Resumo: O presente trabalho teve por objetivo estudar alguns aspectos comportamentaisde Spodoptera frugiperda (J. E. SMITH)em variedades de algodoeiro. Emcasa-de-vegetação, determinou-se a densidade adequada de adultos por planta, a distribuiçãovertical de ovos, nas diferentes partes da planta, e a idade adequada das plantaspara infestação, visandoa realizaçãode estudos de resistênciae a melhoria das práticasde manejo de S. frugiperdaem algodoeiro.Emlaboratório,avaliou-sea atratividadee a não-preferência alimentar, com e sem chance de escolha, de lagartas recém-eclodidas por partes de plantas e por variedades de algodoeiro. O desenvolvimentobiológico do inseto foi também acompanhado confinando-se lagartas às diferentes variedades. Os resultados permitiram concluir que S. frugiperda tem preferênciapor ovipositar em plantas com 60 dias de idade, preferencialmente na face abaxialdas folhas, localizadas no terço superior das plantas. A densidade de 3 ou 4 casais de adultos por planta foi suficiente para a realização de testes de nãopreferência para oviposição. As variedades Coodetec 408, BRS Aroeira, BRS Araçá, BRS Ita 90 e DeltaPenta apresentaram resistência do tipo não-preferência para Qviposição.Afolha foi a parte da planta mais atrativa e preferida para alimentação por lagartas recém-eclodidas. Em condições de livre escolha, Coodetec 410 foi a mais atrativacomparativamente à Acala 90, FiberMax 966 e DeltaPenta. As lagartas demonstraram maior preferência para alimentação por BRS Araçá em relação à BRS Cedro,BRS Ita 90, DeltaPenta, Coodetec 408 e BRS Aroeira. 46 minutos foi o tempo maisadequadopara avaliação da atratividadede plantas a lagartas recém-eclodidasde S. frugiperda.As variedades BRS Cedro, DeltaPenta e Acala 90 expressaram elevado nívelde antibiose sobre as lagartas. As variedades BRS Cedro, DeltaPenta e Acala 90 expressaram elevado nível de antibiose sobre as lagartas. / Abstract: The objective of this research was to study some behavioral aspectsof Spodoptera frugiperda (J. E. SMITH)in cotton varieties. Adequate density of adultsper plant, vertical distribution of eggs in different plant parts, and adequate plant age for infestation were determined in the greenhouse for future resistance studies and improvementof S. frugiperda management practices in cotton. The attractiveness and non-preference for feeding of newly-hatched caterpillars for cotton plant parts and varietieswere evaluated in the laboratory in free-choice and no-choice tests. The insect'sbiologicaldevelopment was also monitored by confining caterpillars to different cottonvarieties.Results allowedto concludethat S. frugiperdaprefers to ovipositon 60- day-oldplants,preferablyon the abaxialsurface of leaves in the upper thirdof plants. A densityof 3 or 4 pairs of adults per plant was sufficient to conduct non-preference-forovipositiontests. Varieties Coodetec 408, BRS Aroeira, BRS Araçá, BRS Ita 90, and DeltaPenta showed the non-preference-for-oviposition type of resistance; the most attractiveand preferred plant part for feeding of newly-hatched caterpillars were the leaves.Under free-choice conditions, Coodetec 410 was the most attractive variety whencompared with Acala 90, FiberMax966, and DeltaPenta. Caterpillars showed greater preference for feeding for BRS Araçá in relation to BRS Cedro, BRS Ita 90, DeltaPenta,Coodetec 408, and BRS Aroeira. The most adequate time to evaluate plant attractivenessto newly-hatched S. frugiperda caterpillars was 46 minutes. Varieties BRS Cedro,DeltaPenta, and Acala 90 expressed a high levei of antibiosis on the caterpillars. IPR20, Coodetec 410, and DeltaOpal also showed this mechanism but in a less intense manner; FiberMax 977, however, was the most susceptible variety for pest developrnent. / Orientador: Arlindo Leal Boiça Junior / Coorientador: Alcebíades Ribeiro Campos / Banca: Antonio Carlos Busoli / Banca: Edson Luiz Lopes Baldin / Banca: José Roberto Scarpellini / Banca: José Carlos Barbosa / Doutor

Insetos vetores de fitopatógenos são responsáveis por causarem perdas significativas na agricultura, principalmente por disseminarem microrganismos que provocam doenças nas plantas hospedeiras. A mosca-branca Bemisia tabaci biótipo B é um inseto praga de relevante importância na cultura do tomate, capaz de transmitir diversos vírus nessa cultura. Assim, os objetivos deste trabalho foram: verificar a resistência à transmissão do begomovírus Tomato severe rugose virus (ToSRV) por B. tabaci biótipo B em genótipos de tomateiro com e sem aplicação do inseticida sintético ciantraniliprole; conhecer melhor acerca das interações inseto-planta através de experimentos de atratividade quanto à alimentação e oviposição do inseto e de comportamento de prova de adultos virulíferos de B. tabaci biótipo B em genótipos de tomateiro monitorados por Electrical penetration graph (EPG). Foram selecionados cinco genótipos de tomateiro: \'Santa Clara\' (suscetível à mosca-branca), \'Ivety\' e \'Carina TY\' (resistentes a ToSRV), LA1335 e LA716 (resistentes à mosca-branca), para um experimento inicial para selecionar o genótipo mais suscetível ao inseto e ao vírus, o mais resistente ao vírus, e o mais resistente ao inseto para condução dos demais experimentos. Selecionou-se \'Santa Clara\' (suscetível a ambos os organismos), \'Ivety\' (resistente a ToSRV) e LA716 (resistente ao inseto). No experimento de atratividade, ficou demonstrado que LA716 repeliu as moscas-brancas, sendo o menos atrativo, seguido de \'Ivety\' e depois \'Santa Clara\' que foi o genótipo que apresentou maior número de moscas-brancas. Os três genótipos tratados com o inseticida ciantraniliprole, e o genótipo LA716 não tratado (resistência genética isoladamente) conseguiram reduzir a inoculação de ToSRV pelas moscas-brancas e provocar alta mortalidade dos insetos. Ademais, os genótipos tratados com ciantraniliprole, e \'Ivety\' não tratado apresentaram menor severidade de ToSRV do que \'Santa Clara\', o mais suscetível. Através da técnica de EPG, ficou evidenciado que LA716 e o tratamento dos três genótipos com ciantraniliprole promoveram efeitos semelhantes em B. tabaci biótipo B, em que houve redução do número de eventos da onda np, de prova, da onda C e da onda G por inseto, além de reduzir significativamente a proporção de indivíduos que realizaram as ondas pd, E1, E2, F, G, ingestão no floema por tempo prolongado, provas curtas, e aumentar a duração da onda np e reduzir a duração do tempo de prova, onda C, número de provas curtas e número de E2 prolongado por inseto. Ainda, a integração da resistência de LA716 com ciantraniliprole promoveu efeitos adicionais, como a redução da proporção de moscas-brancas que realizaram prova, onda C e G, em relação aos genótipos não tratados com o inseticida, e aumento da duração da onda np, prova e C por evento. Pela análise dos resultados, pode-se concluir que LA716 é altamente resistente a B. tabaci biótipo B. Além de ser menos atrativo, o genótipo proporciona efeitos semelhantes ao inseticida ciantraniliprole quanto à alta mortalidade de moscas-brancas, à redução da incidência de ToSRV, e à ação negativa sobre o comportamento de prova do inseto. A associação da resistência genética de LA716 com ciantraniliprole é interessante para o manejo da transmissão de ToSRV, por promover efeitos adicionais. / Insect vectors of phytopathogens are responsible by significant losses in agriculture, especially by disseminating microorganisms that cause diseases in host plants. The whitefly Bemisia tabaci B biotype is an important pest in the tomato crop, capable to transmit several virus in this crop. Thus, this study aimed to: verify the resistance to the transmission of the begomovirus Tomato severe rugose virus (ToSRV) by B. tabaci B biotype in tomato genotypes treated and non treated with the insecticide cyantraniliprole; know about insectplant interactions through experiments of feeding and oviposition attractiveness by the insect and probe behaviour of viruliferous adults of B. tabaci biotype B in tomato genotypes monitored by Electrical penetration graph (EPG). It was selected five tomato genotypes: \'Santa Clara\' (susceptible to the whitefly), \'Ivety\' and \'Carina TY\' (resistant to ToSRV), LA1335 and LA716 (resistant to the whitefly), to an initial experiment to select the most susceptible genotype to the insect and to the virus, the most resistant to the virus, and the most resistant to the insect for carry out the others experiments. Thus, it was selected \'Santa Clara\' (susceptible to both organisms), \'Ivety\' (resistant to ToSRV) and LA716 (resistant to the insect). The attractiveness experiment showed that LA716 repelled the whiteflies, which was considered the least attractive, followed by \'Ivety\', and after \'Santa Clara\' which had the largest total number of whiteflies. The three cyantraniliprole treated genotypes, and the nontreated genotype LA716 (genetic resistance isolated) were able to reduce ToSRV inoculation by the whiteflies and to cause high mortality of insects, moreover, the cyantraniliprole treated genotypes, and non-treated \'Ivety\' had less ToSRV severity than \'Santa Clara\', the most susceptible. Through the EPG technique, it was clear that LA716 and the treatment of the three genotypes with cyantraniliprole promoted similar effects in B. tabaci B biotype, in which there was reduction of the number of events of waveform np, probe, waveform C and waveform G per insect, besides of reducting significantly the proportion of individuals that performed the waveforms pd, E1, E2, F, G, ingestion into phloem for long time, short probes, and increase the duration of the waveform np and reduce the duration of probe, waveform C, number of short probes and number of sustained E2 per insect. Moreover, the integration of the LA716 resistance with cyantraniliprole promoted additional effects, such as the reduction of the proportion of whiteflies that performed probe, waveform C and G, in relation to the non-treated genotypes, and an increasing in the duration of waveform np, probe and waveform C per event. Analyzing the results, it can be concluded that LA716 is highly resistant to B. tabaci biotype B. Besides of being less attractive, the genotype promotes similar effects to the insecticide cyantraniliprole considering the high mortality of whiteflies, the reduction of ToSRV incidence, and the negative effects in probing behaviour of the insect. The association of LA716 genetic resistance with cyantraniliprole is interesting to the ToSRV transmission management by promoting additional effects.

A citricultura brasileira concentra-se principalmente no Estado de São Paulo que contribui com 80,4 % da produção nacional, sendo o Brasil um dos maiores produtores mundiais de citros. Um dos problemas enfrentados pela citricultura é a sua vulnerabilidade a pragas e doenças, devido principalmente a baixa diversidade genética nas variedades comerciais utilizadas, associada ao sistema de plantio em áreas extensas. Uma das doenças que vem causando crescentes prejuízos para a citricultura brasileira é a pinta preta ou mancha preta dos citros causada pelo fungo Guignardia citricarpa Kiely. O uso de conhecimentos de biologia molecular e métodos biotecnológicos devem ser considerados como importante alternativa para a produção de plantas geneticamente modificadas expressando genes de resistência. Para se obter plantas de citros resistentes a doenças, se faz necessário identificar genes que estejam relacionados com os mecanismos de defesa da planta. Na tentativa de identificar estes genes, o objetivo geral deste trabalho foi a identificação de genes in silico no banco de dados do Projeto Millenium CitEST e a análise de expressão diferencial de genes envolvidos na defesa. Mais de 7600 sequencias foram identificadas nas buscas no CitEST com similaridade aos genes R e genes envolvidos na HR e defesa, MAPKs e SNF1. Destes, foram selecionados 273 sequencias para experimentos de macroarranjo para análise da interação Citrus sinensis-Guignardia citricarpa. A análise estatística revelou que 171 genes (62,63%) apresentaram expressão diferencial significativa ao nível de 5% de probabilidade. Destes, 80 apresentaram expressão diferencial significativa maior do que duas vezes, dos quais 38 genes foram induzidos e 42 foram reprimidos no tecido infectado. Entre os genes induzidos estão MAPKs, genes de resistência (R), genes envolvidos na resposta de hipersensibilidade (HR) e na defesa da planta. Entre os transcritos reprimidos, há quatro similares a peroxidases e cinco similares a catalases, o que era esperado já que catalases e algumas peroxidases são capazes de remover H2O2, e assim a planta produz espécies reativa de oxigênio capaz de desencadear a ativação de genes de defesa. Os dados do macroarranjo foram validados via transcrição reversa seguida de PCR quantitativo em tempo real (RT-PCRq) de 9 genes. As análises confirmaram a expressão diferencial de 8 deles sendo que somente um apresentou resultado contrastante ao macroarranjo, o que demonstra a eficiência da metodologia de macroarranjos para estudo de muitos genes simultaneamente. Os genes diferencialmente expressos identificados na interação C. sinensis-G. citricarpa são de grande importância, pois são fortes candidatos para serem utilizados na transformação genética de plantas com o objetivo de obter novas variedades de plantas com resistência a patógenos. / The Brazilian citrus industry is concentrated mainly in the State of Sao Paulo which contributes with 80.4% of national production, with Brazil being a leading world producer of citrus. One of the problems facing the citrus industry is its vulnerability to pests and diseases, mainly due to low genetic diversity of the commercial varieties used, linked to the system of planting in extensive areas. A disease that is causing increasing damage to the brazilian citrus industry is the black spot of citrus caused by the fungus Guignardia citricarpa Kiely. The use of knowledge of molecular biology and biotechnological methods should be considered as an important alternative for the production of genetically modified plants expressing genes for resistance. In order to obtain citrus plants resistant to diseases it is necessary to identify genes that are related to the defense mechanisms of the plant. In an attempt to identify these genes, the general aim of this study was to identify genes in silico in the database of the Millennium CitEST Project and to perform differential expression analysis of genes involved in the defense mechanisms. More than 7600 reads were identified in the CitEST search with similarity to R genes, genes involved in HR and defense, MAPKs and SNF1. It was selected 273 reads for macroarray experiments to analysis of Citrus sinensis-Guignardia citricarpa interaction. Statistical analysis revealed that 171 genes (62.63%) showed significant differential expression at the level of 5% probability. From these, 80 showed significant differential expression higher than two fold, in which 38 genes were induced and 42 were repressed in infected tissue. Among the induced genes are MAPKs, resistance (R) genes, genes involved in hypersensitivity response (HR) and plant defense. Among the suppressed transcripts, there are four similar to peroxidases and five similar to catalases, which is expected because catalases and some peroxidases are able to remove H2O2, and so the plant produces reactive oxygen species capable of triggering the activation of defense genes. The macroarray data were validated by reverse transcription followed by quantitative real-time PCR (RT-PCRq) of 9 genes. The analysis confirmed the differential expression of 8 of them, and only one presented different result of macroarray which demonstrate the efficiency of the macroarray methodology to analyze several genes simultaneously. The genes differentially expressed in the interaction of C. sinensis x Guignardia citricarpa identified are of great importance because they are strong candidates for use in genetic transformation of plants with the objective of obtaining new varieties of plants resistant to pathogens.

Espécies de Meloidogyne constituem o principal grupo de fitonematoides causadores de doenças em plantas. São espécies polífagas, distribuídas mundialmente e que se reproduzem profusamente nas mais variadas culturas agrícolas. O fumo não figura exceção, sendo severamente afetado por tais vermes. Globalmente, o controle destas moléstias é realizado essencialmente pela incorporação, em cultivares comerciais, de um único gene de resistência encontrado em Nicotiana tomentosa (gene Rk) e sabidamente efetivo contra às raças 1 e 3 de M. incognita. Todavia, a frequente ocorrência de plantas sintomáticas em cultivos nacionais sugere a existência de outros genótipos de Meloidogyne como agentes etiológicos da doença atualmente. Neste ensejo, realizou-se, no presente estudo, um levantamento das espécies de Meloidogyne ocorrentes em 39 áreas de plantio de fumo na região Sul do Brasil, em cultivares portadoras do gene Rk. Para isto, populações oriundas de raízes infectadas foram estabelecidas e mantidas em plantas de tomate e indivíduos das mesmas foram identificados em nível de espécie por meio da análise de isoenzimas (&alpha;-esterases), da observação de configurações perineais e do sequenciamento da região 18S-ITS1-5.8S do RNA ribossomal. Isolados obtidos a partir destas populações foram estabelecidos a partir de uma única massa de ovos, mantidos em plantas de tomate, agrupados em espécies com base nestes resultados e analisados quanto à patogenicidade em hospedeiros diferenciais e frente a várias características morfométricas. Identificaram-se M. incognita em dezoito amostras (46,2%), M. enterolobii em dez (25,6%), M. javanica em treze (33,3%), M. arenaria em duas (5,1%) e M. inornata em uma (2,6%). Populações mistas também foram encontradas (25,6%). Duas populações (5,1%) morfológica e bioquimicamente atípicas (LGM 27 e LGM 38) foram encontradas, mas com espectro de patogenicidade e sequências de 18S-ITS1-5.8S idênticas a M. enterolobii e M. incognita, respectivamente. Variações intra-específicas foram observadas sob o âmbito patogênico e morfométrico, porém geralmente próximas às suas descrições originais. M. incognita raça 2 foi predominante (79%) e variantes de M. enterolobii foi fato comum (40%). Tendo em vista a elevada ocorrência de M. enterolobii neste levantamento e a escassez de informações acerca de fontes de resistência a esta espécie, buscou-se, também, caracterizar, com base em índice de galhas (IG), a reação (resistência/suscetibilidade) de 97 acessos perante M. enterolobii. Destes, 7 genótipos (J102007T10, J1612008T12, J1612008T34, I112008T97, I112008T98, I112008T99 e I112008T100) foram identificados e avaliados em um segundo experimento. Estes mostraram-se como fontes promissoras de resistência, haja vista que exibiram valores baixos de severidade (IG<1,0). Diante dos resultados, depreende-se a necessidade de incluir resistência a biótipos virulentos, máxime raça 2, de M. incognita, a M. javanica, a M. arenaria e a M. enterolobii em cultivares comerciais. Sob este aspecto, este estudo definiu fontes de resistência para M. enterolobii, constituindo informação inédita na literatura fitonematológica. / Meloidogyne species are undoubtedly the major group of plant parasitic nematodes. Meloidogyne species are polyphagous, worldwide distributed and reproduce profusely in various agronomical crops. Tobacco crop is not an exception, being severely affected by these worms. Worldwide, the control of these diseases is based mainly on the incorporation in commercial cultivars of a single resistance gene found in Nicotiana tomentosa (gene Rk) and known to be effective against races 1 and 3 of M. incognita. However, the frequent occurrence of symptomatic plants in tobacco crops suggests the existence of other genotypes of Meloidogyne as etiological agents of the disease today. Therefore, our study aimed to perform a survey of Meloidogyne species occurring in 39 tobacco growing areas in Southern Brazil, in cultivars carrying the Rk gene. For this, Meloidogyne populations obtained from infected roots were established, maintained on tomato plants and specimens were identified at specific level by isoenzyme analysis (&alpha;-esterase), by analysis of perineal patterns and by sequencing of the 18S- ITS1-5.8S ribosomal RNA region. Isolates obtained from these populations were established from a single egg mass, maintained in tomato plants, grouped into species based on these results and analyzed for host range (pathogenicity) and several morphometric characteristics. From the total of samples, eighteen (46.2%) contained M. incognita, ten (25.6%) M. enterolobii, thirteen (33.3%) M. javanica, one (2.6%) M. arenaria and one (2.6%) M. inornata. Mixed populations were also found in 25.6% of the samples. Two populations (LGM 27 e LGM 38) (5.1%) were morphologically and biochemically atypical, but with host ranges and ITS1-5.8S-18S sequences identical to M. enterolobii and M. incognita, respectively. Intraspecific variations were observed in relation to host range and morphometric characters, but usually close to their original descriptions. M. incognita race 2 was predominant (79%) and strains of M. enterolobii were common fact (40%). Due to the high incidence of M. enterolobii found in this survey and the absence of information about resistance sources to this species, we characterized, based on gall index (GI), the reactions (resistance/susceptibility) of 97 accession of Nicotiana spp. to M. enterolobii. Seven genotypes (J102007T10, J1612008T12, J1612008T34, I112008T97, I112008T98, and I112008T99 I112008T100) were identified and evaluated in a second experiment. These were considered promising sources of resistance, once they exhibited low severity values (IG <1.0). From of our results, it is obviously necessary to include resistance to virulent biotypes (mainly race 2) of M. incognita, M. javanica, M. enterolobii and M. arenaria in commercial cultivars. This study defined resistance sources for M. enterolobii thus providing novel information for the literature related to plant nematodes.

O seqüenciamento em larga-escala e a aplicação da tecnologia de seqüências expressas (ESTs) permitiram a identificação de milhares de seqüências genômicas de vários organismos. Através de projetos como o de obtenção de ESTs de cana-de-açúcar (SUCEST – Sugarcane EST Project), a tecnologia de macro e microarranjos de DNA pôde ser aplicada para o monitoramento da expressão gênica. O primeiro passo deste trabalho foi otimizar a técnica de macroarranjos utilizando-se colônias bacterianas. Os arranjos foram construídos com o auxílio do robô Q-Bot. Células bacterianas contendo clones de cDNA de cana-de-açúcar foram arranjadas em duplicata em membranas de náilon e crescidas por 6 e 12 horas. Como resultado, concluiu-se que é possível estudar a expressão gênica em larga-escala usando macroarranjos de colônias bacterianas crescidas por 6 horas. Na segunda parte, o objetivo foi identificar genes de defesa em duas variedades de cana-de-açúcar (Saccharum sp.), em resposta ao herbívoro Diatraea saccharalis (Lepidoptera: Crambidae). A cana-de-açúcar é de grande importância para a agricultura brasileira e para a economia geral de muitos países em desenvolvimento. Estas plantas são atacadas por D. saccharalis, a mais importante praga desta cultura, causando significativas perdas econômicas. Como é sabido, as plantas desenvolveram complexas estratégias para se protegerem do ataque de insetos-praga. As variedades SP80-3280 (susceptível) e SP81-3250 (tolerante) de cana-de-açúcar foram expostas à D. saccharalis. Plantas submetidas à lagarta e plantas controle foram coletadas em 0.5, 6, 12 e 24 horas de experimento. As análises permitiram a identificação de genes diferencialmente expressos em resposta à lagarta nas duas variedades. Foram analisados o tempo, dinâmica e regulação da expressão de 3.840 clones do SUCEST, os quais foram crescidos na membrana de náilon por 6 horas. Estes ESTs foram agrupados em dezesseis classes: metabolismo de aminoácidos, crescimento/desenvolvimento, metabolismo de proteínas, metabolismo de RNA, metabolismo secundário, resposta à diferentes condições de estresse, transporte, bioenergética, transdução de sinal, dinâmica celular, metabolismo de DNA, metabolismo de lipídios, elementos móveis, metabolismo de nitrogênio, sulfato e fosfato, metabolismo de nucleotídeos e função não determinada (ND). Estes resultados ajudam elucidar as estratégias de defesa desenvolvidas pela cana-de-açúcar para evitar os danos causados por esta praga. Neste estudo, os dados do macroarray revelaram 580 ESTs com expressão aumentada, oriundos das duas variedades. Estes resultados são importantes não somente porque este é o primeiro estudo em larga-escala da expressão gênica de uma monocotiledônea em resposta ao ataque de herbívoros, mas também porque permite a comparação dos perfis de expressão entre genótipos susceptível e tolerante de cana-de-açúcar. Este estudo abre possibilidades para a engenharia genética de plantas de cana-de-açúcar inseto-resistentes e também para o uso destes genes como marcadores moleculares em programas de melhoramento assistido. / Large-scale sequencing and the application of expressed sequence tag (EST) technology has led to the identification of hundreds of thousands of genomic sequences from various organisms. Through projects like the Sugarcane Expressed Sequence Tag (SUCEST), DNA arrays technology could be applied in monitoring gene expression. The first step of this work was to optimize the macroarray technique with bacterial colonies. Arrays were robotically constructed (Q-Bot). Bacterial cells carrying sugarcane cDNA clones were arrayed in duplicate onto nylon membrane and grown for 6 and 12 hours. As a result, we conclude that is possible to study the gene expression in large-scale using macroarray through bacterial colony that grown for 6 hours. The second part of this work was to study how different two sugarcane varieties (Saccharum sp.) are in response resistance against Diatraea saccharalis (Lepidoptera: Crambidae). Sugarcane is of great importance for Brazilian’s agriculture and for the general economy of many tropical developing countries. These plants are attacked by D. saccharalis, the most important insect pest of sugarcane, causing significant economic losses. As it is well known, plants have evolved complex defense strategies to protect them in opposition to attack by insects. The varieties SP80-3280 (susceptible) and SP81-3250 (tolerant) were exposed to D. saccharalis. Plants materials from plants with the borer and control plants (without the borer) were collected at 0.5, 6, 12 and 24 hours time points. The analysis revealed genes differentially expressed in response to sugarcane borer in both varieties. We analyzed the timing, dynamics, and regulation of the expression of 3,840 SUCEST clones, which were grown on a nylon membrane for 6 h. These ESTs was grouped into 16 broad categories: amino-acid metabolism, plant grow and development, protein metabolism, RNA metabolism, secondary metabolism, stress response, transport, bioenergetics, signal transduction, cellular dynamics, DNA metabolism, lipid, fatty-acid metabolism, mobile genetic elements, nitrogen, sulphur and phosphate metabolism, nucleotide metabolism, and the group matched to unable to classify, which no indication of the function of the gene product are known. These results help to elucidate the defense strategies developed by sugarcane in order to prevent against insect damage. In this study, macroarray analyses revealed 580 ESTs with increased expression, from both varieties. These results are important not only because this is the first large-scale gene expression study of a monocot in response to insect attack, but also because it allows the comparison of the gene expression pattern between susceptible and tolerant genotypes. This study opens possibilities for genetically-engineering of insect-resistant sugarcane and for using these genes as molecular markers in conventional breeding programs.

<p dir="ltr">Specialty crop growers in the Great Lakes region of the United States and Canada have been struggling for decades to manage the carrot weevil (<i>Listronotus oregonensis</i>). In carrot, parsley, and celery production, growers may experience 40-100% yield loss, despite the intensive use of insecticides (Boivin, 1999; Jasinski, 2008; Telfer et al., 2019). To improve sustainable management of this native pest in commercial vegetable production, we must address existing knowledge gaps in carrot weevil mate-finding behavior, population genetic diversity and gene flow, and grower adoption and efficacy of integrated pest management (IPM) strategies, like cultivar selection, to combat this pest. To date, these factors have not been evaluated in the carrot weevil system, yet together they have significant implications for developing novel monitoring tools, grower decision making, and the potential evolution of insecticide resistance in this damaging pest.</p><p dir="ltr">To address knowledge gaps in grower adoption and the efficacy of IPM strategies, such as cultivar selection, I investigated the influence of carrot cultivar on the oviposition preference of carrot weevil in the laboratory and quantified the diversity of insect assemblages on carrots in Ohio and Indiana. In tandem, I delivered an online survey to specialty crop growers in Indiana and Ohio to measure their knowledge and use of insect-resistant cultivars as a part of an IPM strategy. I found that carrot cultivar did not impact carrot weevil oviposition preference, but rather the number of petiole trichomes was positively correlated with the cumulative number of egg scars found on a given carrot cultivar. Similarly, cultivar did not influence the diversity of insect assemblages associated with carrot foliage, but hemipterans were the dominant group found across cultivars. Moreover, insect diversity across all carrot cultivars was high, with Shannon-Weiner diversity indices ranging from 2.14 to 2.45.</p><p dir="ltr">I analyzed survey responses from vegetable growers regarding their knowledge, current use, and future use of insect-resistant cultivars and found no significant differences between conventional or organic growers, in either Indiana or Ohio, or across experience levels regarding their knowledge and current or future use of insect-resistant cultivars. Although a greater number of organic growers were aware of insect-resistant cultivars, they were not incorporated into their IPM plans. In contrast, half of responding conventional growers indicated implementing insect-resistant cultivars in current IPM plans. Growers indicated that more information about the efficacy, costs, and benefits of insect-resistant vegetable cultivars would be most helpful in informing their decision to incorporate this IPM tactic on their farms.</p><p dir="ltr">To address knowledge gaps regarding carrot weevil mate-finding behavior, I investigated behavioral response to conspecific frass using Y-tube bioassays and the volatiles of frass samples were analyzed using gas-chromatography mass-spectrometry (GC-MS) as starting points to assess the presence of volatile chemical cues that might function as sex or aggregation pheromones. I found that male and female weevils oriented strongly towards volatiles from male frass. However, conspecifics were not attracted to mixed or female frass, suggesting male frass contains behaviorally relevant chemical cues that are unique or present in greater abundance than in female frass. When I analyzed the chemical abundance and composition of the male frass volatiles, I found 16 common compounds across all samples and no compounds were unique to the male volatile blend. Specifically, (1S)-6,6-dimethyl-2-methylene-Bicyclo[3.1.1]heptane and (1R)-2,6,6-Trimethylbicyclo[3.1.1]hept-2-ene were present in significantly higher abundance in extracts of males frass compared to those of female frass.</p><p dir="ltr">Finally, to address knowledge gaps in the genetic diversity and gene flow of carrot weevil populations in the Great Lakes Region of the U. S., I used single nucleotide polymorphisms (SNPs) to compare genetic diversity and structure of agricultural populations of carrot weevil in Michigan and Ohio. I found limited genetic differentiation between three carrot weevil populations collected from celery and parsley fields located in Michigan and Ohio. I also found high inbreeding coefficients and heterozygote deficiency in all populations, suggesting that all carrot weevil populations are locally confined. These results suggest significant gene flow between populations, which has implications for management, specifically the mitigation and management of insecticide resistance.</p><p dir="ltr">Together, the results from my dissertation can be applied to the improvement of carrot weevil IPM across the Great Lakes region of the United States and Canada. Further investigations into the impact of physical characteristics, specifically petiole trichomes, on carrot weevil oviposition behavior can inform the breeding and development of resistant carrot cultivars. Results from my behavioral bioassay studies represent a critical first step towards the identification of an aggregation or sex pheromone, which would greatly improve detection of resident carrot weevil populations when used in conjunction with current monitoring traps. My results also suggest that monitoring and mitigation of insecticide resistance in carrot weevil populations should be considered at the regional scale, at minimum, with significant gene flow between carrot weevil populations within the Great Lakes Region.</p>

Powdery mildew, caused by the pathogen Blumeria graminis (DC) Speer (Syn. Erysiphe graminis DC) f. sp. tritici, is a major disease of wheat (Triticum aestivum L.). Race-specific resistance is easily identified in the field due to its qualitative phenotype and it is easy to incorporate because it is inherited as a single gene. Unfortunately, this type of resistance is easily overcome by the pathogen. Traits associated with quantitative trait loci (QTL) such as adult-plant resistance (APR), have become popular with plant breeders because of their durability over a wide geographic range and time. Due to the quantitative nature of these genes, they are difficult to study requiring multiple assessments of disease development under natural conditions in more than one location over a period of several weeks. Numerous QTL for APR to powdery mildew have been mapped in independent studies in different wheat backgrounds. The wheat cultivar Massey has been the subject of several studies due to its APR to powdery mildew that has remained effective for several decades. However, it has been difficult to identity simple sequence repeat (SSR) markers that are tightly linked to the QTL for APR in Massey. Such markers give breeders an advantage by allowing them to quickly identify and select for traits that would be difficult to distinguish in the field among breeding progeny from several backgrounds. Therefore, identification of tightly linked markers associated with APR to powdery mildew is necessary so that these traits can be selected for reliably in progeny. / Master of Science

Powdery mildew, caused by Blumeria graminis (DC.) E.O. Speer f. sp. tritici E'm. Marchal (syn. Erysiphe graminis f. sp. tritici), is one of the major diseases of wheat (Triticum aestivum L.) worldwide. The use of cultivars with resistance to powdery mildew is an efficient, economical and environmentally safe way to control powdery mildew. Race-specific resistance has been extensively used in breeding programs; however, it is ephemeral. Adult plant resistance (APR) to powdery mildew is more durable as demonstrated by the cultivar Massey, which has maintained its APR to powdery mildew since its release in 1981. To develop an efficient breeding strategy, it is essential to understand the genetic basis of APR. The objectives of this study were to identify molecular markers associated with APR to powdery mildew in common wheat Massey and to verify their association using recombinant inbred (RI) lines. A cross was made between the powdery mildew susceptible cultivar Becker and Massey. One hundred and eighty F2:3 lines were rated for disease severity under natural pressure of powdery mildew in field. Using both restriction fragment length polymorphism (RFLP) and microsatellite markers, three quantitative trait loci (QTL), designated as QPm.vt-1B, QPm.vt-2A and QPm.vt-2B, were identified in the Becker x Massey F2:3 generation. These loci are located on chromosomes 1B, 2A and 2B, respectively, and explained 17%, 29% and 11% of the total variation among F2:3 lines for powdery mildew resistance, respectively. Cumulatively, the three QTLs explained 50% of the phenotypic variation among F2:3 lines in a multi-QTL model. The three QTLs associated with APR to powdery mildew were derived from Massey and displayed additive gene action. QPm.vt-2B also fits a recessive model for APR to powdery mildew. In the second part of this study, 97 RI lines were developed from the Becker x Massey cross. The RI lines were evaluated for APR to powdery mildew under natural disease pressure for three years. Both single marker analysis and interval mapping confirmed the presence of the three QTLs identified in the F2:3 generation. The three QTLs, QPm.vt-1B, QPm.vt-2A and QPm.vt-2B, accounted for 15%, 26% and 15% of the variation of mean powdery mildew severity of the RI lines over three years. In a multi-QTL model, the three QTLs explained 44% of the phenotypic variation of the RI lines. The RI lines were grouped according to the genotype of the three QTLs, represented by markers GWM304a, KSUD22 and PSP3100, respectively. The RI lines with Massey alleles at all three loci had a mean disease severity of 3.4%, whereas the RI lines with Becker alleles at all three loci had a mean disease severity of 22.3%. These severity values are similar to those of the corresponding parents. The molecular markers identified and verified as to their association with APR to powdery mildew in Massey have the potential for use in marker-assisted selection for resistance to powdery mildew and in pyramiding powdery mildew resistance genes, as well as facilitating a better understanding of the molecular basis of APR to powdery mildew. / Ph. D.

Orientador: Arlindo Leal Boiça Junior / Banca: Antonio Carlos Busoli / Banca: André Luiz Lourenção / Resumo: Este trabalho teve como objetivo avaliar possíveis fontes de resistência de cultivares de amendoim de hábitos de crescimento ereto e rasteiro, a Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae) dos tipos não-preferência para oviposição, alimentação (com e sem chance de escolha) e antibiose. Os experimentos foram conduzidos em laboratório, sob condições controladas de temperatura (25 ± 2°C), umidade (60 ± 10%) e fotofase (12 horas). Foram utilizadas cultivares de amendoim de hábitos de crescimento ereto (IAC 5, IAC 8112, IAC 22 e IAC Tatu ST) e rasteiro (IAC 503, IAC 505, IAC 147, IAC 125, IAC Caiapó e IAC Runner 886). Estas cultivares foram avaliadas separadamente devido ao hábito de crescimento do material em todos os testes. Os testes de não-preferência para oviposição foram realizados com adultos recém-emergidos, os quais foram alimentados com solução de mel a 10%, permanecendo nas gaiolas por quatro dias até o início das avaliações. Estas consistiram na quantificação do número de ovos e posturas, com posterior cálculo do número de ovos por postura. Para os testes de não-preferência para alimentação foram colocados discos foliares das cultivares em placas de Petri, liberando uma lagarta de terceiro ínstar por cultivar. Avaliou-se a atratividade a 1; 5; 10; 15; 30; 60; 120; 360; 720; 1440 minutos, contando o número de lagartas que se alimentavam nas cultivares e, ao término do experimento, quantificou-se a área foliar consumida por cultivar. No teste de antibiose, além da identificação de cultivares de amendoim resistentes a S. frugiperda, determinaram-se, também, os graus de resistência através da análise multivariada. As cultivares foram semeadas em campo. Lagartas de primeiro ínstar foram individualizadas em placas de Petri forradas com papel filtro umedecido. Diariamente, foram fornecidos folíolos novos. Avaliaram-se... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study aimed to evaluate possible sources of resistance of peanut cultivars of the upright growth and runner growth habit, to Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae) by non-oviposition and non-feeding preference (tests choice and no-choice) and antibiosis. The experiments were conducted in laboratory under controlled conditions of temperature (25 ± 2°C), humidity (60 ± 10%) and photophase (12 hours). Peanut cultivars of upright growth habit (IAC 5, IAC 8112, IAC 22 and IAC Tatu ST) and runner growth habit (IAC 503, IAC 505, IAC 147, IAC 125, IAC Caiapó and IAC Runner 886) were evaluated. These cultivars were evaluated separately due to the material growth habit in all tests. Tests of non-oviposition preference were conducted with newly emerged adults, and they were fed with a 10% honey solution, remaining in the cages for four days until the start of evaluations. Evaluations consisted of quantifying the eggs and egg masses numbers, followed by calculation the number of eggs per egg masses. To perform non-feeding preference tests, leaves discs from different cultivars were placed in disposable Petri dishes followed by releasing of a third instar caterpillar for each cultivar. The attractiveness at 1; 5; 10; 15; 30; 60; 120; 360; 720 and 1,440 minutes was evaluated by counting the caterpillars number which were fed on cultivars, quantified the leaf area consumed by cultivar was evaluated at the end of the experiment. Additionally, identification of peanuts cultivars resistance to S. frugiperda, and the resistance degree was evaluated by multivariate analysis of the antibiosis test. The cultivars were planted in plots on the field. First instar larvae were placed individually in disposable Petri dishes plates, over a moistened filter paper. New leaflets were provided every day. The duration (days), weight (mg) and viability... (Complete abstract click electronic access below) / Mestre

L'utilisation de molécules stimulatrices de défense des plantes (SDP), également appelées inducteurs de résistance, constitute une alternative possible aux traitements fongicides conventionnels pour contrôler les maladies dues aux chanpignons phytopathogènes. Dans cette étude, nous avons mis en évidence que trois produits à caractère SDP (FSOV2, FSOV7 et FSOV10) protègent le blé (Triticum aestivum L.) contre la septoriose (Mycosphaerella graminicola, anamorphe Zymoseptoria tritici) lorsqu'ils sont utilisés de façon préventive, mais cette protection dépend fortement du cultivar considéré. Les cultivars Alixan, Premio et Altigo testés ici présentaient au départ des niveaux de résistance distincts à la septoriose. Les protections obtenues ne sont pas liées à un effet direct sur la germination des spores du champignon, mais à l'induction des mécanismes de défense chez le blé qui réduisent la nécrose foliaire et la sporulation du champignon. Ainsi, l'observation des différents stades du processus infectieux de M. graminicola en microscopie et le dosage des activités enzymatiques fongiques de dégradation des parois (CWDE) in planta révèlent que le niveau de protection induite varie avec le SDP appliqué et avec le cultivar traité. L'expression de neuf gènes impliqués dans différentes voies de défense, suivi par RTqPCR, et les activités enzymatiques peroxydase et phénylalanine ammonia lyase ont été mesurée au cours du temps, depuis le traitement par les SDP jusqu'à 5 jours après infection. Les résultats obtenus montrent, que les mécanismes de défense sont induits différemment en fonction du cultivar et en fonction du SDP appliqué. Ces résultats suggèrent que la réussite au champ des SDP est conditionnée de façon déterminante par le choix du couple SDP-cultivar. / The use of resistance inducers (RI) is a potential alternative to conventional fungicide treatments against plant fungal diseases. In the present study, we revealed that preventive applications of three RI conferred protection efficacies against M. graminicola, with protection levels varying with the wheat cultivar. Alixan, Premio and Altigo cultivars were previously known to exhibit distinct resistance levels to M. graminicola. The observed protections did not result from a direct effect on spore germination, but were related to the induction of wheat defense mechanisms. The induced resistances reduced foliar necrosis, as well as the sporulation level of the fungus. Microscopic observations of the infection process of M. graminicola and cell wall degrading enzymes (CWDE) activities measured in planta showed that the applied RI as well as the considered treated wheat cultivar influences the impact on the infection process and the protection efficacy. We investigated from the time of treatment until 5 days after inoculation plant peroxidase and phenylalanine ammonia lyase activities and the expression of nine genes involved in distinct defense pathways. Our results indicated that defense mechanisms are differently induced according both to the wheat cultivar and the RI. Therefore, the successful use of RI at the field level strongly depends on the RI-cultivar combination.