Dissertationen zum Thema „Plant viruses Control“

Thesis (MSc)--Stellenbosch University, 2003.<br>ENGLISH ABSTRACT: Ribosome inactivating proteins (RIPs) are currently classified as rRNA N-glycosidases, but also have polynucleotide: adenosine glycosidase activity. RIPs are believed to have anti-viral and anti-fungal properties, but the exact mechanism of these proteins still need to be elucidated.The mechanism of resistance however, appears to be independent of the pathogen. For resistance the RIP terminates virus infected plant cells and stops the reproduction and spread of the virus. Transgenic plants containing RIPs should thus be resistant to a wide range of viruses. The ultimate goal of the larger project of which this forms part is the development of virus resistant plants. To monitor the expression of a RIP in a transgenic plant a detection method had to be developed. Antibody detection of the RIP was decided upon as the most cost effective method. The RIP, Dianthin 30 from Dianthus caryophyllus (carnation), was used and expressed in bacterial and insect expression systems. The bacterial expression experiments were done using the pET expression system in BL21(DE3)pLysS cells. The expression in this system yielded recombinant protein at a very low concentration. Expression experiments were also performed in insect tissue culture with the baculovirus vector BAC-TO-BAC™.With this system the expression was also too low to be used for the production of antibodies. A Dianthin 30 specific peptide was then designed and then produced by Bio-Synthesis. This peptide was then used to raise antibodies to detect Dianthin 30. These antibodies were tested on Dianthus caryophyllus proteins. To establish if this detection method was effective to monitor the expression in plants, tobacco plants were transformed with Agrobacterium tumefaciens containing Dianthin 30 in the pART27 plant expression vector. The putative transformed plants were analysed with peR and Southern blots.<br>AFRIKAANSE OPSOMMING: Tans word Ribosomale-inaktiverende proteïene (RIPs) geklassifiseer as rRNA N-glikosidase wat ook polinukleotied: adenosien glikosidase aktiwiteit bevat. Daar word geglo dat RIPs anti-virale en anti-fungus eienskappe bevat, maar die meganisme van beskerming word nog nie ten volle verstaan nie. Dit is wel bewys dat die meganisme van weerstand onafhanklik is van die patogeen. Virus geinfekteerde plantselle word deur die RIP gedood om die voortplanting en verspreiding te bekamp en sodoende word weerstand bewerkstellig. Transgeniese plante wat dan 'n RIP bevat sal dus weerstandbiedend wees teen 'n wye spektrum virusse. Die hoofdoel van die breër projek, waarvan die projek deel uitmaak: is die ontwikkeling van virusbestande plante. Om die uitdrukking van die RIP in die transgeniese plante te kontroleer, moes 'n deteksie metode ontwikkel word. Die mees koste effektiewe deteksie metode is met teenliggame. Die RIP, Dianthin 30 from Dianthus caryophyllus (angelier) was gebruik vir uitdrukking in bakteriele- en insekweefselkultuur. Die bakteriele uitdrukkingseksperimente was gedoen met die pET uitdrukkings sisteem III BL21(DE3)pLysS selle. Die uitdrukking in die sisteem het slegs rekombinante proteïene gelewer in uiters lae konsentrasies. Uitdrukkingseksperimente was ook gedoen in insekweefselkultuur met die baculovirus vektor BAC-To- BACTM. Met die sisteem was die uitdrukking ook veels te laag om bruikbaar te wees vir die produksie van teenliggame. Daar is toe 'n peptied ontwerp wat Dianthin 30 kan verteenwoordig vir die produksie van teenliggame. Die teenliggame is getoets teen Dianthus caryophyllus proteïene. Om vas te stel of die deteksiemetode wel die uitdrukking van Dianthin 30 sal kan monitor, is tabak ook getransformeer met Dianthin 30. Die transformasies is gedoen met die hulp van Agrobacterium tumefaciens en die pART27 plant uitdrukkings vektor. Die plante is getoets met die polimerase ketting reaksie en Southern klad tegnieke.

Este trabalho teve como objetivo avaliar a infectividade de estirpes fracas do Papaya ringspot virus - type W (PRSV-W) em plantas de melancia (Citrullus lanatus), em função da origem da estirpe fraca, da concentração e da espécie doadora do inóculo e da idade da planta-teste de melancia, inoculada mecanicamente. Também foi avaliado o efeito protetor dessas estirpes em plantas de melancia em casa de vegetação e em campo. A seleção de estirpes fracas do PRSV-W foi feita a partir de bolhas de folhas de melancia infectadas naturalmente em campo. A infectividade da estirpe fraca selecionada foi comparada com a das estirpes fracas PRSV-W-1 e PRSV-W-2, previamente selecionadas de bolhas de folhas de abobrinha de moita (Cucurbita pepo) 'Caserta' com mosaico. Como controle foi utilizada uma estirpe severa do vírus obtida de abobrinha de moita (PRSV-W-C). A avaliação do efeito da concentração e da espécie da planta fonte do inóculo na infectividade de plantas de melancia foi feita com extratos de 4, 8, 12 e 16 discos de folhas de abobrinha de moita e de melancia, infectadas separadamente com as estirpes fracas e severa, e diluídos em 2 mL de tampão fosfato. O efeito da idade da planta-teste de melancia na infectividade das estirpes fracas foi estudado comparando-se plantas inoculadas em quatro estádios de desenvolvimento, a partir do estádio cotiledonar, com inóculos das diferentes estirpes do PRSV-W extraídos de 12 discos foliares/2 mL de tampão. O efeito protetor da estirpe fraca obtida de bolhas de folhas de melancia com mosaico foi avaliado em plantas premunizadas e desafiadas com a estirpe PRSV-W-C, em casa de vegetação e em campo. Plantas de melancia premunizadas com as estirpes fracas PRSV-W-1 e PRSV-W-2 e plantas não protegidas também foram avaliadas no teste em campo. Foram avaliadas a proteção, com base nos sintomas, a produção e o conteúdo de açúcares (grau brix) dos frutos colhidos das plantas premunizadas e não premunizadas. Uma estirpe fraca do vírus, denominada PRSV-W-3, foi selecionada de bolhas de folhas de melancia com mosaico. Em todos os testes de infectividade em plantas de melancia, independente da concentração e da planta fonte do inóculo e do estádio de desenvolvimento da planta-teste inoculada, a estirpe fraca PRSV-W-3 apresentou taxas de infectividade semelhantes as das estirpes PRSV-W-1 e PRSV-W-2, chegando a 100% em alguns casos. A infectividade da estirpe severa PRSV-W-C foi de 100% em todos os testes. Aparentemente, a infectividade das três estirpes fracas foi mais diretamente afetada pela intensidade de fricção das folhas no momento da inoculação mecânica do que pelas variáveis estudadas. A estirpe fraca PRSV-W-3 protegeu as plantas de melancia contra a infecção e/ou manifestação da estirpe PRSV-W-C em casa de vegetação. Em campo, todas as plantas de melancia premunizadas com as três estirpes fracas também ficaram protegidas contra a estirpe severa utilizada no desafio. A produção das plantas premunizadas não diferiu estatisticamente entre si, nem mesmo daquelas inicialmente sadias infectadas em campo. O conteúdo de açúcares e a aparência da polpa dos frutos também foram semelhantes em todos os tratamentos.<br>The purpose of this work was to evaluate the infectivity of three mild strains of Papaya ringspot virus - type W (PRSV-W) on watermelon (Citrullus lanatus). The effect of the origin of the mild strain, the concentration of the inoculum, the species of the source of the inoculum and the age of the test-plant on the infectivity of mechanically inoculated watermelon were also evaluated. The protective effect of these mild strains on preimmunized watermelon plants was evaluated under greenhouse and field conditions. Mild strains were selected from blisters formed on mosaic leaves of naturally infected watermelon plants. The infectivity of the selected mild strain was compared with that of mild strains PRSV-W-1 and PRSV-W-2, which were previously obtained from blisters formed on mosaic leaves of zucchini squash (Cucurbita pepo cv. Caserta). A severe strain isolated from zucchini squash (PRSV-W-C) was used as control. The effect of the concentration and the species source of inoculum of the mild strains on the infectivity of watermelon plants was studied with inoculum extracted from 4, 8, 12 and 16 leaf discs of zucchini squash and watermelon plants, separately infected with the mild and severe strains, diluted in 2 mL of phosphate buffer. Four stages of development of watermelon plants, starting at the cotyledonal stage, were tested for the infectivity with the mild strains. Inocula were prepared with extracts of 12 leaf discs diluted in 2 mL of phosphate buffer. The protective effect of the mild strain selected from blisters on mosaic leaves of watermelon plants was evaluated on preimmunized plants challenge inoculated with severe strain PRSV-W-C, under greenhouse and field conditions. Watermelon plants preimmunized with mild strains PRSV-W-1 and PRSV-W-2 and unprotected plants were also included in the field trial. Protection was evaluated based on plant simptons, yield and sugar content in the fruits. One mild strain, named PRSV-W-3, was obtained from blisters on mosaic leaves of watermelon plants. The rate of infection of watermelon plants with mild strain PRSV-W-3 was similar to that with mild strains PRSV-W-1 and PRSV-W-2 in all infectivity tests, independently of the concentration of the inoculum, species source of the inoculum and stage of development of the inoculated test-plant, reaching 100% in some cases. Rate of infectivity with severe strain PRSV-W-C was always 100%. Apparently, the infectivity of the mild strains on watermelon was more directly related with the intensity of the abrasion produced by mechanical inoculation than with the above studied variables. The selected mild strain PRSV-W-3 protected watermelon plants against superinfection with the severe strain in the greenhouse tests. Protection was also effective under field conditions. Yield of plants preimmunized with all three mild strains and unprotected plants were statistically similar. The sugar content and the quality of the pulp of the fruits were similar for all treatments.

Thesis (MSc)--Stellenbosch University, 2003.<br>ENGLISH ABSTRACT: The aim of this study was to combine two common diagnostic tools: serological kits and genetic fingerprinting to identify cherry leaf-roll nepovirus (CLRV), and to establish a marker system to characterize walnut germplasm. The detection of plant viruses is difficult. Restrictions are imposed for quarantine purposes on the importation of plant material from foreign countries. Modern techniques such as a PCR based screening method for CLRV are required to ensure material do not harbour viruses. A primer pair was designed to amplify a 430 bp non-coding homologous region. For the choice of primers, consensus sequences were considered and areas where the sequence data shared 98.5% homology, were chosen. The sensitivity of this detection method was 100-fold higher when compared to the ELISA. The PCR fragment was verified by nucleotide sequencing. AFLP technology was used to identify polymorphic fragments for 6 walnut cultivars and a rootstock, and SCARs were developed from AFLP specific bands. The AFLP technique distinguished all the walnut cultivars and the rootstock. However, conversion of AFLP fragments to SCAR markers for the development of a simple robust technique for cultivar discrimination, was not successful. Using 27 AFLP primer combinations, polymorphic fragments as high as 47.8% were scored. The reason for the lack of efficient conversion was as the result of the AFLP technique. The SCAR primers were generated from sequences internal to the AFLP primers but the specificity of the markers was in the AFLP primers not the internal sequence. In this study using AFLP, walnut cultivars were found to be closely related. The AFLP primer pairs used, provided polymorphic fragments. From these fragments, 7 SCAR markers were developed. It was expected that these SCARs derived from the AFLP markers would detect slight differences between cultivars. The Paradox SCAR marker was the only one that could divide the cultivars into two groups. When Chandler SCAR products were digested with the restriction enzyme Rsal, the same banding pattern as that of Paradox SCAR products was observed.<br>AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om twee algemene opsporingstegnieke te kombineer: serologiese toetsstelle en genetiese vingerafdrukke om cherry leaf-roll nepovirus (CLRV) te eien en om In merkersisteem te ontwikkel wat okkerneut kiemplasma kan karakteriseer. Die opsporing van plant virusse is baie moeilik. As gevolg van kwarantyn vereistes, word daar beperkinge geplaas word op die invoer van plant materiaal vanuit die buiteland. Moderne tegnieke soos hierdie een wat op PKR berus, word benodig om te verseker dat CLRV nie in plantmateriaal teenwoordig is nie. In Stel inleiers is ontwerp wat In 430 bp nie-koderende homoloë area amplifiseer. Hiervoor is konsensus volgordes bestudeer en slegs die volgordes wat 98,5% homologie getoon het, is gekies. In vergelyking met ELISA was die sensitiwiteit van hierdie deteksie metode 100 maal beter. DNA volgordebepaling is op die resulterende fragment gedoen om die PKR produk te verifieer. AFLP tegnologie is gebruik om polimorfiese fraqmente vir 6 okkerneut kultivars en 'n onderstok te identifiseer en SCARs is uit hierdie fragmente ontwikkel. Die AFLP tegniek kon tussen al die okkerneut kultivars en die onderstok onderskei. Die omskakeling van die AFLP fragmente in SCAR merkers om sodoende In eenvoudige kragtige tegniek vir kultivar onderskeiding te ontwikkel, was egter nie suksesvol nie. Met die gebruik van 27 AFLP inleier kombinasies, kon polimorfiese fragmente van so hoog as 47.8% verkry word. Die rede hoekom omskakeling onsuksesvol was lê by die aard van die AFLP tegniek. Die SCAR inleiers is ontwikkel uit volyordes intern tot die AFLP inleiers, maar die spesifisiteit van die merkers het juis in die AFLP inleiers gelê en nie in die interne volgordes nie. In hierdie studie, met die gebruik van AFLP, is gevind dat okkerneut kultivars baie naby verwant is. Die AFLP inleierstelle wat gebruik is, het polimorfiese fragmente gelewer. Uit hierdie fragmente is 7 SCAR merkers ontwikkel. Daar is verwag dat die SCARs wat uit die AFLP merkers ontwikkel is, klein verskille tussen kultivars sou opspoor. Dit was egter net die Paradox SCAR merker wat die kultivars in twee groepe kon verdeel. Restriksie ensiem vertering met Rsalop die Chandler SCAR produkte het dieselfde bandpatrone as die van die Paradox SCAR produkte gelewer.

As algas produzem uma grande diversidade de compostos com atividade biológica, inclusive que agem diretamente sobre vírus ou como indutores de fitoalexinas. Em vista disso, foi investigada a redução de sintomas causados por Tobacco mosaic vírus (TMV) em plantas de fumo tratadas com cianobactérias ou algas eucarióticas, além de se tentar elucidar o modo de ação das algas no patossistema estudado. Quando as plantas de fumo foram tratadas dois dias antes da inoculação, foi verificado que suspensões dos isolados 004/02, 008/02, 061/02, Anabaena sp. e Nostoc sp. 61, bem como as preparações do conteúdo intracelular do isolado 004/02 (4 C) e do filtrado do meio de cultivo do isolado 061/02 (61 M) apresentaram efeito na redução dos sintomas de TMV em plantas de fumo, cultivar TNN. Além disso, foi estudado o efeito direto das algas sobre as partículas de vírus. Os resultados mostraram que os isolados Anabaena sp., Nostoc sp. 21, Nostoc sp. 61 e 090/02 apresentam compostos que agem diretamente sobre o TMV. Para tentar elucidar o mecanismo de ação das algas no patossistema estudado, diversos parâmetros bioquímicos foram investigados. Foi detectado que a preparação 4 C aumentou a atividade de peroxidases e que todos os tratamentos analizados reduziram a atividade de &#946;-1,3-glucanase em folhas de fumo a partir do quarto dia após o tratamento das plantas. Por sua vez, as suspensões dos isolados 008/02 e 061/02 e a preparação 61 M proporcionaram maior acúmulo de superóxido, enquanto que a preparação 4 C reduziu o acúmulo de peróxido de hidrogênio, em relação aos controles água destilada e meio de cultura BG 11, 37 horas após a inoculação do vírus. Em vista disso, as algas podem ser utilizadas como agentes de controle biológico, por apresentar ação direta sobre fitopatógenos ou alterarem o metabolismo de plantas, o que pode estar associado com a sintese de compostos de defesa.<br>Algae produce several different compounds that show biological activity, including ones with antiviral activity or that act as phytoalexin inducers. Thus, it was investigated the reduction of Tobacco mosaic virus (TMV) symptoms on tobacco plants treated with cyanobacteria or eukaryotic algae, and it was studied the way of action of algae on the studied pathosystem. When the tobacco plants were treated two days before the inoculation, it was verified that the suspension of 004/02, 008/02, 061/02 Anabaena sp., and Nostoc sp. 61 strains as well as the intracellular preparation of 004/02 strain (4 C) and the medium filtrated from 061/02 strain (61 M) reduced TMV symptoms on tobacco plants, cultivar TNN. Furthermore, it was studied the direct effect of the algae suspensions on virus particles. The results showed that Anabaena sp., Nostoc sp. 21, Nostoc sp. 61 and 090/02 strains have compounds with direct activity on TMV. To try to elucidate the way of the action of algae, on the studied pathosystem, several biochemical parameters were investigated. It was seen that the preparation 4 C increase peroxidase activity and all treatments decrease &#946;-1,3-glucanase activity on tobacco leaves from the forth day on after the treatment. Moreover, 008/02 and 061/02 strains and the 61 M preparation caused higher superoxide accumulation, and the preparation 4 C decreased hydrogen peroxide accumulation when compared to the controls distilled water and BG 11 medium 37 hour after virus inoculation. In this way, the algae could be a biocontrol agents, because it shows direct action on phytopathogens and/or change the metabolism of the plants, that could be associated with the synthesis of deffence compounds.

Este trabalho teve por objetivo selecionar estirpes fracas do Passion fruit woodiness virus (PWV) e avaliar o seu efeito protetor para o controle do endurecimento dos frutos do maracujazeiro. Foram selecionadas seis estirpes fracas do PWV. Três a partir de plantas de elite, encontradas em pomares severamente afetados pelo vírus (F-101, F-102 e F-103) e três a partir de bolhas formadas em folhas de maracujazeiro com mosaico (F-99, F-144 e F-145). O efeito protetor das estirpes fracas foi avaliado em maracujazeiros, em casa de vegetação e em campo. Em casa de vegetação foi observada uma proteção parcial das estirpes F-101, F-102 e F-144, contra a estirpe severa PWV-SP. Em campo, num primeiro experimento, as seis estirpes fracas selecionadas foram avaliadas e aproximadamente 4 meses após o desafio com a estirpe PWV-SP, todas as plantas apresentaram sintomas severos da doença. Diante da proteção parcial em casa de vegetação e da ausência total de proteção no experimento de campo, duas hipóteses foram apresentadas para explicar a intensificação de sintomas em maracujazeiros premunizados e desafiados com a estirpe severa do virus: a) a ocorrência de baixa concentração e/ou distribuição irregular das estirpes fracas nos tecidos das plantas premunizadas permite a infecção e estabelecimento da estirpe severa posteriormente inoculada e b) as estirpes fracas selecionadas são de uma espécie diferente de Potyvirus, serologicamente relacionada com o PWV, mas que não oferecem proteção contra a estirpe severa deste último. A primeira hipótese foi estudada repetindo-se o experimento com maracujazeiros premunizados com as estirpes F-101 e F-144, separadamente, e cultivados em campo sob condições de telado. Antes do desafio, foram feitos estudos quantitativos das estirpes F-101 e F-144, em diferentes folhas das plantas, através do DAS-ELISA indireto. Foi observada uma grande variação na concentração das estirpes fracas nos tecidos de diferentes folhas da mesma planta. Em 68,3 %, de 300 discos foliares, as estirpes fracas não foram detectadas pelos critérios adotados nessa investigação. Mais uma vez todas as plantas premunizadas e desafiadas apresentaram sintomas severos da doença, quatro meses após o desafio. A segunda hipótese foi estudada através de testes de proteção em plantas de crotalária premunizadas com as estirpes F-101 e F-144 e da análise da seqüência de nucleotídeos do gene da capa protéica das estirpes F-101, F-103 e PWV-SP. Nos testes de proteção, todas as plantas premunizadas com as estirpes fracas ficaram protegidas contra a infecção e/ou manifestação dos sintomas causados pela estirpe severa PWV-SP. Estudos quantitativos das estirpes fracas nessa hospedeira revelaram uma maior uniformidade na concentração do vírus nos tecidos foliares. A análise da seqüência de nucleotídeos do gene que codifica a capa protéica, apontaram identidade de 99,7 % entre as estirpes fracas e de 97,5 % destas com a estirpe severa, mostrando tratarem-se de estirpes do mesmo vírus. Esses resultados mostram que a premunização não parece ser uma alternativa adequada para o controle do endurecimento dos frutos do maracujazeiro, devido à falha na proteção. Essa quebra de proteção parece estar relacionada com a baixa concentração e/ou distribuição irregular das estirpes fracas nas folhas do maracujazeiro, que propiciam a existência de sítios de infecção para a estirpe severa posteriormente inoculada.<br>The main purpose of this work was to select mild strains of Passion fruit woodiness virus (PWV) and to evaluate their protective effect in passion flower (Passiflora edulis f. flavicarpa Deg.) challenged with a severe strain of the virus. Three mild strains were selected from outstanding plants found in orchards severely affected by the virus (F-101, F-102 and F-103) and three others were obtained from blisters formed in passion flower leaves with mosaic (F-99, F-144 and F-145). The protective effect of the mild strains was evaluated in passion flower under greenhouse and field conditions. Plants preimmunized with mild strains F-101, F-102 and F-144, under greenhouse conditions, showed partial protection after challenge inoculation with the severe strain PWV-SP. Total absence of protection was observed in passion flower preimmunized with all six mild strains and challenged with PWV-SP in the first field experiment. Due to these results, two hypotheses were raised to explain the intensification of symptoms in passion flower preimmunized with mild strains and challenged with the severe strain of the virus: a) the occurrence of low concentration and/or irregular distribution of the mild strains in the tissues of the preimmunized plants allow the infection and establishment of the later inoculated severe strain and b) the selected mild strains belong to a different species of Potyvirus, serologically related to PWV, but that do not offer protection against the severe strain of PWV. The first hypothesis was studied in a field experiment with passion flower preimmunized with mild strains F-101 and F-144, separately, and cultivated under screenhouse. Before the challenge inoculation, leaf samples were taken from five leaves of all protected plants and the concentration of the mild strains was estimated by indirect DAS-ELISA. A group of plants was challenged in three expanded leaves of the vine and another group was challenged with viruliferous aphids placed on the tip of the vine. All preimmunized plants showed severe symptoms of the disease, four months after the challenge inoculation. A great variation was observed in the concentration of the mild strains in the tissues of different leaves of the same plant. The ELISA test was not able to detect the mild strains in extracts of 205 out of 300 leaf disks. The second hypothesis was tested with crotalaria plants (Crotalaria juncea L.) preimmunized with mild strains F-101 and F-144 and analysis of the nucleotide sequence of the coat protein gene of the F-101, F-103 and PWV-SP strains. All preimmunized crotalaria plants were protected against the infection and/or manifestation of the symptoms caused by the severe strain PWV-SP. Quantitative studies of the mild strains in crotalaria revealed a larger uniformity in the concentration of the virus in the leaves. The analysis of the nucleotide sequence of the coat protein gene pointed out identity of 99.7% among the mild strains. The severe strain shared 97.5 % identity with both mild strains, showing that they are all strains of the same virus. These results showed that preimmunization does not seem to be an appropriate alternative for the control of the passion fruit woodiness disease in passion flower due to the breakdown in protection. Failure in protection seems to be related to the low concentration and/or irregular distribution of the mild strains in the leaves of the passion flower, which allow the occurrence of infection sites available for superinfection with the severe strain.

O mosaico rugoso, causado pelo begomovirus ToSRV, é uma das principais doenças do tomateiro. Neste trabalho avaliou-se a eficácia de quatro inseticidas, ciantranilliprole foliar, ciantraniliprole solo, espiromesifeno e tiametoxam no controle das infecções primária e secundária do ToSRV, em tomateiros, transmitido por Bemisia tabaci biótipo B. Os tratamentos, confinados separadamente em gaiolas a prova de insetos, foram: controle, representado por tomateiros sadios e infectados, pulverizados com água, mais insetos avirulíferos; infecção primária, simulada com tomateiros sadios pulverizados com inseticida, mais insetos virulíferos e infecção secundária, simulada com tomateiros sadios e infectados com o ToSRV, pulverizados com inseticida, mais insetos avirulíferos. Nenhum inseticida foi eficiente no controle da infecção primária. No caso da simulação da infecção secundária, 4% e 16% respectivamente, dos tomateiros tratados com os inseticidas ciantraniliprole solo e ciantraniliprole foliar foram infectados, contra 84% e 74% de tomateiros infectados nos respectivos controles. Para os tratamentos com os inseticidas tiametoxam e espiromesifeno, na simulação da infecção secundária, 58% e 62% dos tomateiros foram infectados, respectivamente, contra 66% e 74% de plantas infectadas nos respectivos controles. O uso racional de inseticidas que reduzem a infecção secundária associado com a eliminação de fontes externas de inóculo poderá contribuir para o manejo da doença.<br>The severe mosaic, caused by ToSRV begomovirus, is a major disease of tomato. This study evaluated the efficacy of four insecticides, sprayed cyazypyr, drench cyazypyr, sprayed spiromesifen and thiamethoxam on controlling primary and secondary infections by ToSRV to tomato plants, transmitted by Bemisia tabaci biotype B. Treatments were confined separately in proof insects cages, which were: control, represented by healthy and infected tomato plants sprayed with water and aviruliferous insects releasing; primary infection, simulated by healthy tomato plants with insecticide spraying and viruliferous insects releasing, and secondary infection, simulated with healthy tomato plants and ToSRV infected tomato plants, sprayed with insecticide, and aviruliferous insects releasing. None of the insecticides was effective in controlling primary infection. In the case of secondary infection simulation, 4% and 16% of the tomato plants treated with soil and foliar cyazypyr insecticides, respectively, were infected; against 84% and 74% of infected tomato plants in their respective controls. For treatments with thiamethoxam and spiromesifen insecticides spraying, in secondary infection simulation, 58% and 62% of tomato plants were infected, respectively, versus 66% and 74% of infected plants in their respective controls. The rational use of insecticides to reduce secondary infection associated with elimination of external sources of inoculum may contribute to disease management.

Analysis of biological performance parameters of Aphis craccivora on groundnut variety ICG12991 in laboratory and field trials demonstrated that ICG12991 was resistant to the aphid vector of groundnut rosette diseases and that this resistance was stable over time and under high aphid pressure. Feeding experiments related slow population development and high aphid mortality on ICG 12991 to an inhibition of phloem feeding from the sieve elements. Consequently, virus transmission of all three agents of rosette disease was almost totally absent even under very high pressure of viruliferous aphids. Further investigation of the underlying mechanism of resistance in ICG 12991 showed that the resistance was induced by aphid probes rather than constitutive. The induced plant response was described as a hypersensitive response around the aphid feeding sites. Aphid-resistance and hypersensitivity were identified in at least 7 groundnut varieties and it is proposed that more varieties are likely to express these characteristics. The results led to the recommendation to evaluate groundnut varieties for aphid resistance by evaluating aphid population development. The identification of vector resistance and its efficacy in reducing virus spread of rosette disease in the field has opened the way for novel approaches for groundnut disease management.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-11-07T17:02:36Z No. of bitstreams: 2 Dissertação - Míriam de Almeida Marques - 2011.pdf: 1500273 bytes, checksum: d34aa41a75621b87445ae291524bb2e7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5)<br>Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2014-11-07T17:02:51Z (GMT) No. of bitstreams: 2 Dissertação - Míriam de Almeida Marques - 2011.pdf: 1500273 bytes, checksum: d34aa41a75621b87445ae291524bb2e7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5)<br>Made available in DSpace on 2014-11-07T17:02:51Z (GMT). No. of bitstreams: 2 Dissertação - Míriam de Almeida Marques - 2011.pdf: 1500273 bytes, checksum: d34aa41a75621b87445ae291524bb2e7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2011-08-01<br>As an alternative to synthetic insecticides, mineral oils and vegetable oils have been shown to be promising in controlling Bemisia tabaci biotype B. This study aimed to evaluate the effect of mineral and vegetable oils on eggs, nymphs and adults of B. tabaci biotype B, in the transmission of the bean golden mosaic virus and on dry production. The experiments were conducted at screenhouse at Embrapa Rice and Beans, Santo Antônio de Goiás, Goiás. The following oils in the concentration of 1% were used: Assist® mineral oil, Neem Max® neem oil, Veget'oil® vegetable oil, Citrole® orange oil, Castor Oil®, Sesame Oil®. The two last oils were emulsified with detergent Zupp® at 2%. Thiamethoxam at 200 g pc/ha (GD - dispersible granule) was used as a standard for comparison of chemical insecticide and distilled water as control. In all experiments, the experimental unit consisted of a polyethylene pot (34 cm diameter x 12 cm) containing two seedlings of common bean cv. Pearl. The experimental design was completely randomized with four replications / treatment. In the first experiment was evaluated the effect of oils on the mortality of eggs of B. tabaci. The second experiment was conducted to verify that the treatment of eggs with the oils would affect first instar nymphs. In the third experiment was evaluated the mortality of nymphs of the second to fourth instar after spraying of oil on the second instar nymphs. In these experiments, seedlings of bean cv. Pearl infested with eggs or nymphs of the whitefly were sprayed with 250 μL of each treatment on the abaxial surface of leaves. In the fourth experiment, four seedlings infested with adults and distributed in one m2 were sprayed with 30 ml of treatment. In the fifth experiment the effect of oils on adult mortality, the incidence of bean golden mosaic virus and crop production were evaluated by spraying only vegetable oils before the infestation of the seedlings with adults. All treatments caused mortality of eggs lesser or equal to 9.5%. Vegetable oils, when applied to eggs, caused mortality of newly-hatched nymphs lesser or equal to 22.3%. All treatments caused significant mortality of second instar nymphs, ranging from 46.5% for thiamethoxan to 81.6% for to castor oil. The treatments also affected the development of the nymphs until the fourth instar. Mortalities of nymphs greater than 80% were observed with castor, neem, sesame and orange oils. Only castor, sesame and mineral oils caused significant adult mortalities of 41.2%, 25.7% and 38.0%, respectively, one day after spraying. The orange, castor, neem and sesame oils, when sprayed prior to insect infestation of beans seedlings, caused mortality of whitefly adults ranging from 32.1% to 53.6%, one day after application. The castor and orange oils reduced the incidence of golden mosaic virus in bean. Castor oil favored greater pod yield, while the lowest pod yield was observed on sesame oil. The total number of seeds, mean number of seeds per pod and seed weight was not increased with the use of vegetable oils. The use vegetable and mineral oils have the potential to control B. tabaci biotype B.<br>Como alternativa aos inseticidas sintéticos, óleos vegetais e óleos minerais têm se revelado promissores no controle da Bemisia tabaci biótipo B. Esse trabalho teve como objetivo avaliar o efeito de óleos vegetais e mineral sobre ovos, ninfas e adultos da B. tabaci biótipo B e sobre a transmissão do vírus do mosaico dourado no feijoeiro e na sua produtividade. Os experimentos foram conduzidos em casa telada na Embrapa Arroz e Feijão, Santo Antônio de Goiás, Goiás. Utilizaram-se os seguintes óleos na concentração de 1%: óleo mineral Assist®, óleo de nim Max Neem®, óleo vegetal Veget’oil®, óleo de laranja Citrole®, óleo de Mamona®, óleo de Gergelim®, sendo os dois últimos emulsificados com detergente neutro Zupp® a 2%. Como padrão de comparação de inseticida químico utilizou-se o thiamethoxam a 200 g p.c./ha (granulado dispersível-GD) e como testemunha água destilada. Em todos os experimentos, a unidade experimental foi constituída por um vaso de polietileno (34 cm de diâmetro x 12 cm de altura) contendo duas plântulas do feijoeiro comum cv. Pérola. O delineamento experimental utilizado foi o inteiramente casualizado com quatro repetições/tratamento. No primeiro experimento foi avaliado o efeito dos óleos sobre a mortalidade dos ovos da B. tabaci. O segundo experimento foi instalado para verificar se o tratamento de ovos com os óleos afetaria as ninfas de primeiro ínstar da mosca-branca. No terceiro experimento foi avaliada a mortalidade de ninfas do segundo ao quarto ínstar após a pulverização dos óleos sobre ninfas do segundo ínstar. Nestes experimentos, plântulas de feijão cv. Pérola infestadas com ovos ou ninfas da mosca-branca foram pulverizadas com 250 μL de cada tratamento na face abaxial das folhas. No quarto experimento, quatro plântulas infestadas com adultos e distribuídas em um m2 foram pulverizadas com 30 ml dos tratamentos. No quinto experimento avaliou-se o efeito dos óleos na mortalidade dos adultos, na incidência do vírus do mosaico dourado do feijoeiro e na produtividade dessa cultura, pulverizando-se apenas os óleos vegetais antes da infestação dos adultos nas plântulas. Todos os tratamentos causaram mortalidade de ovos menor ou igual a 9,5%. Os óleos vegetais quando aplicados sobre os ovos, apresentaram efeito residual, causando mortalidade de ninfas recém-eclodidas menor ou igual a 22,3%. Todos os tratamentos causaram mortalidades significativas de ninfas do segundo ínstar, variando de 46,5% para o thiamethoxan até 81,6% para o óleo de mamona. Os tratamentos também afetaram o desenvolvimento das ninfas até o quarto ínstar. Foram observadas mortalidades de ninfas maiores que 80% com o óleo de mamona, nim, gergelim e laranja. Somente os óleos de mamona, gergelim e mineral causaram mortalidades significativas de adultos de 41,2%, 25,7% e 38%, respectivamente, um dia após a pulverização. Os óleos vegetais de laranja, mamona, nim e gergelim, quando pulverizados antes da infestação do inseto em plântulas de feijão, causaram mortalidade de adultos da mosca-branca variando de 32,1% a 53,6%, um dia após aplicação. Os óleos de mamona e laranja reduziram a incidência do vírus do mosaico dourado no feijoeiro. O óleo de mamona favoreceu maior produção de vagens, enquanto que o gergelim proporcionou menores valores de produção. O número total de grãos, número de grãos por vagem e massa de grãos, não foi incrementado com o uso de óleos vegetais. O uso de óleos vegetal e mineral apresenta potencial no controle da B. tabaci biótipo B.

Master of Science<br>Entomology<br>C. Michael Smith<br>The bird cherry oat aphid, Rhopalosiphum padi, and one of the viruses it vectors, barley yellow dwarf virus (BYDV), form a very damaging pest unit on cereals. Understanding how people can better manage crops to prevent damage or recognize environmental or geographic factors that put their crops at risk for BYDV could lead to improved virus aphid management strategies. One of the most successful methods for mitigating pest damage is using pest-resistant varieties of crops. Seven candidate wheat varieties were screened for resistance to R. padi, by testing aphid population densities and aphid host choice. Results of this research identified six varieties of wheat that show resistance to R. padi. One of the major knowledge gaps in BYDV management is forecasting potential damage. Our objective was to create viral presence maps, to start building the foundation of correlations between persistent and changing frequency of viruliferous R. padi. The results of BYDV assays in R. padi in wheat fields across Kansas indicated that the amount of BYDV infection in viruliferous aphids changes rapidly from year to year, and differs considerably between geographic regions and field landscape characteristics in Kansas. Neonicotinoid wheat seed treatment is a management technique that reduces R. padi populations. However, a common question among producers is whether or not seed treatments stop viral transmission. Results of greenhouse seed treatment experiments with plants from neonicotinoid treated and untreated seed infested with viruliferous R. padi indicated no significant difference in viral transmission due to seed treatment. This new information about R. padi wheat varietal resistance, geographic distribution of BYDV in Kansas, and neonicotinoid seed treatment creates a better understanding of aphid-virus-wheat interactions. Results from this thesis directly enhance producer ability to forecast risk from BYDV, select wheat varieties that effectively reduce R. padi as a BYDV vector, and to make better decisions about the use of insecticide to reduce BYDV infection.

Las aves son susceptibles a una serie de enfermedades infecciosas que afectan su productividad, dentro de ellas la enfermedad de Newcastle es considerada como una de las más importantes porque las cepas velogénicas del virus tienen un gran impacto sanitario sobre la industria avícola, siendo una enfermedad notificable a la Organización Mundial de Salud Animal (OIE). Los programas de vacunación para prevenir y controlar la enfermedad causada por este tipo de cepas velogénicas son diversos y varían en función del riesgo sanitario de la granja, muchos de ellos requieren de varias aplicaciones de vacunas durante la campaña de crianza de las aves, usualmente al 1,10 y 28 día de edad, este manejo podría generar un stress en los pollos y originar mermas en el rendimiento productivo del lote en el largo plazo. La genética actual ha hecho que los pollos de engorde de conformación sean altamente susceptibles a condiciones de estrés, por lo que existe la necesidad de inmunizar a las aves contra todos los agentes en planta de incubación evitando el stress de la vacunación y los riesgos que ésta conlleva. La hipótesis de la tesis es que la vacuna contra la enfermedad de Newcastle vectorizada en el virus HVT de la enfermedad de Marek aplicada a pollos al primer día de edad en planta de incubación protege contra cepas patógenas del vENC. Se realizó un Ensayo Clínico que probó la vacuna vectorizada en Marek comparándola con otros esquemas de vacunación y un grupo control. Las aves se desafiaron a los 26 días post vacunación, con una cepa velogénica de campo y se registraron los efectos adversos y la eficacia de las vacunas. El esquema con la mejor protección y el menor número de eventos adversos fue la vacuna vectorizada con Marek.<br>Tesis

No estado do Espírito Santo (ES), uma das principais áreas produtoras de mamão do país, a eliminação sistemática de plantas doentes tem sido aplicada desde a década de 1980 para o controle do mosaico do mamoeiro (Papaya ringspot virus - type P; PRSV-P). O uso permanente dessa prática nos últimos 25 anos levou a uma aparente seleção e predominância de isolados fracos do vírus. Os objetivos deste trabalho foram: investigar a prevalência desses isolados fracos, bem como a estabilidade e o efeito protetor contra isolados severos do vírus; estudar a infecção natural de abobrinha de moita (Cucurbita pepo cv. Caserta) e abóbora moranga (C. maxima cv. Exposição) com o PRSV-P quando plantadas ao lado de mamoeiros infectados e caracterizar um isolado do Alfalfa mosaic virus (AMV) em infecção natural em mamoeiro. A detecção de possíveis isolados fracos do vírus foi realizada por PTAELISA, microscopia eletrônica e RT-PCR. Todos os isolados também foram inoculados mecanicamente em mamoeiro cv. Golden para avaliação de sintomas. Sequências de nucleotídeos e de aminoácidos deduzidos do gene da proteína capsidial de alguns isolados fracos mostraram identidades superiores a 89% e 90%, respectivamente, com isolados do PRSV-P. De 119 amostras de mamoeiros analisadas, 86 estavam infectadas com o PRSV-P, mas somente 75 induziram sintomas fracos em mamoeiros. Quatro isolados fracos foram selecionados ao acaso para estudos de estabilidade, e de proteção em casa de vegetação. Apenas dois isolados fracos induziram sintomas estáveis em mamoeiros até a oitava transferência. A proteção total só foi obtida com plantas premunizadas com dois isolados fracos e desafiados com o isolado PRSV-PES. Plantas de mamoeiros cv. Golden premunizadas com vários isolados fracos do PRSV-P foram expostas em condições de campo na ESALQ/USP, Piracicaba, SP, em dois experimentos independentes. Poucas plantas permaneceram com sintomas fracos de mosaico até o final dos experimentos. Uma terceira exposição foi realizada em Linhares, ES, com mamoeiros cvs. Sunrise Solo e Golden premunizados com oito isolados fracos, coletados nos experimentos em campo na ESALQ/USP. Apenas uma planta premunizada com um isolado fraco permaneceu com sintomas leves da doença até a última avaliação. Tentativas de detectaçao de infecções naturais de cucurbitáceas com o PRSV-P foram realizados em dois plantios de abobrinha de moita e dois de abóbora moranga, na ESALQ/USP, Piracicaba, SP. A detecção do vírus foi feita por meio da inoculação de extratos foliares das cucurbitáceas em mamoeiros cv. Golden. Os mamoerios foram avaliados por meio de sintomas, PTA-ELISA e RT-PCR. Nenhuma planta de mamoeiro inoculada com extratos foliares das duas cucurbitáceas exibiu sintomas de mosaico, embora o gene ci, mas não o cp, tenha sido detectado em uma amostra de folhas de mamoeiro, indicando que ao menos uma planta de abobrinha de moita estava infectada. Finalmente, no decorrer dos ensaios de campo na ESALQ/USP, constatou-se uma planta de mamoeiro apresentando sintomas severos de mosaico amarelo, deformação foliar e necrose sistêmica, diferente daqueles induzidos pelo PRSV-P. Análises biológicas, sorológica e moleculares confirmaram tratar-se do AMV. Este é o primeiro relato de infecção natural de mamoeiro com esse vírus.<br>Papaya ringspot virus type P (PRSV-P) causes the major disease in Brazilian papaya orchards that result in significant yield losses. In Espírito Santo state systematic rouging of infected plants has been applied since early 1980s for the control of this disease. Its permanent use over the last 25 years has lead to an apparent selection and predominance of mild strains throughout papaya orchards. The objectives of this work were to investigate the prevalence of mild isolates, as well the stability and protective effect against severe isolates of the virus; The aim of this work was to study the natural infecction of zucchini squash (Cucurbita pepo cv. Caserta) and pumpkin (C. maxima cv. Exposição) grown near to papaya trees infected with PRSV-P and characterize an isolate of Alfalfa mosaic virus (AMV) in natural infection in papaya. The detection of possible mild isolates of the virus was performed by PTA-ELISA, electron microscopy and RT-PCR. All isolates were inoculated mechanically in papaya cv. Golden for symptoms evaluation. Nucleotides and deduced amino acids sequences of the coat protein gene of some mild isolates showed identities above 89% and 90%, respectively, with isolates of PRSV-P. Of 119 samples from papaya plants analyzed, 86 were infected with PRSV-P and 75 induced mild symptoms on papaya. Four mild isolates were randomly selected for stability and protection studies under greenhouse. Only two isolates induced mild symptoms on papaya and remained stables until the eighth transference. Full protection was obtained with preimmunized plants with two mild isolates and challenged with the isolate PRSV-P-ES. Plants of papaya cv. Golden preimmunized with several mild isolates of PRSV-P were exposed under field conditions at ESALQ/USP, Piracicaba,SP, in two independent experiments. Few plants remained with mild mosaic symptoms at the end of the experiments. A third field exposition was held in Linhares,ES, with papaya cvs. Golden and Sunrise Solo preimmunized with eight mild isolates, collected in field experiments at ESALQ/USP. Only one plant preimmunized with a mild isolate remained with mild symptoms of the disease until the last evaluation. Attempts of detection of natural infections of cucurbits with PRSV-P were carried out in two plantations of zucchini squash and pumpkin at ESALQ/USP, Piracicaba,SP. The detection of the virus was made by inoculation of leaf extracts of cucurbits in papaya cv. Golden. The papaya plants were assessed by symptoms, PTAELISA and RT-PCR. None of papaya plants exhibited symptoms of mosaic, while the ci gene, but not the cp, was detected in a sample of leaves of papaya, indicating that at least one clump of zucchini squash plant was infected. Finally, during the field test at ESALQ/USP, a papaya plant was found showing severe symptoms severe yellow leaf mosaic, leaf distortion and systemic necrosis, different from those induced by PRSV-P. Biological, serological and molecular tests confirmed the infection with AMV. This is the first report of natural infection of papaya with this virus.

Au champ, le contrôle de la transmission non persistante du virus Y de la pomme de terre repose sur l’utilisation d’huiles minérales blanches en épandage hebdomadaire. Si ces traitements sont efficaces, leurs modes d’action ne sont pas complètement élucidés. Au cours de ces travaux de thèse, nous avons été amenés à évaluer l’impact du traitement sur chacun des 3 partenaires du pathosystème : la plante, le virus et son vecteur, le puceron. Nous avons montré que le traitement des plants de pomme de terre avant leur infestation par des pucerons virulifères, réduit significativement la transmission du virus YNTN. De plus, celui-ci affecte le comportement et la physiologie du puceron colonisateur M. Euphorbiae. Nous avons caractérisé des effets négatifs sur l’orientation du puceron adulte et la survie des stades larvaires. Des effets probiotiques se traduisent par l’amélioration de la survie et de la reproduction des pucerons adultes, ainsi que de certains paramètres de la prise alimentaire. Etonnement, nous montrons que l’exposition directe du puceron au traitement suffit à déclencher des effets similaires : aphicides et probiotiques. Enfin, nous montrons que le traitement de la plante entraine des événements cellulaires précoces et tardifs similaires aux processus de perception et de signalisation en réponse à un bioagresseur. Les résultats obtenus placent l’huile minérale parmi les méthodes de lutte alternatives des plus prometteuses, pour son efficacité et qui répond aux préoccupations environnementales actuelles<br>Mineral oil weekly spray has been shown to efficiently reduce non persistent PVY transmission in the field. To date, its mode of action is poorly understood. In this project we assessed mineral oil effect on each partner of the plant – aphid – virus system. We showed that potato plant treatment prior to viruliferous aphid infestation, led to significantly reduced virus YNTN transmission. Besides, it affects M. Euphorbiae behaviour and physiology. Indeed, we characterized negative effects on adult orientation and nymphal survival. Other probiotic effects characterized were: enhanced longevity and reproduction of surviving adults, and some parameters related to feeding behaviour as well. Surprisingly, we demonstrated that direct aphid exposure to mineral oil is sufficient to trigger similar aphicid and probiotic effects. Eventually, we show that plant treatment triggers cellular events similar to pathogen perception and signalling response. Our results highlight the high-potential use of mineral oil as alternative method in virus transmission control, for its efficacy and environment friendly use as well

Thesis (PhD)--University of Stellenbosch, 2003.<br>ENGLISH ABSTRACT: Grapevine leafroll-associated virus-3 is one of ten members of the C/osteroviridae that are known to infect grapevine. Nine of these viruses are associated with grapevine leafroll disease, of which GLRaV-1 and GLRaV-3 are the most important and widespread. Members of the C/osteroviridae are unique amongst the viruses, as it is the only known family whose members encode a heat shock protein 70 kOa homolog (Hsp70h). The Hsp70h is a movement protein (MP) that is required for the active translocation of the virion structure through the plasmodesmata into adjacent cells. Broad-spectrum resistance to unrelated viruses can be obtained by a pathogen-derived resistance (POR) strategy that is based on the expression of a dysfunctional MP in plants. The Hsp70h has two distinct domains. The N-terminal two thirds of the protein is an ATPase domain and shares high homology with the ATPase domains of all Hsp70h proteins from the C/osteroviridae and Hsp70 proteins from the prokaryote and eukaryote kingdoms. Conserved amino acids are found in the ATPase domain and are required for the positioning of the ATP at the catalytic site for ATP hydrolysis. The C-terminal domain is variable and the function of this domain in the Closteroviridae is not known. In prokaryote and eukaryote Hsp70 proteins, the C-terminal domain is required for protein-protein interactions. The American NY-1 isolate of GLRaV-3 has been sequenced and POR strategies have been attempted with the coat protein, divergent coat protein and replicase genes, but not with a dysfunctional form of the hsp70h gene. In this study, double-stranded RNA was isolated from a commercial vineyard with unknown virus status, but with distinct grapevine leafroll symptoms, and from two grapevine sources of known virus status, one with mild and one with severe symptoms. The GLRaV-3 hsp70h gene was amplified by RT-PCR from the dsRNA and the gene sequence was analysed. The hsp70h gene from the three virus sources contained more than 94% nucleotide sequence homology to the NY-1 isolate and the conserved amino acids required for ATPase activity were present. The hsp70h gene isolated from GLRaV-3 from a commercial Stellenbosch vineyard showing clear leafroll symptoms was selected for further work and was subjected to site-directed mutagenesis to engineer four point mutations in the gene. These four mutations resulted in the substitution of Asn for Asp", Gly for Thr1O, Lys for Glu 174 and Asn for Asp 197. The wild type (WT) and mutated (Mut) forms of the hsp 70h genes were cloned into a bacterial expression vector. Expression of both the WT- and Mut-Hsp proteins was achieved, and the protein was expressed in the insoluble inclusion bodies. All attempts to refold and isolate active proteins from the inclusion bodies were unsuccessful. Attempts to increase the concentration of soluble protein within the expressing bacteria were unsuccessful. Due to the lack of active protein, biochemical tests on the ATPase activity of the WT- and Mut-Hsp proteins could not be conducted. The wt- and mut-hsp genes were cloned into a plant expression vector for transformation into tobacco plants. These transformations were successful and gave rise to 22 Km' and 18 Km' plants from the WT- and Mut-Hsp constructs respectively. Two plant lines, M5 and M10, transformed with the mut-hsp transgene construct, appeared to have a high level of resistance to the challenging potato X potexvirus, whereas all the other tested plants were susceptible to the challenging virus. It was thus shown that a dysfunctional form of the GLRaV-3 Hsp70h could provide resistance to an unrelated virus in tobacco.<br>AFRIKAANSE OPSOMMING: Wingerdrolblaar-geassosieerde virus 3 (GLRaV-3) is een van 10 lede van die Closteroviridae wat wingerd kan infekteer. Nege van die virusse is met wingerdrolblaar geassosieer. Die GLRaV-1 en GLRaV-3 is die belangrikste en mees wyd verspreide lede van die rolblaar-geassosieerde Closteroviridae. Lede van die Closteroviridae is uniek in die opsig dat die virusse vir 'n 70 kDa-homoloë hitteresponsproteïen (Hsp70h) kodeer. Die Hsp70 is 'n bewegingsproteïen (MP) wat belangrik is vir die translokasie van die virus deur die plasmodesmata na die naasliggende sel. Breë-spektrum weerstand teen onverwante virusse kan behaal word deur 'n patogeen-afgeleide weerstandstrategie (POR), wat op die uitdrukking van 'n disfunksionele MP wat in plante uitgedruk word, gebaseer is. Die Hsp70hproteïen het twee gebiede. Die N-terminale gebied is In ATPase-gebied en toon hoë homologie met ander ATPase-gebiede van Hsp70h-proteïene van die Closteroviridae, asook die prokariotiese en eukariotiese koninkryke. Gekonserveerde aminosure wat belangrik is vir die posisionering van ATP in die katalitiese domein vir ATP-hidrolise is in die ATPase-gebied gevind. Die C-terminale gebied is variërend en die funksie van die gebied in die Closteroviridae is onbekend. In prokariotiese en eukariotiese Hsp70h-proteïene is die C-terminale gebied belangrik vir proteïenproteïen interaksies. Die nukleotiedvolgorde van die Amerikaanse NY-1-isolaat van GLRaV-3 is al bepaal en POR-strategieë is ook op die kapsiedproteïen, uiteenlopende kapsiedproteïen en die replikasie-proteïen uitgevoer, maar nog nie op 'n disfunksionele vorm van die Hsp70h-geen nie. In hierdie studie is dubbelstring-RNA (dsRNA) van 'n kommersiële wingerd met onbekende virusstatus wat rolblaarsimptome toon, geïsoleer, asook van twee wingerde met 'n bekende virusstatus, een met ligte en een met strawwe simptome. Die GLRaV-3 hsp70h-geen is met hulp van die polimerasekettingreaksie-metode (PKR) vanaf die dsRNA geamplifiseer en die geen se nukleotiedvolgorde is bepaal. Die hsp 70-gene van drie verskillende wingerde het meer as 94% homologie met die NY-1-isolaat getoon. Die gekonserveerde aminosure wat vir ATPase-aktiwiteit belangrik is, was teenwoordig. Die hsp70h-geen van GLRaV-3, wat uit 'n kommersiële wingerd met duidelike rolblaarsimptome in die Stellenbosch-gebied geïsoleer is, is vir verdere navorsing gekies en dit is aan setel-gerigte mutagenese blootgestelom vier mutasies van die geen te bewerkstellig. Die gevolg van hierdie vier mutasies was die verandering van Asn na Asp", Gly na Thr1o, Lys na Glu174 en Asn na Asp197. Die wilde (WT) en veranderde (Mut) vorms van die hsp-gene is in 'n bakteriese uitdrukkingsvektor gekloneer. Uitdrukking van beide die WT- en die Mut-Hspproteïene is behaal, maar die proteïene was in die onoplosbare fraksie geleë. Pogings om die onoplosbare proteïene te isoleer en in 'n aktiewe oplosbare vorm te verkry, was onsuksesvol. Verdere pogings om die proteïene in die oplosbare fraksie van die bakteriese ekspressiesisteem uit te druk, was ook onsuksesvol. As gevolg van die gebrek aan aktiewe proteïen kon biochemiese toetse nie op die ATPaseaktiwiteit van die WT- en Mut-Hsp proteïne gedoen word nie. Die wt- en mut-hsp-gene is ook in In plantekspressievektor gekloneer vir transformasie in tabakplante. Hierdie transformasies was suksesvol en het aanleiding gegee tot 22 kanamisienbestande (Km') en 18 Km' plante vanaf die WT- en Mut-Hspkonstrukte onderskeidelik. Twee plantlyne, M5 en M10, wat met die mut-hsptransgene getransformeer is, het 'n hoë vlak van weerstand teen die infekterende aartappelvirus X getoon in vergelyking met ander plante wat met die virus geïnfekteer is. Daar is dus bewys gelewer dat 'n disfunksionele vorm van die GLRaV-3 Hsp70h weerstand kan bied teen 'n onverwante virus in tabak.

A cultura do tomateiro (Solanum lycopersicum L.) é importante mundialmente devido ao alto consumo de seus frutos. Nos últimos anos surgiram nesta cultura no Brasil alguns vírus emergentes com altas taxas de disseminação, como begomovírus e crinivírus, transmitidos pela Bemisia tabaci biótipo B, que podem causar danos à produção do tomateiro. A espécie de begomovírus atualmente mais encontrada no Brasil, em plantios de tomateiro, é o Tomato severe rugose virus (ToSRV). De 2002 a 2004, pesquisadores relataram incidências desse vírus em mais da metade das amostras com sintomas de geminiviroses coletadas em vários estados brasileiros e sua presença continua sendo verificada frequentemente. No ano de 2006, um crinivírus, o Tomato chlorosis virus (ToCV), foi relatado no Brasil, infectando plantas de tomate no Estado de São Paulo e atualmente encontra-se presente em diveros estados brasileiros. Os objetivos desse trabalho foram: determinar os períodos mínimos de acesso à aquisição e à inoculação do ToSRV e do ToCV pela B. tabaci biótipo B; identificar o período de retenção do ToSRV no inseto e a interação do ToSRV e do ToCV na aquisição e na transmissão por esse aleirodídeo. Também foi avaliada a eficiência do inseticida cloridrato de cartape no controle da disseminação primária e secundária do ToSRV pela B. tabaci biótipo B em tomateiros em gaiolas em casa de vegetação. Finalmente avaliou-se a eficiência do aleirodídeo Trialeurodes vaporariorum na transmissão de um isolado brasileiro do ToCV. Os períodos mínimos de acesso à aquisição e à inoculação de ambos os vírus pela B. tabaci biótipo B foram de cinco minutos. O tempo de retenção do ToSRV em B. tabaci biótipo B foi de 25 dias. A eficiência de um único adulto de B. tabaci na transmissão simultânea do ToSRV e do ToCV para tomateiros foi de 44,7%, similar àquela da transmissão isolada do ToRSV (47,4%) e do ToCV (44,7%). A eficiência de T. vaporariorum na transmissão do ToCV foi inferior à da B. tabaci biótipo B. Usando 40 insetos por vaso com duas plantas as eficiências de transmissão foram 57,7% e 100%, respectivamente. O inseticida cloridrato de cartape reduziu a infecção secundária do ToSRV pela B. tabaci biótipo B, mas não foi eficiente para reduzir a infecção primária em tomateiros.<br>Tomato (Solanum lycopersicum) is one of the leading vegetables grown and consumed in Brazil and in the world, after potato. The importance of tomato is related to its high consumption worldwide and also its nutritive value. Presently the most important virus diseases responsible for yield losses on tomato crops in Brazil are those caused by begomovirus and crinivirus, both transmitted by Bemisia tabaci biotype B. At the moment the prevalent species of begomovirus is Tomato severe rugose virus (ToSRV). From 2002 to 2004, researchers reported incidence of this virus in more than half of the symptomatic tomato samples collected in several Brazilian states. In 2006, a crinivirus, Tomato chlorosis virus (ToCV), was reported for the first time in Brazil, infecting tomato plants in the State of São Paulo and at present the virus occurs in several Brazilian states. The objectives of this study were to determine the minimum acquisition and inoculation access periods of ToSRV and ToCV by B. tabaci biotype B; identify the retention period of ToSRV in the insect; and the interaction of ToSRV and ToCV on the transmission by this aleyrodidae. It was also evaluated the effectiveness of the insecticide cartap hydrochloride in controlling the primary and secondary spread of ToSRV by B. tabaci biotype B on tomato plants in a greenhouse. Finally, it was evaluated the efficiency of Trialeurodes vaporariorum in the transmission of a Brazilian isolate of ToCV. The minimum acquisition and inoculation access periods for both viruses by B. tabaci biotype B were five minutes. The maximum retention time of ToSRV in B. tabaci biotype B was 25 days. The efficiency of a single adult of B. tabaci to simultaneously transmit ToSRV and ToCV to tomato plants was 44.7%, similar to the transmission of ToRSV (47.4%), and ToCV (44.7%) separately. T. vaporariorum was less efficient than B. tabaci on the transmission of ToCV. Using 40 insects per pot with two plants, transmission efficiencies were 57.7% and 100%, respectively. The insecticide cartap hydrochloride reduced secondary infection of ToSRV transmitted by B. tabaci biotype B, but was not effective in reducing the primary infection in tomato.

Made available in DSpace on 2015-12-10T14:24:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-08-07. Added 1 bitstream(s) on 2015-12-10T14:30:35Z : No. of bitstreams: 1 000851956.pdf: 1446006 bytes, checksum: d0fd0cae1f3cd364c6ad457c27e8870b (MD5)<br>A cultura do pimentão (Capsicum annuum L.) tem elevada importância econômica e social para o Equador. Vários fatores têm contribuído para a baixa produtividade dessa cultura no país e dentre eles destacam-se as doenças de diversas etiologias. Poucas informações sobre a ocorrência de doenças nessa cultura estão disponíveis nas para as condições equatorianas. Neste trabalho foi realizado levantamento da ocorrência da doença pata seca e de viroses na cultura do pimentão, em plantios comerciais, durante anos de 2013 e 2014; a identificação dos agente(s) causal(ais) da pata seca; avaliações do eficácia da pulverização da aplicação de produtos fitossanitários (sulfato de cobre + fosetil alumínio; iprodione + clorotalonil; Trichoderma harzianum + T. koingii) em condições de campo, no controle da doença nos híbridos Nathalie e Quetzal e a sensibilidade in vitro do(s) patógeno(s) dessa doença a fungicidas (tebuconazole, benomil, clorotalonil, fosetil alumínio, iprodione, metalaxil + propanocarb e tebuconazole). A pata seca foi constatada em um 79,2% das propriedades, com incidência entre 5% a 53,6%, em quanto as viroses, foram detectada em 62,5% das propriedades, com incidência entre 9,6% a 61,2%. Os fungos S. clerotium rolfsii e Fusarium spp. foram isolados com maior freqüência das plantas com sintomas de pata seca. Entretanto, S.rolfsii foi mais virulento, sendo considerado o principal agente causal da doença pata seca. Não foi observada diferença dos produtos fitossanitários, aplicados no colo das plantas, na incidência de pata seca, nos dois ensaios realizados em campo. A aplicação de iprodione + clorotalonil propiciou uma maior quantidade de frutos comerciais nos dois ensaios e uma maior produção em um dos ensaios. Os Isolados de S. rolfsii apresentaram maior sensibilidade aos fungicidas tebuconazole, benomil e iprodione, ...<br>Sweet pepper (Capsicum annuum L.) has high economic and social importance for Ecuador. Several factors have contributed to the low productivity of this crop in the country and among them there are the diseases of different etiologies. Little information on the occurrence of diseases in this culture is available to the Ecuadorian conditions. This study was carried out survey of the occurrence of the disease pata seca and viruses in sweet pepper in commercial plantations, during the years 2013 and 2014; the identification of the agent (s) causal (es) of pata seca; evaluations of the effectiveness of spray application of pesticides (copper sulfate + fosetyl aluminum, iprodione + chlorothalonil; Trichoderma harzianum + T. koingii) in field conditions in controlling the disease in hybrid Nathalie and Quetzal and in vitro sensitivity ( s) pathogen (s) of the disease to fungicides (tebuconazole, benomyl, chlorothalonil, fosetyl aluminum, iprodione, metalaxyl + propanocarb and tebuconazole). The pata seca was found in a 79.2% of the properties, with an incidence of 5% to 53.6%, as in the viruses were detected in 62.5% of the properties, with an incidence of 9.6% to 61.2%. The fungus Sclerotium rolfsii and Fusarium spp. were isolated more frequently plants with symptoms of pata seca. However, S. rolfsii was more virulent, being considered the main causative agent of the disease pata seca. There was no difference of pesticides applied in the base of the plants, the incidence of pata seca in the two trial carried out in the field. The application of iprodione + chlorothalonil provided a larger amount of fruits in the two ...

Orientador: Antonio Carlos Maringoni<br>Banca: Maria José de Marchi Garcia<br>Banca: Margarida Fumiko Ito<br>Banca: Marcelo Agenor Pavan<br>Banca: Tadeu Antonio Fernandes da Silva Júnior<br>Resumo: A cultura do pimentão (Capsicum annuum L.) tem elevada importância econômica e social para o Equador. Vários fatores têm contribuído para a baixa produtividade dessa cultura no país e dentre eles destacam-se as doenças de diversas etiologias. Poucas informações sobre a ocorrência de doenças nessa cultura estão disponíveis nas para as condições equatorianas. Neste trabalho foi realizado levantamento da ocorrência da doença "pata seca" e de viroses na cultura do pimentão, em plantios comerciais, durante anos de 2013 e 2014; a identificação dos agente(s) causal(ais) da "pata seca"; avaliações do eficácia da pulverização da aplicação de produtos fitossanitários (sulfato de cobre + fosetil alumínio; iprodione + clorotalonil; Trichoderma harzianum + T. koingii) em condições de campo, no controle da doença nos híbridos Nathalie e Quetzal e a sensibilidade in vitro do(s) patógeno(s) dessa doença a fungicidas (tebuconazole, benomil, clorotalonil, fosetil alumínio, iprodione, metalaxil + propanocarb e tebuconazole). A "pata seca" foi constatada em um 79,2% das propriedades, com incidência entre 5% a 53,6%, em quanto as viroses, foram detectada em 62,5% das propriedades, com incidência entre 9,6% a 61,2%. Os fungos S. clerotium rolfsii e Fusarium spp. foram isolados com maior freqüência das plantas com sintomas de "pata seca". Entretanto, S.rolfsii foi mais virulento, sendo considerado o principal agente causal da doença "pata seca". Não foi observada diferença dos produtos fitossanitários, aplicados no colo das plantas, na incidência de "pata seca", nos dois ensaios realizados em campo. A aplicação de iprodione + clorotalonil propiciou uma maior quantidade de frutos comerciais nos dois ensaios e uma maior produção em um dos ensaios. Os Isolados de S. rolfsii apresentaram maior sensibilidade aos fungicidas tebuconazole, benomil e iprodione, ...<br>Abstract: Sweet pepper (Capsicum annuum L.) has high economic and social importance for Ecuador. Several factors have contributed to the low productivity of this crop in the country and among them there are the diseases of different etiologies. Little information on the occurrence of diseases in this culture is available to the Ecuadorian conditions. This study was carried out survey of the occurrence of the disease "pata seca" and viruses in sweet pepper in commercial plantations, during the years 2013 and 2014; the identification of the agent (s) causal (es) of "pata seca"; evaluations of the effectiveness of spray application of pesticides (copper sulfate + fosetyl aluminum, iprodione + chlorothalonil; Trichoderma harzianum + T. koingii) in field conditions in controlling the disease in hybrid Nathalie and Quetzal and in vitro sensitivity ( s) pathogen (s) of the disease to fungicides (tebuconazole, benomyl, chlorothalonil, fosetyl aluminum, iprodione, metalaxyl + propanocarb and tebuconazole). The "pata seca" was found in a 79.2% of the properties, with an incidence of 5% to 53.6%, as in the viruses were detected in 62.5% of the properties, with an incidence of 9.6% to 61.2%. The fungus Sclerotium rolfsii and Fusarium spp. were isolated more frequently plants with symptoms of "pata seca". However, S. rolfsii was more virulent, being considered the main causative agent of the disease "pata seca". There was no difference of pesticides applied in the base of the plants, the incidence of "pata seca" in the two trial carried out in the field. The application of iprodione + chlorothalonil provided a larger amount of fruits in the two ...<br>Doutor

A broca da cana-de-açúcar, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) é naturalmente infectada por diversos patógenos, sendo os mais importantes Metarhizium anisopliae (Metsch.) (Ascomycota: Clavicipitaceae), Beauveria bassiana (Bals.) (Ascomycota: Cordycipitaceae) e vírus da granulose. Esses microrganismos podem co-infectar um mesmo indivíduo em condições de campo, mas os resultados das interações desses patógenos não são conhecidos. O presente trabalho objetivou determinar a virulência desses patógenos aplicados de forma isolada e em infecções mistas na fase larval de D. saccharalis em condições de laboratório sobre dieta artificial e em plantas de milho. A maioria das combinações resultou em efeito aditivo na mortalidade dos insetos (M. anisopliae + B. bassiana, M. anisopliae + DsGV e M. anisopliae + B. bassiana + DsGV), entretanto ficou evidenciado antagonismo entre B. bassiana e o granulovírus. Todos os cadáveres oriundos das aplicações associadas apresentaram sintomas de apenas um dos patógenos envolvidos na infecção. A produção de conídios de uma espécie de fungo nos cadáveres submetidos à co-infecção foi semelhante à produção de conídios da mesma espécie na infecção isolada. Na infecção mista, os dois fungos se desenvolveram na hemolinfa do hospedeiro, entretanto em um estágio tardio do processo infectivo ocorreu a exclusão de um dos patógenos. A infecção por B. bassiana diminuiu drasticamente a densidade de hemócitos circulantes na hemolinfa das lagartas, efeito não observado para M. anisopliae. Duas populações de laboratório de D. saccharalis, uma oriunda de Piracicaba-SP e outra de Araras-SP, foram testadas quanto à suscetibilidade aos fungos M. anisopliae e B. bassiana. A população de insetos de Piracicaba foi menos suscetível em 2009 do que a mesma população em 2008 e também em relação à população de Araras. Dessa forma, foi realizado um bioensaio comparando a suscetibilidade de insetos das duas populações e da progênie (F1) dos cruzamentos diretos entre elas, criadas nas mesmas condições bióticas e abióticas, para determinar se a suscetibilidade diferencial estaria relacionada a fatores genéticos. Não foram observadas diferenças significativas na suscetibilidade das duas populações e dos cruzamentos aos fungos. A suscetibilidade diferencial observada nos dois primeiros bioensaios provavelmente esteja relacionada às variações no sistema de criação, em componentes nutricionais e antimicrobianos da dieta e não a variações entre as populações dos hospedeiros.<br>The sugarcane borer, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) is naturally infected by various pathogens, the most important are Metarhizium anisopliae (Metsch) (Ascomycota: Clavicipitaceae), Beauveria bassiana (Bals.) (Ascomycota: Cordycipitaceae) and granulovirus. These microorganisms may coinfect the same host under field conditions, but the result of these pathogens interactions is not known. This study investigated the virulence of these pathogens applied separately and together on larvae of D. saccharalis in the laboratory on artificial diet and on corn plants. Most combinations resulted in additive effect on insect mortalities (M. anisopliae + B. bassiana, M. anisopliae + DsGV and M. anisopliae + B. bassiana + DsGV), however it became apparent antagonism between B. bassiana and the granulovírus. In all cadavers resulted from mixed applications, only one pathogen sporulated or externalized their symptoms in the host. Production of conidia of one fungal species on the cadavers subjected to co-infection was similar to the production of conidia of the same species in a single infection. In mixed infection, the two fungi have developed in the hemolymph of the host, and the exclusion of one pathogen occurred at a later stage of the infective process. Infection with B. bassiana has drastically reduced the density of circulating hemocytes in the hemolymph of the larvae, which was not observed for M. anisopliae. Two laboratory populations of D. saccharalis, one coming from Piracicaba-SP and other from Araras-SP, were tested for susceptibility to M. anisopliae and B. bassiana. The insect population from Piracicaba was less susceptible to application of fungi in 2009 compared to the same population in 2008 and compared to the Araras population. Therefore, a bioassay was performed comparing the susceptibility of the two populations and the progeny (F1) of direct crossings between them, using insects reared in the same conditions. This assay was performed to determine if the differential susceptibility would be related to genetic factors. No significant differences were observed in the susceptibility of insect populations and crossings to both fungi. The differential susceptibility observed in the first two assays was probably related to variations in the rearing system and nutritional and antimicrobial components in the diet and not due to variations among host populations.

L'importance des degats occasionnes en zone tropicale par le maize streak virus (msv) transmis par cicadulina mbila (naude) et par le maize stripe virus (mstpv) ou le maize mosaic virus (mmv) transmis tous deux par peregrinus maidis (ashmead) a motive la creation de varietes resistantes. A cette fin, des recherches sur la biologie des vecteurs, les modes de transmission et le determinisme genetique de la resistance ont ete entreprises. C. Mbila est plus sensible aux temperatures basses et p. Maidis aux temperatures elevees. Ils sont oligophages. Le mil et le mais doux sont leurs plantes preferentielles respectives. A la reunion, les niveaux de populations augmentent fortement durant la saison chaude. C. Mbila peut accumuler rapidement le msv et le transmettre meme a de tres faibles concentrations. La multiplication du mstpv, mise en evidence par la technique elisa, est possible des l'incubation des ufs. Une barriere a la penetration de la paroi intestinale est mise en evidence pour les trois virus; une seconde barriere limite l'acces aux sites d'inoculation pour le mmv et le mstpv. Enfin, une troisieme barriere affecte la transmission ovarienne de mstpv, des determinismes simples chez l'insecte controlent chacune d'elles. Un gene recessif et deux genes dominants controlent la transmission du mstpv. Le composite irat 297 a exprime une excellente resistance aux trois viroses. Le niveau des resistances partielles polygeniques est tres eleve pour la striure; pour le stripe, la resistance complete, est predominante. L'identification de trois serotypes et de deux groupes de virulences montre la variabilite du msv

Peregrinus maidis (ashmead, 1890) (homoptera: delphacidae), est vecteur sur mais et sorgho de deux virus circulants, le maize mosaic virus (mmv) et la maize stripe virus (mstpv). L'utilisation de la technique d'electropenetrographie (epg) sur le modele peregrinus maidis/mais a permis (i) de decrire le comportement alimentaire de cet insecte sur genotype sensible, et trois classes de signaux (1, 2 et 3) ont ete identifiees et correlees respectivement aux activites de sondage, a l'ingestion dans le xyleme et a l'ingestion dans le phloeme, (ii) d'etudier l'activite alimentaire sur genotypes sensibles et resistants. Sur trois lignees issues du composite virose resistant cvr#3 - c#3 (epi 23 / 3. 7. 2. 1. Et 3. 7. 3. 1. , et a211), p. Maidis effectue une duree totale de sondage, un nombre de piqures individuelles et un temps d'acces au phloeme plus importants que sur genotypes et lignees sensibles dont b73 ; il ne presente pas d'ingestion soutenue dans le phloeme durant les huit premieres heures de contact sur ces lignees resistantes. Sur hi40, lignee resistante au virus s. S. , l'insecte presente un comportement alimentaire identique a celui observe sur genotypes sensibles, tandis que sur l'hybride f#1, resultant du croisement d'a211 par b73, il s'alimente de la meme facon que sur genotypes resistants a la transmission. Ce mecanisme de resistance est donc heritable et a caractere dominant. Cette resistance a la transmission du mmv est contournable par l'augmentation du niveau d'infestation. L'electropenetrogaphie est une excellente methode pour cribler des genotypes presentant ce type de resistance

A thesis presented to The Faculty of Science, University of the Witwatersrand, Johannesburg in fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular and Cell Biology 2015<br>Cassava brown streak disease (CBSD) ranks among the top seven biological threats to global food security and is considered to be a major risk to food security in tropical Africa. In Uganda, overall CBSD incidence has increased by c. 20% since 2004, and persistently reduces cassava yields and storage root quality. Presently the disease negatively impacts the livelihoods of over 80% of the farming families who rely on cassava as a staple food and source of income. Two distinct ipomoviruses, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) cause CBSD. The viruses systemically infect primary host plants and accumulate, and cause severe disease symptoms as the plant matures, reducing yields through the induction of necrotic lesions in the storage roots and suppressing utility of cassava stems for subsequent vegetative propagation. Effective control strategies require screening of available germplasm for sources of natural resistance in combination with improved understanding of host-virus interaction to facilitate targeted breeding. Due to a lack of known sources of resistance to CBSD in the cassava germplasm, incorporating new virus resistance into existing cassava genotypes through transgenic RNA interference (RNAi) approaches offers an additional, relevant avenue to reduce the increasing impact of CBSD. The research presented in this thesis provides insights into the complex mechanisms of virus-host interactions linking genotype to phenotype in CBSV- and UCBSV-cassava pathosystems and provides proof of principle for CBSD control by RNAi-mediated technology. Both are contributions to progress towards potential control of the CBSD epidemic in East Africa. To correlate CBSD symptoms with virus titer, within-host CBSV and UCBSV accumulation was studied in leaf, stem and storage root samples collected from 10 genotypes of field-grown cassava with varied levels of resistance to CBSD. CBSV was found to be present in 100% of CBSD samples collected from symptomatic plants. Presence of both CBSV and UCBSV was seen in 45.3% of the samples. Quantitative PCR (RT-qPCR) analysis showed that tolerant genotypes were infected with CBSV alone and accumulated lower virus titer compared to susceptible genotypes, which were co-infected with CBSV and UCBSV. To further comprehend the molecular interaction between CBSD viruses and cassava, deep sequencing was performed to compare profiles of virus-derived small RNAs (vsRNAs) in CBSV- and UCBSV-infected cassava genotypes of NASE 3 (CBSD tolerant), TME 204 and 60444 (CBSD susceptible). The results showed an abundance of 21-24 nt sized vsRNAs which when mapped were shown to cover the entire CBSV and UCBSV genomes. The 21- and 22-nt sizes were predominant compared to the 23- and 24-nt size classes. CBSV-infected plants accumulated higher populations of vsRNAs across the genotypes compared to UCBSV-infected plants, which accumulated moderate amounts of UCBSV-derived sRNAs in TME 204 and 60444, and insignificant amounts in UCBSV-challenged NASE 3, respectively. Quantitative RT-PCR analysis was performed to determine transcript levels of cassava homologues of Dicer (DCL) proteins, particularly DCL4 and DCL2, which are involved in the biogenesis of 21- and 22-nt small RNAs, and to correlate to the abundance of 21- and 22-nt vsRNAs in CBSV- and UCBSV-infected cassava. Similarly, RT-qPCR was performed to determine the expression of Argonaute (AGO) proteins, specifically AGO2 which preferentially sort and bind sRNAs with 5’ adenine (A) or uracil (U) to effector complexes to target mRNAs repression or cleavage, since in this study a major proportion of the vsRNAs were found to have A or U at the first 5’-end. Expression levels of cassava homologues of AGO2, DCL2 and DCL4, which are core components of the gene-silencing pathway, were found to be affected in virus-infected plants across all three genotypes. The levels of viral RNA and vsRNAs correlated with disease phenotype in infected plants. CBSV-infected plants showed more severe CBSD symptoms compared with UCBSV-infected plants of the same genetic background. These results showed that CBSV is more aggressive compared to UCBSV and supports the hypothesis of occurrence of genotype-specific resistance to CBSD viruses. The abundance of 21- and 22-nt vsRNAs in CBSV- and UCBSV-infected plants signifies the viruses activated the RNA-silencing mechanism, referred to as transcriptional or post-transcriptional gene silencing (TGS or PTGS). To test efficacy of RNAi-mediated resistance to control CBSD under field conditions, 14 lines of cassava plants transgenically modified to express, as inverted repeats, two RNAi constructs p718 and p719 targeting near full-length (894 bp) and N-terminal (402 bp) portions of UCBSV coat protein sequence were tested under confined field trial conditions at Namulonge, Uganda. Transgenic plants expressing p718 showed a 3-month delay in CBSD symptom development, while 100% of non-transgenic plants (n = 60) developed CBSD shoot symptoms. Over the 11-month trial duration, 98% of clonal replicates within line 718-001 were found to remain free of CBSD symptoms. RT-PCR analysis detected UCBSV within leaves of 57% of non-transgenic plants compared to only 0.5% across the 14 transgenic lines. Presence of the non-homologous CBSV was detected in all transgenic plants that developed CBSD symptoms. However, 93% of plants of line 718-001 were free of CBSV and UCBSV. At harvest, 90% of storage roots of non-transgenic plants showed severe necrosis, whereas plants of lines 718-001 and 718-005 showed significant suppression of CBSD. Line 718-001 had 95% of roots free from necrosis and was RT-PCR negative for presence of both viral pathogens. To determine durability of RNAi-mediated resistance to CBSD, stem cuttings were obtained from mature plants of lines p718-001, p718-002 and p718-005, replanted and monitored for 11 more months. CBSV but not UCBSV was detected in tissues of plants of lines p718-002 and p718-005, whereas all leaves and roots of p718-001 plants were free of CBSV and UCBSV. Thus, RNAi constructs conferred durable CBSD resistance across the vegetative cropping cycle, providing proof of concept for application of RNAi technology to control CBSD in farmers’ fields. The findings presented in this thesis contribute to understanding the complex interconnected mechanisms involved in CBSV- and UCBSV-host interactions and will contribute to the long-term goals of devising new methods of CBSD control.

The objective of this study was to characterize an unknown agent associated with decline in black raspberry (Rubus occidentalis) in Oregon. A virus was found consistently associated with decline symptoms of black raspberries and was named Black raspberry decline associated virus (BRDaV). Double stranded RNA extraction from BRDaV-infected black raspberry revealed the presence of two bands of approximately 8.5 and 7 kilobase pairs, which were cloned and sequenced. The complete nucleotide sequences of RNA 1 and RNA 2 are 7581 nt and 6364 nt, respectively, excluding the 3' poly(A) tails. The genome structure was identical to that of Strawberry mottle virus (SMoV), with the putative polyproteins being less than 50% identical to that of SMoV and other related sequenced viruses. The final 189 amino acids of the RNA-dependent- RNA-polymerase (RdRp) reveal an unusual indel with homology to AlkB-like protein domains, suggesting a role in repair of alkylation damage. This is the first report of a virus outside the Flexiviridae and ampeloviruses of the Closteroviridae to contain these domains. An RT-PCR test was designed for the detection of BRDaV from Rubus tissue. BRDaV is vectored non-persistently by the large raspberry aphid Amphorophora agathonica, the green peach aphid Myzus persicae, and likely nonspecifically by other aphid species. Phylogenetic analysis of conserved motifs of the RdRp, helicase, and protease regions indicate that BRDaV belongs to the Sadwavirus genus. To assess the rate of spread BRDaV, four newly planted fields of black raspberries (Rubus occidentalis) in Oregon were studied for three years. In an effort to characterize the suspected complexity of synergistic interactions between BRDaV and other Rubus-infecting viruses, the prevalence of ten additional Rubus viruses was also monitored in the study fields. The timing of BRDaV infection as it relates to aphid populations and flights was also determined. Testing of nearby vegetation identified several symptomless Rubus hosts of BRDaV, as well as detection in multiple cultivars of black raspberry and several non-Rubus weeds. It was determined that BRDaV spreads rapidly with a low aphid threshold and consistently is associated with decline of black raspberries in Oregon.<br>Graduation date: 2006

A dissertation submitted in partial compliance with the requirements for the Masters Degree in Technology: Homoeopathy, Technikon Natal, 1997.<br>Most economically important crop plants may become infected with viruses. Several of these virus diseases are limiting factors in agricultural production and have contributed to serious economic and social hardship in many countries, especially in tropical and subtropical regions. Homoeopathic microdoses have been investigated for their role in the control of virus diseases, with good results. However, few of the studies contain statistical analyses. The object of this study was to assess the effect of a homoeopathic preparation of a leaf infected with tobacco mosaic virus (TM Viricum) in the contol of tobacco mosaic virus (TMV). The potencies used were 6CH, 12CH, 30CH and 200CH. iv Trays of 24 tomato seedlings per tray were the subjects of this study. Tomato plants were systemically infected with TMV. Four trays were used per treatment. There was an uninoculated and an inoculated control group. The rest of the test population was divided into two groups. The<br>M

Maize (Zea mays L.) streak virus disease (MSD) is the most important virus disease in Africa but farmers are unaware of its status. A project was initiated to assess the current status of MSD and to breed for its resistance. Four populations comprised of two BC1F1 and two F2 progenies developed by backcrossing and selfing the F1 progenies of two crosses between a donor line (CMl 202) and two susceptible lines (CMl 321 and CMl 384) were developed. Conventional and molecular marker assisted selection (MAS) methods were used to screen for resistance to MSD in each of the four populations. To facilitate unbiased comparison, separate screening nurseries were established for MAS and conventional screening. The objectives of the study were five-fold; 1) to assess the status of MSD in Uganda and understand farmers' preferences and varietal selection criteria for maize using a participatory rural appraisal (PRA), 2) to screen for MSD resistance in early generations of segregating maize populations using conventional method, 3) to screen for resistance to MSD using SSR marker assisted selection , 4) to compare the effectiveness of marker assisted selection and conventional methods for selection for resistance to MSD, and 5) to compare costs associated with MAS and conventional selection methods. Results of PRA showed that unreliable rainfall and insect pests were the dominant constraints to maize productivity in Uganda. Diseases were ranked fifth among the production constraints . Maize streak virus disease was considered the most important disease constraint. Farmers showed common preference for high yielding and early maturing cultivars. However, farmers had other special preferences which were diverse and included large, white and high test density kernels for marketing, and sweet taste, particularly for home consumption. Farmers' research priorities included tolerance to drought, resistance to insect pests and diseases, sweetness, prolificacy, resistance to lodging, and drooping leaves because theyt cover the soil fast and prevent weed growth. Conventional screening for resistance to MSD showed that backcross and selfing populations segregated in 1:1 and 3:1 Mendelian ratios confirming the presence of one major gene with simple inheritance . Severity and incidence of disease were positively correlated suggesting a non-reference by the insects. In the selfing populations, the presence of complete esistance against MSD was suggested because frequency distribution patterns were highly skewed in favour of resistance. There was a decrease in disease severities with selection from BC1F1 to BC2F1 and from F2 to F3 generations indicating that high response to selection was achieved. On the other hand, one marker, umc1917, consistently polymorphic and eo-dominant was selected and used in MAS protocol. Results showed that the observed outcomes fitted the expected ratio of 1:2:1 for a F2 population and 1:1 for a BC1F1 population (X2 not significant). Evaluation of F3 and BC2F1 progeny selected using markers showed low disease severity suggesting that marker assisted selection was effective. However, the study showed that the presence of the O'Tl, was not consistent with symptom expression in the field. Evaluation of lines in three-way crosses identified ten potential lines that were high yielding, highly resistant to MSD and stable across three locations. Both MAS and conventional selection were equally effective in identifying high yielding lines although resistance was higher under MAS. Costs of MAS and conventional method varied depending on the units for comparison. The total costs of conventional method were higher than that of MAS in both first and second selection cycles. Comparing costs per row for conventional and costs per plant or data point for MAS showed that conventional selection was 2.4 times more expensive than costs per sample for MAS. However, costs per plant for MAS were 6.6 times higher than for conventional selection.<br>Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2006