Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Ribosome inactivating proteins (RIPs) are potent toxins produced by a wide range of evolutionarily diverse plants. These toxins cause cell death by physically dismantling ribosomal RNA and shutting down protein synthesis. They also have a strong antiviral activity. Some believe that the antiviral property of RIPs is a function of ribosomal inactivation, others believe that the two properties are unrelated. RIPs are non-specific in their antiviral activity. Transgenic RIPexpressing plants are resistant to a wide spectrum of viruses. Many different viruses threaten grapevine. It is not practical to design individual remedies for each of these viruses. In this study, we screen the grapevine genome for the presence of a RIP gene using degenerate PCR primers. If a RIP gene does exist in grapevine, it is not being expressed in a useful way. We also clone several well-documented RIP genes from various plants into pGEM-T Easy: dianthin from Dianthus caryophyllus; p-Iuffin from Luffa octandra and mirabilis antiviral protein (MAP) from Mirabilis jalapa. These isolated genes are then subcloned into a selection of expression vectors: dianthin into pKK223-3, a bacterial expression vector; p-Iuffin into pCambia3301, a plant expression vector; and MAP into pFLAG, a yeast expression vector. The constructs prepared in this project may be used for the synthesis of RIP molecules. The exogenous application of RIPs has been shown to protect plants from viruses. Transformation of grapevine with the RIP-containing plant expression vector may result in a variety of vine that is resistant to a wide range viruses. This thesis describes preliminary work in an attempt to impart broad-spectrum antiviral resistance to grapevine. / AFRIKAANSE OPSOMMING: Ribosomale-inaktiverende proteïne (RIPs) is kragtige toksienes wat deur 'n wye verskeidenheid evolusionêr diverse plante verskaf word. Hierdie toksienes veroorsaak die dood van die selle deur fisies die ribosomale RNA af te breek en proteïensintese stop te sit. Hulle toon ook 'n sterk antivirale aktiwiteit. Sommige voel dat die antivirale eienskap van RIPs 'n funksie van ribosomale inaktivering is, terwyl ander glo dat die twee eienskappe onafhanklik optree. RIPs is in hul antivirale aktiwiteit onspesifiek. Transgeniese RIP-weergewende plante toon weerstand teen 'n wye spektrum virusse. Wingerd word deur baie verskillende virusse aangeval. Dit is onprakties om spesifieke teenmiddels vir elk van die virusse te ontwerp. In hierdie studie word die wingerdgenoom vir die voorkoms van 'n RIP-geen ondersoek, deur die gebruik van degeneratiewe PKR primers. As daar wel 'n RIP-geen in wingerd voorkom, word dit nie in 'n nuttige manier uitgedruk nie. Ons het ook 'n groep goedgedokumentêre RIP-gene vanuit verskeie plante in pGEM- T Easy gekloneer: dianthin vanuit Dianthus caryophyllus; p-Iuffin vanuit Luffa octandra; en mirabilis antivirale proteïen (MAP) vanuit Mirabilis jalapa. Hierdie geïsoleerde gene is toe in verskeie uitdrukkingsvektore gesubkloneer: dianthin in pKK223-3, 'n bakterïele uitdrukkingsvektor; p-Iuffin in pCambia3301, 'n plant uitdrukkingsvektor; en MAP in pFLAG, 'n gis uitdrukkingsvektor. Die constructs wat in hierdie projek voorberei is, kan gebruik word vir die sintese van RIP molekules. Dit is gevind dat die eksogeniese toepassing van RIPs plante teen virus-infeksie beskerm. Die transformasie van wingerd met die RIP-bevattende plant ekspressievektor kan 'n wingerd wat teen 'n wye verskeidenheid virusse bestand is tot stand bring. Hierdie tesis beskryf die voorlopige werk in 'n poging om breë-spektrum antivirale weerstand in wingerd deelagtig te maak.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Wheat rust, caused by the Puccinia spp., is a global biotic cause of wheat yield losses. This disease can effectively be combatted by implementing rust resistant wheat cultivars. The release of new resistant wheat cultivars is however prolonged due to the time needed to fix resistance genes in a good quality background and develop pure breeding wheat lines. The aim of this study was the pyramiding of novel species derived leaf and stripe rust resistance genes in bread wheat lines through the utilization of high throughput marker assisted selection and microspore derived doubled haploid technology. / AFRIKAANSE OPSOMMING: Koringroes het wêreldwyd verliese in koringopbrengste tot gevolg. Dit word veroorsaak deur die Puccinia fungi. Hierdie siekte kan effektief beveg word deur die verbouing van roesbestande kultivars. Die vrystel van nuwe weerstandbiedende kultivars is egter ‘n langdurige proses weens die tyd verbonde daaraan om weerstandsgene te fikseer in ‘n genetiese agtergrond met ‘n goeie kwaliteit en om dan suiwertelende lyne te ontwikkel. Die doelwit van hierdie studie was om nuwe spesie-verhaalde blaar- en streeproes weestandsgene in koringlyne te stapel met behulp van merker bemiddelde seleksie en mikrospoor geassosieerde verdubbelde haploïede tegnologie.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Wheat production is globally affected by several different wheat rust diseases. The rust diseases can effectively be controlled by the deployment of multiple resistance genes that confer durable resistance. One of the most effective strategies to incorporate resistance genes is by the implementation of recurrent mass selection as it maximizes opportunities for gene pyramiding. The implementation of a recurrent mass selection program in wheat can effectively be enhanced with the use of genetic male sterility and the incorporation of maker assisted-selection (MAS). The aim of the study was to pyramid wheat rust resistance genes in wheat lines by utilizing a male sterile mediated marker-assisted recurrent selection breeding (MS-MARS) scheme. An existing segregating MS-MARS base population and resistance donor lines carrying genes of interest (Sr26, Sr35 and Sr45) were used as female and male crossing parents. Potential markers for the genes of interest were first identified and validated on the male population. PCR based markers tested for Sr26 and Sr45 easily distinguished between resistant and non-resistant plants in the study, while markers tested for the detection of Sr35 and Sr45 in most instances failed to do so. The identified Sr26 marker (Sr26#43) was successfully added to the SU-PBL’s standardized marker set in a multiplex reaction. The standardized marker set and the co-dominant PCR marker for Sr45 were used to screen male and female populations before and after cross-pollination. Several wheat rust resistance genes were present in various frequencies in both male and female populations prior to the first crossing cycle, except Sr26 and Sr45. Increases in gene frequencies and combinations were obtained after the first crossing cycle, highlighting the effectiveness of the MS-MARS breeding strategy to improve gene frequencies of desirable genes. Two MS-MARS crossing cycles were successfully completed and large numbers of hybrid seeds were produced in a short period of time by selecting male sterile plants based on distinct characteristics induced by the dominant male sterility gene. Future studies will include the wide deployment of Sr26 and Sr45 in the MS-MARS breeding program as markers are now available and can be included in the SU-PBL’s standardized marker set for the effective detection of these genes, the development of gene-specific markers for Sr35 to ascertain the presence of the gene in the MS-MARS population and the specific selection of male sterile plants with wide open glumes to maximize outcrossing rates. / AFRIKAANSE OPSOMMING: Koring produksie word wêreldwyd aansienlik deur koringroes siektes geaffekteer. Die siektes kan doeltreffend beheer word deur die ontplooing van veelvuldige weerstandsgene, wat langdurige weerstand tot gevolg het. Een van die mees doeltreffendste strategieë om weerstandsgene in n koring plant te inkorporeer is deur die implementering van herhalende massa seleksie (HMS), siende dat dit geleenthede vir geen stapeling maksimaliseer. Die implementering van 'n HMS program in koring kan effektief aangewend word met behulp van genetiese manlike steriliteit en merker bemiddelde seleksie (MBS). Die doelwit van hierdie studie was om veelvuldige koringroes weerstandsgene in koring lyne te stapel met behulp van die manlik steriliteits merker bemiddelde herhalende seleksie (MS-MBHS)-telingsskema. ‘n Gevestigde segregerende MS-MARS basis populasie en donor lyne, wat die gene (Sr26, Sr35 en Sr45) van belang dra, was onderskeidelik as vroulike en manlike kruisingsouers gebruik. Potensiële molekulêre merkers vir die gene van belang was eers geidentifiseer in literatuur en op die donor lyne getoets, voordat dit vir die opsporing van die gene in die nageslag gebruik was. Polimerase ketting reaksie (PKR)-gebaseerde merkers wat getoets was vir Sr26 en Sr45, kon maklik tussen weerstand en nie-weerstandbiedende plante in die studie onderskei, terwyl ander merkers vir die opsporing van Sr35 en Sr45 nie so doeltreffend was nie. Die geidentifiseerde Sr26 merker was suksesvol bygevoeg tot die SU-PBL se gestandardiseerde merkerpaneel, in ‘n multipleks reaksie. Die gestandardiseerde merkerpaneel en die ko-dominante PKR merker vir Sr45 was gebruik om die manlike en vroulike populasie te analiseer vir die teenwoordigheid van verskeie weerstandsgene voor en na kruisbestuiwing. Merker analise het die teenwoordigheid van verskeie koringroes weerstandsgene in verskillende frekwensies in beide die manlike en vroulike populasie voor die eerste kruising siklus aangedui. Sr26 en Sr45 was egter afwesig in beide populasies. ‘n Toename in geen frekwensies en kombinasies was waargeneem na die eerste kruising siklus. Dit het gevolglik die doeltreffendheid van die MS-MARS teling strategie beklemtoon. Twee herhalende kruising siklusse was suksesvol voltooi en groot hoeveelhede bastersaad was verkry vanaf steriele plante wat geselekteer was op grond van unieke eienskappe wat hulle vertoon as gevolg van die manlike steriliteits geen. Toekomstige studies sluit in, die groot skaalse gebruik van Sr26 en Sr45 in die MS-MARS teelprogram aangesien merkers nou beskikbaar is en gebruik kan word in die MS-MARS teelprogram vir die doeltreffende opsporing van hierdie gene, die ontwikkeling van ‘n geen-spesifieke merker vir Sr35 om die teenwoordigheid van die geen in die MS-MARS populasie vas te stel, en die selektering van manlike steriele plante met wyd oop kaffies om kruisbestuiwing te verhoog.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Grapevine leafroll disease (GLD), caused by the members of the family Closteroviridae, is one of the most economic important viral diseases affecting grapevine. Grapevine leafroll associated virus 3 (GLRaV-3), of the genus Ampelovirus, is the most widespread member of the leafroll associated virus family. To prevent the spread of GLD, management strategies such as rogueing and insect vector control are required to limit crop losses. Alternative control strategies based on genetic modification of the grapevine genome, such as pathogen-derived resistance (PDR), is proven to be effective in conferring resistance to several viruses. Therefore, the focus of this study was to evaluate pathogen-derived resistance strategies for GLRaV-3 using the following two approaches; 1) evaluation of transgenic plants expressing a dysfunctional GLRaV-3 heat shock protein 70 homolog (HSP70h) in order to confer resistance against GLRaV-3, and 2) the construction of artificial microRNAs (amiRNAs) to use as a tool for silencing specific sequences of GLRaV-3 in the grapevine host and the development of an amiRNA-mediated silencing validation system. In the first part of this study, six transgenic plant lines (plant lines #1, #3, #9, #14, #15 and #17) as well as a non-modified plant line, were inoculated with GLRaV-3 by grafting buds of each onto GLRaV-3 infected plant material. After approximately five months, GLRaV-3 virus titres of all grafted plants were quantified relative to two reference genes using RT-qPCR. Results were evaluated by comparing the relative virus titre of each transgenic plant line to that of the non-modified control plant line. Results showed that resistance levels of plant line #3 was significantly enhanced (>99%) and remarkably, plant line #14, showed to be more susceptible to the virus. The second part of the study was the construction and validation of amiRNAs targeting GLRaV-3 sequences. Two 21 nt regions of GLRaV-3 were successfully incorporated into miRNA backbone vvi167b of grapevine. Moreover, target constructs were developed by incorporating corresponding GLRaV-3 target sequences into the 3’ UTR of a green fluorescence protein (GFP) gene. Subsequently, the target constructs were co-infiltrated with the constructed amiRNA in Nicotiana benthamiana and GFP expression levels were quantified to determine the silencing efficiency of the amiRNAs. Results showed that the amiRNAs were successful in silencing the GFP target construct, however, they were not specific in silencing exclusively their corresponding target. These amiRNA constructs are ideal for further viral studies to determine the efficiency of silencing GLRaV-3 in GLD infected grapevines. / AFRIKAANSE OPSOMMING: Wingerd rolblaar siekte (GLD), wat veroorsaak word deur die lede van die familie Closteroviridae, is een van die ekonomies mees belangrike virus siektes van wingerd. Grapevine leafroll-associated virus 3 (GLRaV-3), van die genus Ampelovirus, is die mees wydverspreide lid van die rolblaar geassosieerde virus familie. Om die verspreiding van GLD te voorkom, is bestuur strategieë, soos die verwydering van geïnfekteerde plante en insekvektor beheer, ’n vereiste om oes verliese te beperk. Alternatiewe beheer strategieë gebaseer op genetiese modifikasie van die wingerdgenoom, soos patogeen-afgeleide weerstand (PDR), is bewys om effektief te wees in die verlening van weerstand teen verskeie virusse. Daarom was die fokus van hierdie studie om patogeen-afgeleide weerstand strategieë vir GLRaV-3 te evalueer met behulp van die volgende twee benaderings; 1) die evaluering van transgeniese plante wat 'n disfunksionele GLRaV-3 hitte-skok proteïen 70 homoloog (HSP70h) uitdruk, ten einde weerstand te verleen teen GLRaV-3, en 2) die konstruksie van kunsmatige mikroRNAs (amiRNAs) om te gebruik as 'n instrument vir die ondrukking van spesifieke genoomvolgordes van GLRaV-3 in die wingerd gasheer en die ontwikkeling van ’n stelsel om amiRNA-bemiddelde onderdrukking te bevestig. In die eerste deel van hierdie studie, is ses transgeniese plant lyne (plant lyne # 1, # 3, # 9, # 14, # 15 en # 17) sowel as 'n nie-gemodifiseerde gesonde plant lyn, geïnokuleer met GLRaV- 3 deur enting van ogies van elk op GLRaV-3 besmette plantmateriaal. Na ongeveer vyf maande, is GLRaV-3 virus konsentrasies van alle ingeënte plante gekwantifiseer relatief tot twee verwysing gene deur gebruik te maak van tru-transkripsie kwantitatiewe PCR (RTqPCR). Resultate is geëvalueer deur die relatiewe virus konsentrasie van elke transgeniese plant lyn te vergelyk met dié van die nie-gemodifiseerde kontrole lyn. Resultate het getoon dat weerstand vlakke van plant lyn # 3 beduidend verbeter is (> 99%) en merkwaardig is plant lyn # 14 bewys om meer vatbaar vir die virus te wees. Die tweede deel van die studie was die konstruksie en bevestiging van kunsmatige mikroRNAs (amiRNAs) wat GLRaV-3 genoomvolgordes teiken. Twee 21 nt streke van GLRaV-3 is suksesvol geïnkorporeer in die ruggraat van wingerd mikroRNA vvi167b. Verder is teiken konstrukte ontwikkel deur die inkorporering van ooreenstemmende GLRaV-3 teiken genoomvolgordes in die 3'UTR (3’ ongetransleerde area) van 'n groen fluoressensie proteïen (GFP) geen. Daarna is die teiken konstrukte gesamentlik geïnfiltreer met die gekonstrueerde amiRNA in Nicotiana benthamiana en GFP uitdrukkingsvlakke is gekwantifiseer deur die onderdrukkingsdoeltreffendheid van die amiRNAs te bepaal. Resultate het getoon dat die amiRNAs suksesvol was in die onderdrukking van die GFP teiken konstruk, maar hulle was egter nie-spesifiek in die eksklusiewe onderdrukking van die ooreenstemmende teiken. Hierdie amiRNA konstrukte is ideaal vir verdere virus studies om die doeltreffendheid van GLRaV-3 onderdrukking in GLD besmette wingerdstokke te bepaal.

Made available in DSpace on 2014-06-11T19:28:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-11Bitstream added on 2014-06-13T19:58:12Z : No. of bitstreams: 1 couto_eb_me_botfca.pdf: 859715 bytes, checksum: 112956f8c222cc32cb4262cfa4afff81 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Empresa Privada / O psilídeo-de-concha Glycaspis brimblecombei é uma praga originária da Austrália, sendo detectado no Brasil em junho de 2003, na região de Mogi-Guaçu, SP. O ataque da praga causa descoloração das folhas, redução da área fotossintética com conseqüente redução no crescimento e secamento dos ponteiros, podendo levar as árvores à morte após ataques sucessivos. Devido à facilidade de dispersão deste inseto e aos danos causados em plantações de Eucalyptus, é considerada uma das principais pragas do eucalipto.Além do controle biológico com o parasitóide, há relatos de uso de inseticidas químicos e de espécies de eucalipto resistentes ou mesmo imunes ao psilídeo-de-concha. Entretanto, os mecanismos dessa resistência ainda são desconhecidos. A indução de resistência, pela ativação de indutores, como o metil jasmonato (MJ) vem sendo estudado intensamente nos últimos anos, principalmente através da seleção de genótipos com elevada capacidade de expressão de MJ. O presente trabalho teve como objetivo estudar a indução de resistência através da aplicação exógena de metil jasmonato como forma alternativa para o controle do psilídeo-de-concha. O trabalho foi realizado em condições de laboratório, utilizando mudas da espécie Eucalyptus camaldulensis e do clone híbrido de E. grandis x E. camaldulensis (gracam), sendo esses materiais escolhidos por serem altamente suscetíveis à praga. Foram realizados quatro experimentos com aplicacao de diferentes concentracoes de MJ (5, 50 e 500 ÊM) em plantas de eucalipto para avaliar o efeito na preferencia e capacidade de oviposicao dos adultos do psilideo-de-concha. No primeiro experimento verificou-se que as maiores concentracoes afetaram alguns parametros da biologia do psilideo, sendo que a testemunha apresentou maior numero de ovos e, consequentemente, maior numero de adultos produzidos. Nos dois experimentos... / Red gum lerp psyllid Glycaspis brimblecombei is an Australian eucalypt pest, being detected in Brazil in June 2003, in municipality of Mogi-Guacu, SP. The pest causes leaf fall, reduction of photosynthetic area and consequent reduction in plant growth and dieback, could carry the trees death, after successive defoliations. Due to the dispersion rates of this insect and the damages caused in eucalypt plantations, it is considered one of the eucalyptus main pests. The biological control is the main control method used, with parasitoid Psyllaephagus bliteus (Hymenoptera: Encyrtidae). Besides the biological control, there are reports of chemical insecticides applications and resistant eucalyptus species against red gum lerp psyllid. However, factors of this resistance are still ignored. The resistance induction, by elicitors activation, as methyl jasmonate (MJ) has been studied intensely in last years, mostly through the genotypes selection with high expression capacity of MJ. The work was accomplished in laboratory conditions, using young plants of Eucalyptus camaldulensis and of E. grandis x E. camaldulensis (gracam-h) hybrid clone, being these materials chosen due the high susceptibility to the pest. It was accomplished four experiments with application of different MJ concentrations (5, 50 and 500 ÊM) in eucalyptus plants to verify the effect in red gum lerp psyllid adults preference and oviposition capacity. In the first experiment it was verified that the highest concentration affected some psyllid biology parameters, and control treatment presented larger egg number and, consequently, larger number of adults produced. In the two experiments with free chance choice, independently of genetic material, it was observed the insect preference by control plants. As larger MJ concentration, less adults and eggs occurred in the leaves. In relation... (Complete abstract, click electronic access below)

Made available in DSpace on 2014-06-11T19:28:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-11-20Bitstream added on 2014-06-13T18:58:01Z : No. of bitstreams: 1 000754745.pdf: 1440461 bytes, checksum: 312188c958d82d03b044626af97f7abe (MD5) / O pimentão é uma hortaliça com grande apreciação no Brasil, tendo elevada importância no mercado de condimentos, temperos e conservas. O Estado de São Paulo é o principal produtor desta hortaliça, com produção aproximada de 85.000 toneladas na safra 2010. Dentre os principais problemas fitossanitários da cultura do pimentão, destaca-se a mancha bacteriana, causada por espécies de Xanthomonas spp. A doença pode ocasionar perdas substanciais na produtividade da cultura, especialmente em períodos de elevadas pluviosidade e temperatura, além da baixa eficácia de controle com produtos químicos, como fungicidas cúpricos. Em vista do exposto, o objetivo do presente trabalho foi caracterizar 59 isolados de Xanthomonas spp. de pimentão obtidos de diferentes regiões produtoras do Brasil, através da utilização de técnicas bioquímicas/fisiológicas, moleculares e verificar a sensibilidade in vitro dos isolados aos sulfatos de cobre e zinco e suas misturas. Para os ensaios bioquímicos/fisiológicos, foram realizados os testes de reação diferencial de Gram, solubilidade em KOH a 3%, hidrólise de amido, atividade pectinolítica e a utilização de 13 fontes de carbono, em microplacas GN2 da Biolog®. A identificação molecular foi realizada através de reações de PCR com os iniciadores específicos para X. euvesicatoria, X.vesicatoria, X. gardneri e X. perforans. A sensibilidade in vitro dos isolados aos sulfatos de cobre e zinco foi avaliada nas concentrações de 50, 100, 200 e 400 μg.mL-1, além da mistura de 50% de cada um dos produtos, nas mesmas concentrações finais no meio de cultura PSA. Com base nos resultados dos testes bioquímicos/fisiológicos e moleculares (PCR) com iniciadores específicos revelaram a prevalência de X. euvesicatoria em pimentão no Brasil. Todos os isolados avaliados foram resistentes ao sulfato de zinco e 85% deles foram resistentes ao... / Pepper is a vegetable with great appreciation in Brazil and high importance for the market of condiments, spices and canned goods. Sao Paulo state is the main producer of pepper in Brazil with almost 85.000 ton in 2010. One of the most important phytosanitary problems for this crop is the bacterial spot, caused by species of Xanthomonas spp. This disease can cause crop productivity losses, especially during periods of high rainfall and temperature, in addition to the low efficacy of chemicals, as copper fungicides. In this study, we characterized 59 strains of Xanthomonas spp. associated with bacterial spot from different producing regions of Brazil, using biochemical/physiological and molecular techniques, and evaluated the in vitro sensitivity of strains to copper and zinc sulfates. For the biochemical/physiological assays, Gram staining, KOH string test, starch hydrolysis, pectinolytic activity and the utilization of 13 carbon sources on Biolog® GN2 microplates were performed. Molecular characterization was performed by PCR with specific initiators for X. euvesicatoria, X.vesicatoria, X. gardneri and X. perforans. In vitro sensitivity of strains to copper and zinc sulfates was evaluated at concentrations 50, 100, 200 and 400 μg.mL-1, together with the mixing of the chemicals, in the same final concentration in culture medium. Based on starch hydrolysis and pectinolytic activity results, strains were divided in two distinct groups: X. vesicatoria with X. perforans (positive pectinolytic and amylolytic activities) and X. euvesicatoria with X. gardneri (negative pectinolytic and amylolytic activities). PCR and carbon sources utilization results revealed the prevalence of X. perforans on pepper in Brazil. All strains were resistant to zinc sulfate and 85% to copper sulfate. The mixing of the chemicals at concentration of 400 μg.ml-1 inhibited the growth of all strains assessed.

Resumo: A cana-de-açúcar (Saccharum spp.) é uma das gramíneas cultivadas mais importantes do mundo e o Brasil é hoje o maior produtor e exportador mundial de cana, de açúcar de cana e etanol de cana, com o estado de São Paulo sendo o maior produtor. Entretanto, esta cultura é ameaçada por várias pragas e doenças. Dentre essas pragas, uma das mais importantes na região centro-sul é a cigarrinha-das-raízes (Mahanarva fimbriolata), que assumiu este posto devido ao aumento da colheita mecanizada. Por meio da técnica de cDNA-AFLP este trabalho teve como objetivo identificar genes possivelmente envolvidos na resistência a ninfas de Mahanarva fimbriolata em cana-de-açúcar. Para isso, foi utilizada uma variedade suscetível (SP80-1816) e uma resistente (SP83-5073). Ambas foram infestadas com ninfas da cigarrinha 77 dias após o plantio e amostras de raízes foram coletadas antes da infestação (controle) e 1, 2 e 7 dias após a infestação, posteriormente foi feita a extração de RNA, síntese de cDNA e o cDNA-AFLP. Após a análise dos fragmentos foram encontrados 16 fragmentos expressos somente na variedade resistente. Nove fragmentos foram encontrados no 2º dia de infestação e sete no 7º dia. Dentre os 16 fragmentos, sete foram recuperados e sequenciados. Seis fragmentos não apresentaram homologia com proteínas já descritas e um fragmento, de 129 pb, apresentou homologia com uma proteína putativa associada à senescência. Todos os fragmentos podem representar genes relacionados com a resistência da planta contra o inseto / Abstract: The sugarcane (Saccharum spp.) is the most important grass cultivated in the world and Brazil is now the world's largest producer and exporter of sugarcane, sugarcane sugar and sugarcane ethanol, with the São Paulo state being the largest producer. However, this culture is threatened by several pests and diseases. Among these pests, one of the most important in south-central region is the froghopper-roots (Mahanarva fimbriolata), that took this position because of increased mechanization. Through cDNA-AFLP technique this study aimed to identify genes possibly involved in resistance to nymphs of Mahanarva fimbriolata in sugarcane. Therefore we used a susceptible variety (SP80-1816) and a resistant (SP83-5073). Both were infested with spitlebugs 77 days after planting and root samples were collected before infestation (control) and 1, 2 and 7 days after infestation, was later made the extraction of RNA, cDNA synthesis and cDNA-AFLP. After fragments analyses were found 16 fragments expressed only in the resistant variety. Nine fragments were found on day 2 of infection and seven on day 7. Among the 16 fragments, seven were recovered and sequenced. Six fragments showed no homology to described proteins and a fragment of 129 bp showed homology to a putative protein associated with senescence. All fragments could represent genes related to the plant resistance against the insect / Orientadora: Maria Inês Tiraboschi Ferro / Coorientador: Miguel Angelo Mutton / Banca: Lucia Maria Carareto Alves / Banca: Marcelo de Almeida Silva / Mestre

Made available in DSpace on 2014-06-11T19:26:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-11-09Bitstream added on 2014-06-13T20:54:10Z : No. of bitstreams: 1 moraes_fe_me_jabo.pdf: 1680634 bytes, checksum: 35809b1bab51b8d182c7d496667f751f (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A cana-de-açúcar (Saccharum spp.) é uma das gramíneas cultivadas mais importantes do mundo e o Brasil é hoje o maior produtor e exportador mundial de cana, de açúcar de cana e etanol de cana, com o estado de São Paulo sendo o maior produtor. Entretanto, esta cultura é ameaçada por várias pragas e doenças. Dentre essas pragas, uma das mais importantes na região centro-sul é a cigarrinha-das-raízes (Mahanarva fimbriolata), que assumiu este posto devido ao aumento da colheita mecanizada. Por meio da técnica de cDNA-AFLP este trabalho teve como objetivo identificar genes possivelmente envolvidos na resistência a ninfas de Mahanarva fimbriolata em cana-de-açúcar. Para isso, foi utilizada uma variedade suscetível (SP80-1816) e uma resistente (SP83-5073). Ambas foram infestadas com ninfas da cigarrinha 77 dias após o plantio e amostras de raízes foram coletadas antes da infestação (controle) e 1, 2 e 7 dias após a infestação, posteriormente foi feita a extração de RNA, síntese de cDNA e o cDNA-AFLP. Após a análise dos fragmentos foram encontrados 16 fragmentos expressos somente na variedade resistente. Nove fragmentos foram encontrados no 2º dia de infestação e sete no 7º dia. Dentre os 16 fragmentos, sete foram recuperados e sequenciados. Seis fragmentos não apresentaram homologia com proteínas já descritas e um fragmento, de 129 pb, apresentou homologia com uma proteína putativa associada à senescência. Todos os fragmentos podem representar genes relacionados com a resistência da planta contra o inseto / The sugarcane (Saccharum spp.) is the most important grass cultivated in the world and Brazil is now the world’s largest producer and exporter of sugarcane, sugarcane sugar and sugarcane ethanol, with the São Paulo state being the largest producer. However, this culture is threatened by several pests and diseases. Among these pests, one of the most important in south-central region is the froghopper-roots (Mahanarva fimbriolata), that took this position because of increased mechanization. Through cDNA-AFLP technique this study aimed to identify genes possibly involved in resistance to nymphs of Mahanarva fimbriolata in sugarcane. Therefore we used a susceptible variety (SP80-1816) and a resistant (SP83-5073). Both were infested with spitlebugs 77 days after planting and root samples were collected before infestation (control) and 1, 2 and 7 days after infestation, was later made the extraction of RNA, cDNA synthesis and cDNA-AFLP. After fragments analyses were found 16 fragments expressed only in the resistant variety. Nine fragments were found on day 2 of infection and seven on day 7. Among the 16 fragments, seven were recovered and sequenced. Six fragments showed no homology to described proteins and a fragment of 129 bp showed homology to a putative protein associated with senescence. All fragments could represent genes related to the plant resistance against the insect

Made available in DSpace on 2014-06-11T19:34:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-28Bitstream added on 2014-06-13T20:25:39Z : No. of bitstreams: 1 zache_rrc_dr_botfca.pdf: 679513 bytes, checksum: 64a578a7d904b5a9f42523044a7451a7 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O manejo de Bemisia tabaci biótipo B tem se tornado um desafio aos agricultores, uma vez que este inseto apresenta elevada capacidade em desenvolver resistência a todas as classes de inseticidas. Na busca por táticas alternativas e menos agressivas para o manejo das moscas-brancas, o uso de genótipos resistentes e a utilização de extratos de vegetais com atividades inseticida e/ou insetistática, têm revelado resultados promissores no combate à B. tabaci biótipo B. Dessa forma, o presente trabalho teve como objetivo avaliar a resistência de genótipos de aboboreiras (Cucurbita spp.) frente a B. tabaci biótipo B, estudar os tipos de resistência envolvidos e avaliar a possível interação no uso desses materiais com extratos aquosos provenientes de espécies da família Meliaceae. Realizou-se também análise bioquímica para confirmação da resistência de alguns genótipos. Para a instalação dos bioensaios, foi mantida uma criação de B. tabaci biótipo B em casa de vegetação. Vinte genótipos de Cucurbita spp. („Alicia AF 9354‟, „Aline AF 9353‟, „Golden Delight‟, „Nova Caravela‟, „Menina Morena‟, „Novita‟, „AF- 6741‟, „Atlas‟, „Bárbara 305‟, „Menina Brasileira‟, „Caserta‟, „Itapuã 301‟, „Tâmara‟, „Samira‟, „Canhão Seca Gigante‟, „Exposição‟, „Novita Plus‟, „Daiane‟, „Formosa‟, „Sandy‟) foram avaliados em ensaios (atratividade, oviposição com e sem chance de escolha, preferência alimentar e antibiose), sob condições de laboratório e casa de vegetação. A atratividade (adultos/cm2) foi avaliada 12, 24, 48 e 72 horas após a liberação. Após a última contagem, foram coletadas folhas para a contagem do número de ovos (com chance de escolha). As... / The management de Bemisia tabaci biotype B has become a challenge to agriculturalists, since this insect presents an elevated capacity to develop resistance to all classes of insecticides. In the search for less aggressive alternative tactics to control whiteflies, both resistant genotypes and vegetal extracts with insecticidal and / or insectistatic activities have revealed promising results for combating B. tabaci biotype B. Thus the present work aimed to evaluate the resistance of squash genotypes (Cucurbita spp.) challenged with B. tabaci biotype B, to study the types of resistance involved and to evaluate possible interactions in the use of these materials with aqueous extracts from species of the family Meliaceae. Biochemical analysis was also performed to confirm the resistance of some genotypes. To install bioassays, a creation of B. tabaci biotype B was maintained in a greenhouse. Twenty genotypes of Cucurbita spp. („Alicia AF 9354‟, „Aline AF 9353‟, „Golden Delight‟, „Nova Caravela‟, „Menina Morena‟, „Novita‟, „AF- 6741‟, „Atlas‟, „Bárbara 305‟, „Menina Brasileira‟, „Caserta‟, „Itapuã 301‟, „Tâmara‟, „Samira‟, „Canhão Seca Gigante‟, „Exposição‟, „Novita Plus‟, „Daiane‟, „Formosa‟, „Sandy‟) were evaluated in assays (attractiveness, oviposition, with and without free choice, feeding preference and antibiosis), under greenhouse and laboratory conditions. Attractiveness (adults/cm2) was evaluated 12, 24, 48 and 72 hours after release. After the final count, leaves were collected to enumerate the number of eggs (with free choice). Areas of leaves were measured to obtain the number of eggs/cm2 and the number of adults/ cm2. An oviposition preference assay was also performed without free choice. Antibiosis assays evaluated... (Complete abstract click electronic access below)

Orientador: Carlos Frederico Wilcken / Banca: Edson Luiz Furtado / Banca: José Djair Vendramim / Resumo: O psilídeo-de-concha Glycaspis brimblecombei é uma praga originária da Austrália, sendo detectado no Brasil em junho de 2003, na região de Mogi-Guaçu, SP. O ataque da praga causa descoloração das folhas, redução da área fotossintética com conseqüente redução no crescimento e secamento dos ponteiros, podendo levar as árvores à morte após ataques sucessivos. Devido à facilidade de dispersão deste inseto e aos danos causados em plantações de Eucalyptus, é considerada uma das principais pragas do eucalipto.Além do controle biológico com o parasitóide, há relatos de uso de inseticidas químicos e de espécies de eucalipto resistentes ou mesmo imunes ao psilídeo-de-concha. Entretanto, os mecanismos dessa resistência ainda são desconhecidos. A indução de resistência, pela ativação de indutores, como o metil jasmonato (MJ) vem sendo estudado intensamente nos últimos anos, principalmente através da seleção de genótipos com elevada capacidade de expressão de MJ. O presente trabalho teve como objetivo estudar a indução de resistência através da aplicação exógena de metil jasmonato como forma alternativa para o controle do psilídeo-de-concha. O trabalho foi realizado em condições de laboratório, utilizando mudas da espécie Eucalyptus camaldulensis e do clone híbrido de E. grandis x E. camaldulensis (gracam), sendo esses materiais escolhidos por serem altamente suscetíveis à praga. Foram realizados quatro experimentos com aplicacao de diferentes concentracoes de MJ (5, 50 e 500 ÊM) em plantas de eucalipto para avaliar o efeito na preferencia e capacidade de oviposicao dos adultos do psilideo-de-concha. No primeiro experimento verificou-se que as maiores concentracoes afetaram alguns parametros da biologia do psilideo, sendo que a testemunha apresentou maior numero de ovos e, consequentemente, maior numero de adultos produzidos. Nos dois experimentos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Red gum lerp psyllid Glycaspis brimblecombei is an Australian eucalypt pest, being detected in Brazil in June 2003, in municipality of Mogi-Guacu, SP. The pest causes leaf fall, reduction of photosynthetic area and consequent reduction in plant growth and dieback, could carry the trees death, after successive defoliations. Due to the dispersion rates of this insect and the damages caused in eucalypt plantations, it is considered one of the eucalyptus main pests. The biological control is the main control method used, with parasitoid Psyllaephagus bliteus (Hymenoptera: Encyrtidae). Besides the biological control, there are reports of chemical insecticides applications and resistant eucalyptus species against red gum lerp psyllid. However, factors of this resistance are still ignored. The resistance induction, by elicitors activation, as methyl jasmonate (MJ) has been studied intensely in last years, mostly through the genotypes selection with high expression capacity of MJ. The work was accomplished in laboratory conditions, using young plants of Eucalyptus camaldulensis and of E. grandis x E. camaldulensis (gracam-h) hybrid clone, being these materials chosen due the high susceptibility to the pest. It was accomplished four experiments with application of different MJ concentrations (5, 50 and 500 ÊM) in eucalyptus plants to verify the effect in red gum lerp psyllid adults preference and oviposition capacity. In the first experiment it was verified that the highest concentration affected some psyllid biology parameters, and control treatment presented larger egg number and, consequently, larger number of adults produced. In the two experiments with free chance choice, independently of genetic material, it was observed the insect preference by control plants. As larger MJ concentration, less adults and eggs occurred in the leaves. In relation... (Complete abstract, click electronic access below) / Mestre