile Ds element. An in vitro propagation strategy was used to produce a population of 25 T0 plants from a single transformed plant regenerated in tissue culture. A T1 population of 10,568 selfed and M82 backcross progeny was produced from the functional T0 line. This population was screened by spraying with 0.05% Liberty® herbicide, followed by a 100 mg/L hygromycin leaf painting procedure to select for Ds only (herbicide tolerant and hygromycin sensitive) individuals. The T-DNA genotype of Ds only plants was confirmed through multiplex PCR and the location of insertions within the genome was determined through TAIL-PCR. Resulting product sequences were blasted against the pre-publication tomato genome browser to determine insertion sites. A population of 309 independent transposants dispersed to all twelve chromosomes from the original insertion site on chromosome five has been developed. The transposon tagged lines are currently being immortalized in seed stocks. / Master of Science