Made available in DSpace on 2015-03-26T13:37:48Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1056837 bytes, checksum: 147af3aea5c0fd4d6961921a210a152c (MD5) Previous issue date: 2012-02-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cowpea mild mottle virus (CpMMV), the causal agent of stem necrosis disease, has drawn attention of soybean producers in recent years due to productivity losses in the main producing regions of Brazil. The disease was first recorded in the Midwest region of Brazil in the 2000/01 soybean season and rapidly spread throughout the country in the following years. CpMMV is usually hard to diagnose due to its wide range of symptoms while the occurrence of asymptomatic soybean cultivars complicates genotypic selection in breeding programs. Serological methods for viral detection require the use of an antiserum of good quality to achieve specificity and sensitivity. The entire coat protein sequence of a Brazilian CpMMV isolate was cloned into a bacterial expression vector and transformed into Escherichia coli BL21::DE3 for in vitro expression. The coat protein, fused to a 6 His-tag, was purified under denaturing conditions by affinity chromatography using a Ni-NTA resin. After renaturation, the integrity and identity of purified recombinant protein was confirmed by SDS-Page and MALDI-ToF/Tof mass spectrometer analyses. New Zealand rabbits were immunized with increasing amounts of the recombinant protein. The specificity and sensitivity of the antisera was shown by Western blot and indirect ELISA assays. The polyclonal antisera raised against recombinant coat protein proved to be a reliable tool for CpMMV detection. / O Cowpea mild mottle virus (CpMMV), agente causal da necrose da haste, tem chamado a atenção dos produtores de soja nos últimos anos devido às perdas de produtividade geradas nas principais regiões produtoras do Brasil. A doença foi relatada pela primeira vez na região Centro-Oeste do Brasil, na safra de 2000/01, e rapidamente se espalhou por todo o país nos anos seguintes. Além disso, tem sido relatada a ocorrência de cultivares de soja assintomáticas, o que dificulta a seleção de genótipos para programas de melhoramento. Os métodos sorológicos para a detecção viral requerem o uso de um antissoro de boa qualidade para alcançar especificidade e sensibilidade. A sequência completa da capa proteica de um isolado brasileiro de CpMMV (Bahia) foi clonado em um vetor de expressão bacteriano e transformado em Escherichia coli BL21: DE3 para expressão in vitro. A proteína capsidial ligada a uma cauda de histidina foi purificada sob condições desnaturantes através de cromatografia de afinidade, utilizando uma resina Ni-NTA. Depois da renaturação, a integridade e a identidade da proteína recombinante purificada foi confirmada por SDS-PAGE e análise de espectrometria de massa (MALDI-ToF/ToF). Dois coelhos da raça Nova Zelândia foram imunizados com quantidades crescentes da proteína recombinante. A especificidade e sensibilidade do antissoro foram avaliadas por Western blot e ELISA indireto. O antissoro policlonal a partir da proteína recombinante da capa proteica provou ser uma ferramenta confiável e eficiente para a detecção CpMMV.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-03-24T12:07:34Z No. of bitstreams: 2 Dissertação - Tássia Tuane Moreira dos Santos - 2017.pdf: 1707301 bytes, checksum: 711b52250570075b4b92d21dd0f3edc0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-03-24T12:08:10Z (GMT) No. of bitstreams: 2 Dissertação - Tássia Tuane Moreira dos Santos - 2017.pdf: 1707301 bytes, checksum: 711b52250570075b4b92d21dd0f3edc0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-24T12:08:10Z (GMT). No. of bitstreams: 2 Dissertação - Tássia Tuane Moreira dos Santos - 2017.pdf: 1707301 bytes, checksum: 711b52250570075b4b92d21dd0f3edc0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-21 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The main damage of whitefly Bemisia tabaci biotype B on beans is virus transmission such as Bean golden mosaic virus (BGMV) and Cowpea mild mottle virus (CpMMV). Different insecticides mechanisms of action, use of selective molecules and alternative methods of control are important techniques for management of this insect. Therefore, this study was conducted to test the efficiency of chemical insecticides on mortality of whiteflies adults and CpMMV transmission on BGMV-resistant transgenic common bean; and to verify the combination of chemical insecticides in sublethal doses with the entomopathogeni fungus Isaria javanica on control of B. tabaci nymhps in commom bean. In first part of the study two experiments were conducted with seed treatments, one with foliar application and another with the combination with seed treatment and foliar application. Experiments were conducted in screenhouse. In first experiment with seeds treatment thiametoxam FS at 300, 500, 700 mL and imidacloprid+thiodicarb FS at 500, 600 e 800 mL were tested. In second experiment were used higher doses of thiametoxam FS (500, 700 e 900 mL) and imidacloprid+thiodicarb FS (600, 800 e 1.000 mL). In foliar application were evaluated the insecticides thiamethoxam+cyantraniliprole WG and flupyradifurone SL at 500 e 750 mL ha-1, thiamethoxam + lambda-cyhalothrin SC and thiamethoxam WG both at 300 mL ha-1. In combination of seed treatment and foliar application the treatments were plants treated only with seed treatment or with ST following 1, 2, 3 or 4 foliar applications with different insecticides at five-days intervals. On second stage, at laboratory the compatibility of spiromesifen and buprofezin with I. javanica was observed. Different insecticides concentrations (12.5; 25; 50 and 100 ppm) were added to conidia suspension. Germination of conidia, mycelial growth and conidial production were evaluated. On screenhouse were determined: 1) the sublethal (CL25) doses of insecticides spiromesifen and buprofezin to 2nd ínstar whitefly nymphs; 2) the bio-efficacy of different isolates of I. javanica to 2nd ínstar whitefly nymphs and 3) the effect of the combination between spiromesifen and buprofezin with I. javanica on control of 2nd ínstar whitefly nymphs. Nymphal mortality and sporulation on cadavers (2 and 3) were evaluated. As result was observed that thiametoxam + ciantraniliprole WG and flupiradifurone SL applied in foliar application resulted in higher mortality of whitefly adults. ST added 3 or 4 foliar application proportioned the highest mortality and reduction on CpMMV transmission. Insecticides are efficient on B. tabaci control; however, it does not avoid the virus transmission. The combinations of ST added 3 or 4 foliar application reduce the CpMMV on 50%. All I. javanica isolates caused nymphal mortalities. Spiromesifen and buprofezin were compatible with I. javanica. Additive and synergistic effects were observed. A management program of whitefly is important to reduction of CpMMV incidence on BGMV-resistant transgenic common bean. Besides, the combination of selective chemical insecticides and I. javanica on B. tabaci might increase the success of control. / O principal dano da mosca-branca Bemisia tabaci biótipo B na cultura do feijoeiro é a transmissão de vírus, como o Bean golden mosaic virus (BGMV) e o Cowpea mild mottle virus (CpMMV). A rotação de mecanismos de ação dos inseticidas, o uso de moléculas seletivas e de métodos de controle alternativos são técnicas importantes para o manejo deste inseto. Os objetivos do estudo foram testar a eficiência de inseticidas químicos na mortalidade de adultos de mosca-branca e na transmissão de CpMMV no feijoeiro geneticamente modificado resistente ao mosaico dourado (RMD) e a associação de inseticidas químicos em doses subletais com o fungo Isaria javanica no controle de ninfas de B. tabaci em feijoeiro-comum. Na primeira etapa do estudo em casa telada, dois experimentos foram realizados com tratamento de sementes, um com aplicação foliar e outro com a combinação de tratamento de sementes e aplicação foliar. No primeiro experimento com tratamento de sementes (TS) os inseticidas thiametoxam FS nas doses 300, 500, 700 mL e imidacloprid+thiodicarb FS nas doses 500, 600 e 800 mL foram testados. Já no segundo TS foram utilizados doses maiores de thiametoxam FS (500, 700 e 900 mL) e de imidacloprid+thiodicarb FS (600, 800 e 1000 mL). Em pulverização foram avaliados os inseticidas thiamethoxam+cyantraniliprole WG (500 e 750 mL ha-1), flupyradifurone SL (500 e 750 mL ha-1), thiamethoxam+lambda-cyhalothrin SC (300 mL ha-1) e thiamethoxam WG (300 mL ha-1). Na combinação de tratamento de sementes e aplicação foliar, as plantas foram tratadas apenas com TS ou com TS seguido de 1, 2, 3 ou 4 pulverizações com diferentes inseticidas a cada 5 dias. Na segunda etapa para verificar a compatibilidade dos inseticidas spiromesifen e buprofezin com o I. javanica, concentrações destes inseticidas (12,5; 25; 50 e 100 ppm) foram adicionadas à suspensão de conídios. Foram avaliados a germinação de conídios, crescimento micelial e produção de conídios. Em casa telada foram determinados: 1) a dose subletal (CL25) dos inseticidas spiromesifen e buprofezin a ninfas de mosca-branca; 2) a bio-eficácia de isolados de I. javanica à ninfas de mosca-branca; e 3) o efeito da combinação de spiromesifen e buprofezin e I. javanica no controle de ninfas de mosca-branca. A mortalidade das ninfas e a esporulação nos cadáveres (2 e 3) foram avaliadas. No primeiro estudo o tiametoxam+ciantraniliprole WG e flupiradifurona SL em pulverização foliar resultaram em maiores mortalidades de mosca-branca. Os inseticidas são eficientes no controle de mosca-branca. Apenas a combinação de TS mais 3 ou 4 pulverizações foliares reduzem a incidência de CpMMV. No segundo estudo, todos os isolados causaram morte de ninfas. Os inseticidas foram compatíveis com I. javanica. Efeitos aditivo e sinérgico foram observados entre I. javanica e spiromesifen e buprofezin no controle de ninfas de mosca-branca. Um programa de manejo de B. tabaci é importante para a redução de CpMMV no feijoeiro RMD. Além disso, a combinação de inseticidas sintéticos seletivos e o fungo I. javanica pode aumentar o sucesso do controle de ninfas de mosca-branca.

Viral diseases are a major limiting factor to cowpea production in many countries of Africa. In Uganda, studies indicated that the cowpea aphid-borne mosaic virus (CABMV) is common and a potential threat to cowpea production in the region. There have been no efforts to develop cowpea cultivars with resistance to CABMV in Uganda. This work focused on the development of cultivars resistant to CABMV. Production of cowpea in Uganda is constrained by several factors, including a lack of awareness of diseases among the majority of farmers. A participatory rural appraisal (PRA) was conducted to elicit farmers’ indigenous knowledge of cowpea production and also to gain insight into their understanding of viral diseases affecting cowpea in Uganda. PRA tools such as group discussions, transect walks, problem listing and ranking were used to gather information. Insect pests, diseases, low yielding cultivars and the high cost of pesticides were perceived to be the most important production constraints. Farmers were not aware of the problem of virus diseases, but provided descriptive names of symptoms. Only three cowpea cultivars (Ebelat, Ecirikukwai and Blackcowpea) were produced in the area. Seed size and colour were seen as important traits in new varieties. Information about the occurrence, distribution and identity of cowpea viruses is limited in Uganda. The objective of this study was to identify the important cowpea virus diseases occurring naturally in the major cowpea growing regions of Uganda. Surveys were conducted to determine the incidence and severity of virus symptoms in four districts (Soroti, Kumi, Pallisa and Tororo) in 2004 and 2005. The incidence ranged from 40.5 to 94.4% and severity ranged from 15.0 to 30.6% (for Kumi and Pallisa districts, respectively) during the 2004 surveys. In 2005, the incidence ranged from 55.9 to 85.4% and severity ranged from 4.7 to 14.5% (for Tororo and Soroti districts, respectively). The CABMV, cowpea mild mottle virus (CPMMV), cowpea severe mosaic virus (CPSMV) and cucumber mosaic virus (CMV) were serologically detected by double antibody sandwich enzyme- linked immunosorbent assay (DAS-ELISA). Fifty four improved cowpea genotypes were screened for resistance to CABMV during the first season of 2004 at Serere Agricultural and Animal Production Research Institute in Uganda. Further screening was conducted in the second season of 2004 using 27 genotypes. The genotypes were planted in single rows between the rows of the susceptible cultivar, Ebelat. This was to provide high pressure of aphid vector (Aphis craccivora Koch) and CABMV inoculum. In addition, the test genotypes were artificially inoculated with a CABMV extract on fully expanded primary leaves of fourteen day-old seedlings. The CABMV incidence and severity was assessed. Disease severity was assessed on a 0-60% visual estimation scale where 0 = with no symptoms and 60 = with severe symptoms. Serological analysis was conducted using DAS-ELISA. Five genotypes showed good levels of resistance to CABMV, namely MU-93, IT82D-889, IT82D-516-2, IT85F-2841 and SECOW-2W. These resistant lines were crossed with three susceptible local landraces, namely Ebelat, Ecirikukwai and Blackcowpea in a North Carolina II mating design. The F1, F2 and BC1F1 populations and the parents were evaluated in the field to assess the response to CABMV and to study the inheritance of resistance to CABMV. The general combining ability (GCA) and specific combining ability (SCA) effects were significant, indicating that both additive and non-additive genetic factors are important in determining the control of CABMV in cowpea. The proportions (%) of the sum of squares for crosses attributable to GCA and SCA for CABMV severity were 51.4% for GCA due to females, 8.4% for GCA due to males and 40.2% for the SCA. The narrow-sense heritability estimates, obtained by regressing F1 on mid-parents was 0.87 and 0.84, F2 on F1 progenies 0.49 and 0.48, and F2 progenies on mid-parents 0.63 and 0.79, for AUDPC and final disease severity, respectively. Single gene conditioned resistance in seven populations, but resistance was quantitatively inherited and involved many genes in eight populations. Observation of transgressive segregation and moderate to high heritability suggests a quantitative mode of gene action and the importance of additive effects. The predominance of GCA variance, high heritability estimates and observation of transgressive segregation suggested that resistance could be improved by selection. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.

MAGALHÃES, Vladimir Gonçalves. Prospecção bioquímica e molecular de fatores possivelmente envolvidos na defesa de feijão-de-corda [Vigna unguiculata (L.) Walp] ao vírus do mosaico severo do caupi (CPSMV). 2011. 109 f. : Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza-CE, 2011. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-15T14:36:03Z No. of bitstreams: 1 2011_dis_vgmagalhaes.pdf: 4392892 bytes, checksum: 571bbf928e7a06498a9e935694d2ab1d (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:26:15Z (GMT) No. of bitstreams: 1 2011_dis_vgmagalhaes.pdf: 4392892 bytes, checksum: 571bbf928e7a06498a9e935694d2ab1d (MD5) / Made available in DSpace on 2016-08-02T20:26:15Z (GMT). No. of bitstreams: 1 2011_dis_vgmagalhaes.pdf: 4392892 bytes, checksum: 571bbf928e7a06498a9e935694d2ab1d (MD5) Previous issue date: 2011 / Cowpea [(Vigna unguiculata (L.) Walp.)] has a major socioeconomic importance in Northeastern Brazil. However, its production is low due abiotic and biotic factors. Amongst the biotic factors the cowpea severe mosaic virus (CPSMV, Secoviridae family) has a great importance because it causes the most prevalent and serious virus disease that affects this crop in the country. Although there are resistant cultivars to CPSMV, the defense mechanisms involved is not understood. For this reason, a comparative study was conducted between resistant and susceptible cultivars, using two experimental approaches. In the first one, the biochemical approach, possible differences of enzyme activities related to oxidative stress (superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase) and pathogenesis (β-1,3-glucanase and chitinase), in addition of phenyl amonium lyase, and H2O2 generation in the leaves of the cultivars Pitíuba (susceptible) and Macaibo (resistant) were analyzed. The secondary leaves were harvested at 6, 12, 24, 48, 72 h after treatment with CPSMV or carborundum (controls) and these above parameters were measures in the protein extracts obtained. It was shown that, in general, the response amongst the cultivars did not differ significantly, suggesting that the defense mechanisms of cowpea are different from the classic response of defense observed for several plant species. In the second approach, molecular, the nucleotide sequences of the genes that code for the translation initiation factors (eIF4E, eIF(iso)4E, eIF4G, eIF(iso)4G and nCBP) and the primary strucuture of the correspondent putative proteins were analyzed in order to search patterns of polymorphis between the studied cowpea cultivars that could be related to a constitutive defense conferred by recessive genes. After sequence analysis, it was found that eIF4E showed polymorphisms between cultivars, and, in at least two positions (68 and 108), there were differences between susceptible and resistant cultivars (Arg68/Pro68; Val108 or Pro108/Ala108). The molecular modeling revealed that differences in amino acid are located in two external loops close to the cap (m7G) binding domain, well reported in cases of recessive resistance within the Potyviridae family. Through immunodetection studies with the leaf extracts and the protein fractions obtained after the affinity chromatography on a Sepharose-7-metil-guanosina column, it was found that the amino acid mutations found did not impair the ability of eIF4E to bind to M7G in vitro. However, as it was observed two variants for eIF4E comparing the resistant and susceptible cultivars to CPSMV, at spatially neighboring regions, it could not be ruled out the hypothesis that this constitutive/recessive resistant trait is correlated with these mutations detected, which could impair, consequently, the in vivo interaction of eIF4E with the viral VPg. / O feijão-de-corda [Vigna unguiculata (L.) Walp.] tem grande importância socioeconômica no Nordeste brasileiro. Entretanto, sua produção é baixa devida a diversos fatores abióticos e bióticos. Dentre os fatôres bióticos, o vírus do mosaico severo do caupi (CPSMV, família Secoviridae) apresenta grande destaque, por causar a virose que mais acomete essa cultura no país. Embora existam cultivares resistentes ao vírus, não se sabe quais os mecanismos de defesa envolvidos. Por essa razão, foi elaborado um estudo comparativo entre cultivares resistentes e susceptíveis, utilizando duas abordagens experimentais. Na abordagem bioquímica, possíveis diferenças de atividades de enzimas relacionadas ao estresse oxidativo (dismutase do superóxido, peroxidase do ascorbato, peroxidase) e à patogênese (β-1,3-glucanase e quitinase), além da fenilamônia liase, e teores de H2O2 foram estudadas nos cultivares Pitiúba (susceptível) e Macaibo (resistente). Após tratamento com o CPSMV ou com apenas carborundum (plantas controles), foram realizadas coletas nos tempos de 6, 12, 24, 48 e 72 h, tendo sido realizadas as análises bioquímicas nos extratos protéicos obtidos das folhas secundárias. Foi verificado que, de maneira geral, a resposta entre os cultivares não diferiram significamente, sugerindo que os mecanismos de defesa de feijão-de-corda sejam diferentes da resposta clássica de defesa. Na segunda abordagem, molecular, as sequências nucleotídicas dos genes codificantes para os fatores de iniciação de tradução (eIF4E, eIF(iso)4E, eIF4G, eIF(iso)4G e nCBP) e as sequências primárias putativas das proteínas correspondentes foram analisados, no intuito de se averiguar a existência de padrões de polimorfismos entre cultivares resistentes e susceptíveis, que pudessem estar relacionados à defesa constitutiva conferida por genes recessivos. Após análise das sequências, foi observado que eIF4E apresentava polimorfismos entre os cultivares, sendo que, em pelo menos duas posições nas sequências primárias putativas do fator (68 e 108), existiram diferenças entre cultivares susceptíveis e resistentes (Arg68/Pro68; Val108 ou Pro108/Ala108). A modelagem molecular revelou que as diferenças em aminoácidos situam-se em dois loops externos, próximos ao domínio de ligação ao capacete (m7G), bastante relatados em casos de resistência recessiva para a família Potyviridae. Através de estudos de imunodectecção posterior ao passo cromatográfico em coluna de afinidade, foi observado que as mudanças de aminoácidos não comprometiam, a capacidade de eIF4E em se ligar ao m7G in vitro. Entretanto, como foram observadas duas variantes para eIF4E, entre cultivares resistentes e susceptíveis ao CPSMV, em regiões próximas espacialmente, não se pode descartar a hipótese de que a resistência recessiva constitutiva esteja associada com essas mutações detectadas nessas sequências, que iriam modificar, consequentemente, a interação da VPg viral com eIF4E in vivo.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O feijÃo-de-corda [Vigna unguiculata (L.) Walp.] tem grande importÃncia socioeconÃmica no Nordeste brasileiro. Entretanto, sua produÃÃo à baixa devida a diversos fatores abiÃticos e biÃticos. Dentre os fatÃres biÃticos, o vÃrus do mosaico severo do caupi (CPSMV, famÃlia Secoviridae) apresenta grande destaque, por causar a virose que mais acomete essa cultura no paÃs. Embora existam cultivares resistentes ao vÃrus, nÃo se sabe quais os mecanismos de defesa envolvidos. Por essa razÃo, foi elaborado um estudo comparativo entre cultivares resistentes e susceptÃveis, utilizando duas abordagens experimentais. Na abordagem bioquÃmica, possÃveis diferenÃas de atividades de enzimas relacionadas ao estresse oxidativo (dismutase do superÃxido, peroxidase do ascorbato, peroxidase) e à patogÃnese (β-1,3-glucanase e quitinase), alÃm da fenilamÃnia liase, e teores de H2O2 foram estudadas nos cultivares PitiÃba (susceptÃvel) e Macaibo (resistente). ApÃs tratamento com o CPSMV ou com apenas carborundum (plantas controles), foram realizadas coletas nos tempos de 6, 12, 24, 48 e 72 h, tendo sido realizadas as anÃlises bioquÃmicas nos extratos protÃicos obtidos das folhas secundÃrias. Foi verificado que, de maneira geral, a resposta entre os cultivares nÃo diferiram significamente, sugerindo que os mecanismos de defesa de feijÃo-de-corda sejam diferentes da resposta clÃssica de defesa. Na segunda abordagem, molecular, as sequÃncias nucleotÃdicas dos genes codificantes para os fatores de iniciaÃÃo de traduÃÃo (eIF4E, eIF(iso)4E, eIF4G, eIF(iso)4G e nCBP) e as sequÃncias primÃrias putativas das proteÃnas correspondentes foram analisados, no intuito de se averiguar a existÃncia de padrÃes de polimorfismos entre cultivares resistentes e susceptÃveis, que pudessem estar relacionados à defesa constitutiva conferida por genes recessivos. ApÃs anÃlise das sequÃncias, foi observado que eIF4E apresentava polimorfismos entre os cultivares, sendo que, em pelo menos duas posiÃÃes nas sequÃncias primÃrias putativas do fator (68 e 108), existiram diferenÃas entre cultivares susceptÃveis e resistentes (Arg68/Pro68; Val108 ou Pro108/Ala108). A modelagem molecular revelou que as diferenÃas em aminoÃcidos situam-se em dois loops externos, prÃximos ao domÃnio de ligaÃÃo ao capacete (m7G), bastante relatados em casos de resistÃncia recessiva para a famÃlia Potyviridae. AtravÃs de estudos de imunodectecÃÃo posterior ao passo cromatogrÃfico em coluna de afinidade, foi observado que as mudanÃas de aminoÃcidos nÃo comprometiam, a capacidade de eIF4E em se ligar ao m7G in vitro. Entretanto, como foram observadas duas variantes para eIF4E, entre cultivares resistentes e susceptÃveis ao CPSMV, em regiÃes prÃximas espacialmente, nÃo se pode descartar a hipÃtese de que a resistÃncia recessiva constitutiva esteja associada com essas mutaÃÃes detectadas nessas sequÃncias, que iriam modificar, consequentemente, a interaÃÃo da VPg viral com eIF4E in vivo. / Cowpea [(Vigna unguiculata (L.) Walp.)] has a major socioeconomic importance in Northeastern Brazil. However, its production is low due abiotic and biotic factors. Amongst the biotic factors the cowpea severe mosaic virus (CPSMV, Secoviridae family) has a great importance because it causes the most prevalent and serious virus disease that affects this crop in the country. Although there are resistant cultivars to CPSMV, the defense mechanisms involved is not understood. For this reason, a comparative study was conducted between resistant and susceptible cultivars, using two experimental approaches. In the first one, the biochemical approach, possible differences of enzyme activities related to oxidative stress (superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase) and pathogenesis (β-1,3-glucanase and chitinase), in addition of phenyl amonium lyase, and H2O2 generation in the leaves of the cultivars PitÃuba (susceptible) and Macaibo (resistant) were analyzed. The secondary leaves were harvested at 6, 12, 24, 48, 72 h after treatment with CPSMV or carborundum (controls) and these above parameters were measures in the protein extracts obtained. It was shown that, in general, the response amongst the cultivars did not differ significantly, suggesting that the defense mechanisms of cowpea are different from the classic response of defense observed for several plant species. In the second approach, molecular, the nucleotide sequences of the genes that code for the translation initiation factors (eIF4E, eIF(iso)4E, eIF4G, eIF(iso)4G and nCBP) and the primary strucuture of the correspondent putative proteins were analyzed in order to search patterns of polymorphis between the studied cowpea cultivars that could be related to a constitutive defense conferred by recessive genes. After sequence analysis, it was found that eIF4E showed polymorphisms between cultivars, and, in at least two positions (68 and 108), there were differences between susceptible and resistant cultivars (Arg68/Pro68; Val108 or Pro108/Ala108). The molecular modeling revealed that differences in amino acid are located in two external loops close to the cap (m7G) binding domain, well reported in cases of recessive resistance within the Potyviridae family. Through immunodetection studies with the leaf extracts and the protein fractions obtained after the affinity chromatography on a Sepharose-7-metil-guanosina column, it was found that the amino acid mutations found did not impair the ability of eIF4E to bind to M7G in vitro. However, as it was observed two variants for eIF4E comparing the resistant and susceptible cultivars to CPSMV, at spatially neighboring regions, it could not be ruled out the hypothesis that this constitutive/recessive resistant trait is correlated with these mutations detected, which could impair, consequently, the in vivo interaction of eIF4E with the viral VPg.

Thesis (MSc. Agriculture (Agronomy)) -- University of Limpopo, 2019 / The traditional practice of farmers in Limpopo Province is to mixed and broadcast crops at planting without definite row arrangement. This practice hinders farm input application and results in low crop yields. Strip intercropping, where crops are planted with definite row arrangement, has the advantage of reducing inter-species competition, optimise plant population and increasing crop yield. This study aimed at improving cowpea-maize cropping systems using strip intercropping. The experiment was conducted at University of Limpopo farm and Ga-Thaba village. Five cowpea varieties (Glenda (check)), IT86K-499-35, IT82E-16, IT86D -1010, TVu-13464 and maize (PAN 6479) were evaluated using randomised complete block design with three replications. Data collected were days to flowering, days to maturity, plant height, canopy width, peduncle length, pod length, number of pods/plant, 100 seed weight, grain yield, fodder weight and land equivalent ratio was also determined. Data were analysed using the Statistix 9.0. The results revealed that in both locations TVu 13464 flowered early (50 days) respectively. At University of Limpopo farm TVu 13464, IT82E-16 and IT86D-1010 matured early (89, 88 and 91 days). At University of Limpopo farm, IT82E-16 had high cowpea grain yield (2230 kg/ha) under monocropping and also produced high grain yield of 1373 kg/ha during 2016/17 season. At Ga-Thaba, IT86D-1010 produced high cowpea grain yield of 1085 kg/ha under monocropping and during 2015/16 (660 kg/ha) while IT86K-499-35 also produced high grain yield of 915 kg/ha during 2016/17. The varieties showed yield stability depending on different locations. At University of Limpopo farm, strip intercropping achieved high maize grain yield of 3961 kg/ha during 2016/17. At Ga-Thaba, strip intercropping produced high maize grain yield 747 and 1024 kg/ha during 2015/16 and 2016/17, respectively. Monocropping produced low maize grain yield during 2015/16 with a mean of 425 kg/ha and mixed intercropping had mean of 499 kg/ha during 2016/17. The calculated LER for two crops over two seasons under strip intercropping ranged from 1.25 and 2.14, whereas under mixed intercropping, it ranged between 0.73 and 1.05 over two seasons at University of Limpopo farm. TVu 13464, IT82E-16 and IT86D-1010 are promising varieties for strip intercropping in low rainfall areas because of their early maturity and high grain yield. The calculated LER for two crops over two seasons xx under strip intercropping ranged from 1.62 and 2.98, whereas under mixed intercropping, it ranged between 0.76 and 1.67 in both seasons at Ga-Thaba. / National Research Foundation (NRF) and Water Research Commission (WRC)

Thesis (M.Sc. Agriculture (Agronomy)) -- University of Limpopo, 2019 / Limpopo Province is a semi-arid region prone to drought. Crop yields continue to decline due to low soil fertility and poor cropping systems. Cowpea is nutritionally rich in proteins essential for human consumption and livestock feeding. It fixes N2 which becomes available for the succeeding crop in rotation. For this reason, it is used as a companion crop in cereal-legume intercropping systems. Maize is one of the most important grain crops in South Africa, it serves as the major staple food for many households. Phosphorus is one of the macro-nutrient elements required by crops to produce satisfactory yields. The interactions between different rates of P fertilisation and cowpea-maize strip intercropping have not been studied in detail under rain-fed maize-cowpea strip intercropping in Limpopo Province. Many smallholder farmers in Limpopo Province obtain low yields due to the practice of mixed intercropping. Two season (2014/15 and 2015/16) experiments were laid out in a split-split plot design at Syferkuil farm to determine the performance of cowpea and maize varieties in cowpeamaize strip intercropping at varying P application rates. Treatments consisted of factors namely, P levels (0, 15, 30, 45 kg/ha), cropping system (monocropping and intercropping), maize varieties (WE3127 and ZM1423) and cowpea varieties (PAN311, TVu13464, IT86D-1010 and IT82D-889). Data were collected from growth and yield parameters that included (number of days to flowering, plant height, number of days to physiological maturity, root weight, number of pods per plant, unshelled net pod weight, number of cobs per plant, unshelled net cob weight and grain yield) in order to determine their performance. Results obtained revealed that P application levels significantly influenced most of the measured growth and yield parameters of both crops. PAN311 flowered earliest (49 days) across P levels. Increasing P application hastened the maturity of the varieties of PAN 311 and TVu13464 in both seasons. The P levels of 30 and 45 kg/ha reduced the number of days to maturity as compared to 0 and 15 kg/ha. TVu13464 variety produced more pods per plant (30) than other varieties. PAN311 yielded more grains (2491 kg/ha) than other varieties. Maize varieties performed well between P applications of 30 and 45 kg/ha. WE3127 yielded 3462 kg/ha whereas ZM1423 yielded 3306 kg/ha. Intercropping vi system performed better than monocropping system based on the measured growth and yield parameters. Two promising cowpea varieties (PAN311 and TVu13464) performed well and were selected based on their early maturity, drought tolerance and high yielding. Increasing P application levels increased crop yield. Optimum P levels for cowpea-maize strip intercropping were between 30 and 45 kg/ha. The calculated LER values were greater than one which indicates that intercropping was advantageous in land utilisation. The study showed the importance of P application in improving cowpea yield in cowpeamaize strip intercropping. / National Research Foundation (NRF) and University of Missouri, USA

Thesis (M.Sc. Agriculture (Agronomy) -- University of Limpopo, 2019 / Erratic rainfall and insect infestation are some of the factors that limit cowpea production in Limpopo (Asiwe, 2009). Improved cowpea varieties available for dryland production in Limpopo do not meet the farmer’s needs. Specific trait crosses are needed to be made with adapted varieties for the purpose of developing high yielding, pest tolerant and adapted varieties. Varieties with desired specific traits were planted in isolation plots for outcrossing by insects. F1 lines derived from parental lines that were half sib seeds were harvested and planted as F2, and seeds obtained from F2 generation were used to develop F3 segregating population at University of Limpopo Experiential Farm, Syferkuil, and planted along with parents. This F3 generation forms the current evaluation on which this report is based. Data collected included; aphid severity (measured on a 9-point scale), number of days to 90 % maturity, grain yield, 100 seed weight and number of genotypes selected for advancement. Results showed significant variation among progeny over parents therefore expressing transgressive segregation. Genotypes derived from pedigree TX08-30-5 were five while pedigree IT98K-205-8 and IT97K-499-35 had four genotypes each that expressed early maturity (80-90 days) which are desirable for evading drought. Twenty-three genotypes from pedigree GEC and three from pedigree IT97K-499-35 obtained grain yield (>101 g/plot and >161 g/plot) that was better than their parents. Only one genotype derived from pedigree TX08-30-1 expressed high resistance to aphids better than the parent with a mean aphid score range of 1-2. Four genotypes from pedigree TX08-30-5 obtained bigger 100 seed weight (>18.6 g) compared to parent. These progeny also proved to be high yielders as they expressed heterosis for all yield attributes than their parents. Eighty-two genotypes were selected for advancement. Significant variations were observed among progeny giving opportunity to make selections. In most cases, genotypes outperformed their parents which indicate positive heterosis. The 82 promising genotypes selected will be advanced for further selections from multi location testing for stability and adaptation.

4 pp. / Summer cover crops can add nitrogen to the soil, build up and maintain soil organic matter, suppress pest populations, mitigate soil erosion, and reduce nutrient leaching when they are used in Arizona vegetable systems. However, careful management is required since cover crops can modify the availability of soil nitrogen and other critical nutrients. The ratio between carbon to nitrogen (C:N) in decomposing cover crop biomass is a critical indicator of the overall process of breakdown and eventual release of nutrients. This article introduces five cover crops that could improve vegetable systems in Arizona. The mixtures of a legume and a non-legume cover crop species can also be planted to obtain desired C:N ratios to optimize the benefits of cover crops.

Introdução: As proteínas alimentares são fontes de peptídeos atuantes em vários processos metabólicos. Existem evidências de que a proteína do feijão caupi (Vigna unguiculata L. Walp) é capaz de reduzir o colesterol em hamsters e em humanos, porém, a permeabilidade após a digestão, o mecanismo de ação e a evidência direta da participação de peptídeos no metabolismo colesterol não são claros. Objetivo: Investigar a permeabilidade intestinal e avaliar o efeito nas vias luminal e endógena do metabolismo do colesterol de peptídeos provenientes de feijão caupi (Vigna unguiculata L. Walp). Metodologia: A permeabilidade dos peptídeos provenientes do caupi produzidos por hidrólise enzimática foi testada em linhagens de células Caco-2 usando placas Transwell®. Para investigar o efeito na via luminal, três peptídeos identificados na fração 3 kDa do hidrolisado (LLNPDDEQL; FFFGQDGGSKGEE e LNL) foram testados na solubilização de colesterol e fosfatidilcolina, no tamanho das micelas de colesterol e interação com ácidos biliares in vitro. Para verificar o efeito no metabolismo endógeno, linhagens de células HepG2 foram incubadas com peptídeos sintéticos (MELNAVSVVHS e MELNAVSVVSH) identificados como resultado do ensaio de permeação nas células Caco-2. A expressão de RNAm dos transportadores de colesterol NPC1L1, ABCA1 e ABCG1 foi realizada nas células Caco-2 e a expressão de HMGCR, SREBP2, LDLR, LXR, AMPK1 foi avaliada nas células HepG2. Resultados: A exposição das células Caco-2 à fração 3 kDa do hidrolisado (2,5 e 5 mg/mL) aumentou a expressão de ABCG1 nos tempos 6 h e 12 h. O níveis de RNAm dos genes SREBP2, HMGCR e LDLR reduziram nas HepG2 após 24h do tratamento com o peptídeo MELNAVSVVHS (50 M e 100 M). A fração 3 kDa do hidrolisado e os peptídeos LLNPDDEQL; FFFGQDGGSKGEE e LNL foram capazes de reduzir a solubilidade do colesterol micelar in vitro em no máximo 42 por cento , bem como, provocaram mudanças estruturais ao interagirem com a fosfatidilcolina, com destaque ao peptídeo LNL (50 por cento de ligação). O peptídeo LNL foi o único capaz de promover a precipitação do colesterol em forma de cristais devido à interação com os ácidos biliares. Conclusões: A fração 3 kDa do hidrolisado e todos os peptídeos testados foram capazes de insolubilizar o colesterol in vitro. Constata-se que o mecanismo de competição pelo espaço intramicelar com o colesterol se dá pela interação com os componentes micelares e não diretamente com o colesterol. O peptídeo do feijão caupi MELNAVSVVHS foi permeável e foi capaz de reduzir a expressão do fator de transcrição SREBP2 (consequentemente reduzindo HMGCR e LDLR) / Introduction: Food proteins are sources of peptides acting in sevral metabolic processes. There is evidence that cowpea (Vigna unguiculata L. Walp) protein is able to lower cholesterol levels in hamsters and humans, but its permeability after digestion, mechanism of action and direct evidences of peptide participation in cholesterol metabolism are not clear. Objective: To investigate the intestinal permeability and to evaluate the effect on the luminal and endogenous cholesterol metabolism pathways of peptides from cowpea (Vigna unguiculata L. Walp). Methods: The permeability of the cowpea peptides produced by enzymatic hydrolysis was tested on Caco-2 cell lines using Transwell® plates. To investigate the effect on the luminal pathway, three peptides identified in the 3 kDa hydrolyzate fraction (LLNPDDEQL; FFFGQDGGSKGEE and LNL) were tested for in vitro cholesterol and phosphatidylcholine solubilization, cholesterol micelle size changing and interaction with bile acids. To verify the effect on endogenous metabolism, HepG2 cell lines were incubated with synthetic peptides (MELNAVSVVHS or MELNAVSVVSH) identified as a result of the permeation assay on Caco-2 cells. The mRNA expression of the cholesterol transporters NPC1L1, ABCA1 and ABCG1 was performed on Caco-2 cells and the expression of HMGCR, SREBP2, LDLR, LXR, AMPK1 was evaluated in HepG2 cells. Results: Exposure of Caco-2 cells to the 3 kDa hydrolyzate fraction (2.5 and 5 mg/mL) increased ABCG1 expression at 6 h and 12 h times. The mRNA levels of the SREBP2, HMGCR and LDLR genes were reduced in HepG2 after 24h of treatment with the MELNAVSVVHS peptide (50 M and 100 M). The 3 kDa of the hydrolyzate fraction and the peptides LLNPDDEQL; FFFGQDGGSKGEE and LNL were able to reduce the solubility of micellar cholesterol in vitro in a maximum of 42 per cent , as well as, caused structural changes when interacting with phosphatidylcholine, with emphasis on the LNL peptide (50 per cent of binding). The LNL peptide alone was able to promote cholesterol precipitation in the form of crystals due to interaction with bile acids. Conclusions: The 3 kDa hydrolysate fraction and all peptides tested were able to insolubilize cholesterol in vitro. It was observed that the mechanism of competition for the intramicellar space with cholesterol is given by the interaction with the micellar components and not directly with the cholesterol. The MELNAVSVVHS cowpea peptide was permeable and was able to reduce the expression of the SREBP2 transcription factor (thereby reducing HMGCR and LDLR)