Orientador: Maria Lígia Rodrigues Macedo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T23:07:55Z (GMT). No. of bitstreams: 1 Silva_DesireeSoaresda_D.pdf: 3168437 bytes, checksum: b863b3429e08cd7ff7926b540a4d69b5 (MD5) Previous issue date: 2014 / Resumo: Inibidores de peptidases (IPs) são proteínas vegetais envolvidas nos mecanismos de defesa contra herbívoros e patógenos. São considerados agentes antimetabólicos, uma vez que sua presença na dieta de insetos compromete seu desenvolvimento normal. Este efeito se dá através da formação de complexos entre os IPs e as enzimas digestivas, comprometendo assim o processo digestivo dos insetos. Neste trabalho, investigamos efeitos do inibidor de tripsina de sementes de Adenanthera pavonina (ApTI) sobre o desenvolvimento de dois insetos-praga, a broca-da-cana (Diatraea saccharalis) e a lagarta-do-cartucho (Spodoptera frugiperda). Bioensaios, estudos bioquímicos e de proteômica, foram realizados a fim de determinar o efeito de ApTI sobre o desenvolvimento destes insetos. A exposição aguda de larvas de D. saccharalis ao ApTI diminuiu a atividade das tripsinas e quimotripsinas em 87% e 63%, respectivamente. ApTI afetou o desenvolvimento deste inseto como observado através da diminuição na viabilidade larval (55%) e pupal (20,6%). Larvas oriundas da geração F1 apresentaram efeitos similares aos observados na geração F0, como redução no peso (23%) e na taxa de sobrevivência (33%). Análises bioquímicas revelaram que o inibidor foi refratário à digestão pelas peptidases de D. saccharalis e que o perfil de enzimas digestivas permaneceu sensível à inibição por quantidades crescentes de ApTI. Entretanto, não foi possível realizar a identificação das proteínas do intestino desta lagarta por espectrometria de massas. Larvas de S. frugiperda foram criadas em dieta contendo 0,5% de ApTI. Ao final dos experimentos, uma redução no peso médio larval de 30% foi observada. Porém, diferentemente dos resultados obtidos para D. saccharalis, as enzimas das larvas alimentadas com ApTI mostraram-se resistentes à inibição, sugerindo que esta espécie adapta-se à dieta contendo inibidor. O sequenciamento das proteínas presentes nos fluidos intestinais de S. frugiperda permitiu a identificação de 41 proteínas. Dentre elas foi possível identificar serinopeptidases do tipo tripsina e quimotripsina, proteínas estruturais, enzimas quinases e fosfatases. Nossos resultados demonstram que são necessários novos estudos para obtenção de mais informações acerca das proteínas presentes no fluido intestinal de S. frugiperda, para correlacionarmos sua expressão diferencial em larvas submetidas a diferentes tratamentos, conhecendo desta maneira um novo indício sobre os mecanismos adaptativos desta espécie contra os IPs / Abstract: Peptidase inhibitors (PIs) are plant proteins involved in defense mechanisms against herbivores and pathogens. They are considered antinutritional compounds, since their presence in insect diet hinder the normal larval development. This effect is due the complexation between PIs and peptidases, affecting the insect digestive process. In this work, we investigated the effects of trypsin inhibitor from Adenanthera pavonina seeds (ApTI) on the development of two pest insects: the sugarcane borer (Diatraea saccharalis) and the fall armyworm (Spodoptera frugiperda). Biochemical and proteomic studies were carried out in order to determine the ApTI effects on the development of these insects. The acute exposure of sugarcane borer to ApTI reduced the trypsin and chymotrypsin activities in 87% and 63%, respectively. The effect of ApTI on development of sugarcane borer was noticed by reduced larval (23%) and pupal viability (33%). Biochemical analysis revealed that the inhibitor was refractory to digestion by D. saccharalis peptidases and the enzymes maintained sensibility to inhibition by increasing amounts of ApTI. However, it was not possible to identify proteins from sugarcane borer gut through mass spectrometry approach. S. frugiperda larvae were reared in artificial diet containing 0.5% ApTI and a reduction in average larval weight of 30% was noticed. But, differently of D. saccharalis, S. frugiperda midgut enzymes became resistant to inhibition by ApTI, showing that this species adapted to ApTI-diet. From the sequencing of S. frugiperda gut fluid proteins we identified 41 proteins. Among them, we identified trypsin and chymotrypsin enzymes, structural proteins and quinases and phosphatases enzymes. Our results demonstrated that further researches are necessary in order to obtain more information about proteins from S. frugiperda gut fluid. The correlation between differential expressed proteins in larvae submitted to different treatments is crucial to reach new indicatives about the adaptive mechanisms this species evolve against PIs / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Plants respond to insect and pathogen attack by inducing and accumulating a large set of defense proteins. Colonization of sugarcane stalk by opportunistic fungi, such as Fusarium verticillioides and Colletotrichum falcatum, usually occurs after Diatraea saccharalis (Lepidoptera: Cambridae) caterpillars attack increasing the damage caused by the borer. Two homologous of BARWIN protein were identified in sugarcane, SUGARWIN1 and SUGARWIN2. Their gene expression is induced in response to wound and Diatraea saccharalis damage. However, the recombinant SUGARWIN protein does not affect insect development; but promotes significant morphological and physiological changes in Fusarium verticillioides and Colletotrichum falcatum, which lead to fungal cell death via apoptosis, indicating that SUGARWINs may work as a first layer of defense against the fungi infection. In this study, we deepen our understanding of the role of SUGARWINs in plant defense and the molecular mechanisms by which these proteins affect fungi by elucidating their molecular targets. Our results show that SUGARWINs play an important role in plant defense against opportunistic pathogens. We demonstrated that SUGARWINs are induced by C. falcatum, and the induction of SUGARWINs can vary among sugarcane varieties. The sugarcane variety exhibiting the highest level of SUGARWIN induction exhibited a considerable reduction in C. falcatum infection. Furthermore, SUGARWIN1 exhibited ribonuclease and chitinase activity, whereas SUGARWIN2 exhibited only chitinase activity. This variable enzymatic specificity seems to be the result of divergent amino acid composition within the substrate-binding site. Additionally, plants attacked by insects and pathogens display profound physiological, morphological and chemical changes or adaptations, which may result in organism attraction or avoidance. In this study, we also aimed to understand the insect-fungi association in sugarcane and the role of fungal volatile compounds in this association. Our results have shown that D. saccharalis positively influences C. falcatum infection on sugarcane, inducing a fast growing when compared to C. falcatum treatment without D. saccharalis attack. In addition, both fungi, C. falcatum and F. verticillioides, have been shown a double effect on D. saccharalis caterpillar, they promoted a strong attraction for insects due volatile organic compound emission and positively influenced D. saccharalis feeding and weight gain in diets supplemented with fungi. Fungal volatile organic compounds from C. falcatum and F. verticillioides were identified and quantified; acoradiene and acorenol were specifically induced by the fungi. These data suggest a synergistic interaction, mediated by organic volatile compounds, between D. saccharalis and the fungi C. falcatum and F. verticillioides in sugarcane. / As plantas respondem ao ataque de insetos e patógenos induzindo e acumulando um grande conjunto de proteínas de defesa. A colonização do caule de cana por fungos oportunistas, como Fusarium verticillioides e Colletotrichum falcatum, geralmente ocorre após o ataque de lagartas de Diatraea saccharalis (Lepidoptera: Cambridae), resultando no aumento do dano causado pelo inseto. Dois homólogos da proteína BARWIN foram identificados em cana-de-açúcar, SUGARWIN1 e SUGARWIN2. A expressão desses genes é induzida em resposta ao ferimento mecânico e ao ataque de Diatraea saccharalis, entretanto, a proteína não afeta o desenvolvimento do inseto, mas promove alterações morfológicas e fisiológicas significativas em Fusarium verticillioides e Colletotrichum falcatum, causando a morte destes fungos por apoptose. Esses dados indicam que as SUGARWINs podem funcionar como uma defesa inicial contra a infecção fúngica. Neste estudo, aprofundamos nosso entendimento do papel das SUGARWINs na defesa de plantas e os mecanismos moleculares pelos quais essas proteínas afetam os fungos, elucidando seus alvos moleculares. Nossos resultados mostraram que as SUGARWINs desempenham um papel importante na defesa da planta contra patógenos oportunistas. Foi demonstrado que essas proteínas também são induzidas por C. falcatum em cana-de-açúcar, e sua indução pode variar entre as variedades de cana-de-açúcar. A variedade de cana-de-açúcar que apresentou o maior nível de indução de SUGARWINs apresentou uma redução considerável na infecção por C. falcatum. Além disso, SUGARWIN1 exibiu atividade de ribonuclease e quitinase, enquanto que SUGARWIN2 exibiu apenas atividade de quitinase. Esta especificidade enzimática parece ser o resultado da composição divergente de aminoácidos no sítio de ligação do substrato. Além disso, as plantas atacadas por insetos e patógenos exibem profundas alterações fisiológicas, morfológicas e químicas ou adaptações, que podem resultar em atração ou repelência do organismo, dessa forma, estudamos também a associação inseto-fungos na cana-de-açúcar, e o papel dos compostos voláteis fúngicos nessa associação. Nossos resultados mostraram que D. saccharalis influencia positivamente a infecção por C. falcatum em cana-de-açúcar, induzindo crescimento rápido do fungo quando comparado ao tratamento com C. falcatum sem ataque de D. saccharalis. Além disso, ambos os fungos, C. falcatum e F. verticillioides, mostraram um efeito duplo sobre lagartas de D. saccharalis, promovendo uma forte atração desses insetos devido à emissão de compostos orgânicos voláteis e influenciando positivamente a alimentação de D. saccharalis e ganho de peso em dietas suplementadas com fungos. Os compostos orgânicos voláteis fúngicos de C. falcatum e F. verticillioides foram identificados e quantificados; acoradieno e acorenol foram especificamente induzidos pelos fungos. Estes dados sugerem uma interação sinergistica, mediada por compostos orgânicos voláteis, entre D. saccharalis e os fungos C. falcatum e F. verticillioides em cana-de-açúcar.

Orientadores: Maria Imaculada Zucchi, Anete Pereira de Souza / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T12:00:33Z (GMT). No. of bitstreams: 1 Pavinato_VitorAntonioCorrea_D.pdf: 6829608 bytes, checksum: 7fdb8c74593eb4dc2fe78e2302d3995f (MD5) Previous issue date: 2014 / Resumo: A associação entre subpopulações de insetos e plantas-hospedeiras pode ocorrer por adaptação e esta, pode ser uma etapa anterior ao surgimento de raças-hospedeiras e especialização. Pouco se sabe sobre o papel da mudança da composição da paisagem, mediada pela atividade agrícola recente, na divergência adaptativa e fluxo gênico de insetos fitófagos. Dessa forma, o presente trabalho teve como objetivos: i) desenvolver marcadores moleculares microssatélites para o estudo genético de um inseto fitófago, Diatraea saccharalis; ii) quantificar e caracterizar a estrutura genética, o fluxo gênico e os fatores que contribuem para a divergência genética das subpopulações da espécie; iii) identificar variação genética sob seleção natural que possa estar contribuindo para a divergência genética de subpopulações associadas a cana-de-açúcar e milho e; iv) desenvolver recurso genômico através de bibliotecas genômicas RADtag para a busca, desenvolvimento e caracterização de marcadores moleculares associados a genes candidatos. Dos 20 locos microssatélites, dez foram selecionados para serem utilizados no estudo de ecologia molecular. Os índices de diferenciação mostraram que, tanto a estruturação genética espacial, quanto a determinada pelo hospedeiro, foram significativas. Dos 301 locos AFLP utilizados para a genotipagem de quatro subpopulações, 19 foram identificados como outliers nas comparações par-a-par e desses, cinco locos foram identificados pelos dois métodos empregados na detecção de outliers, e podem, desta forma, estar associados à adaptação à planta-hospedeira. Os resultados das análises de agrupamento utilizando os locos outliers mostraram o agrupamento dos indivíduos em grupos que representam a planta-hospedeira onde foram coletados. Os mesmos resultados foram obtidos com uma amostra dos SNPs isolados através do protocolo de RADtag. Os dados genômicos obtidos até o momento estão sendo utilizados, juntamente com o estudo de genômica comparativa, na identificação e desenho de primers específicos para genes candidatos. Os resultados mostraram os efeitos da expansão e mudança recente da paisagem agrícola na diversidade genética de uma espécie de inseto fitófago. A atividade agrícola pode ser fonte de seleção divergente suficiente para levar à especialização e especiação de insetos. Os resultados deste trabalho sugerem estar havendo divergência ecológica entre as subpopulações de D. saccharalis coletadas em milho e cana-de-açúcar e que esta divergência, por ser recente, não é completa. Além disso, esses resultados mostram a necessidade de estudos complementares para isolar as fontes de seleção divergente, os mecanismos de isolamento reprodutivo, e a arquitetura genética que liga a seleção divergente ao isolamento reprodutivo / Abstract: The association between subpopulations of insects and their host plants can occur by adaptation and this may lead to host-races formation and specialization. Little is known about the role of the changing landscape composition mediated by recent agricultural activity in adaptive divergence and gene flow of phytophagous insects. This study aimed to: i) develop microsatellite markers for the genetic study of a phytophagous insect, Diatraea saccharalis; ii) quantify and characterize the genetic structure, gene flow and the factors that contribute to the genetic divergence of subpopulations of the species; iii) identify genetic variation that may be experiencing natural selection and thus be contributing to genetic divergence of subpopulations associated with sugarcane and maize; iv) develop genomic resource through RADtag libraries to search, characterize and develop molecular markers linked to candidate genes. Ten of 20 microsatellite loci were selected for use in the study of molecular ecology. The genetic differentiation showed that both spatial genetic structure and that determined by the host-plant were significant. Of the 301 AFLP loci used for genotyping four subpopulations, 19 were identified as outliers in pairwise comparisons and five were identified by the two methods employed for outliers detection and thus, can be associated with host-plant adaptation. Cluster analysis using outlier loci showed the grouping of individuals into groups that represent the host plant where they were collected. Data from a sample of SNPs isolated by RADtag protocol showed the same results. The genomic data obtained so far are being used together with the study of comparative genomics for the identification and design of specific primers for candidate genes. The results showed the effects of expansion and recent changes in agricultural landscape in genetic diversity of phytophagous insect species. Farming can generate enough source for ecologically based divergence that could lead to specialization and speciation in insects species. The results of this study suggest that there is ecological divergence between subpopulations of D. saccharalis collected from corn and sugarcane, but it is not complete. In addition, these results showed the need for further studies to isolate the sources of divergent selection, the mechanisms of reproductive isolation, and the genetic architecture linking the divergent reproductive isolation with selection / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

As is the case in many other regions in the world, sugarcane production in southern Africa is affected by a wide range of climatic conditions, which can vary considerably from location to location and from year to year. As a result, the season length and growth cycles of sugarcane in southern Africa differ greatly. Such conditions include the hot and dry regions of northern KwaZulu-Natal, Swaziland and Mpumalanga, where sugarcane is mostly irrigated, to the humid sub-tropical coastal belt extending from the far north coast of KwaZulu-Natal to areas in the Eastern Cape, as well as the cool frost prone midlands regions of KwaZulu-Natal. Owing to the wide range of climatic conditions in which sugarcane is grown in southern Africa, there are many different external factors that affect sugarcane production, including a range of pests and diseases, frost occurrences and variations in soil water. The objective of this research was to (1) identify a number of important variables that affect cane production in southern Africa, (2) employ suitable models to reflect these variables, and (3) simulate and map the extent and severity of these variables at a high spatial resolution over southern Africa. Such variables include the Eldana saccharina and Chilo sacchariphagus stalk borers, sugarcane rust fungus, heat units with selected base temperatures, frost, soil water content, soil compaction, irrigation water demand, conducive and non-conducive growing conditions, flowering proficiencies for sugarcane, sugarcane yields and yield increments per unit of irrigation. The distribution patterns of the above-mentioned variables relied greatly upon the various models employed to represent them, as well as the accuracy of the temperature and rainfall databases to which the various models were applied. Although not definitive, the models used to reflect the variables which had been identified were considered to be generally satisfactory. The resolution at which the variables which had been identified in this study were mapped, was also found to be adequate. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

The use of entomopathogenic fungi to control agriculturally important pests, both in greenhouses and in the field, has been demonstrated by various authors for a number of years. This has been brought about by the development of resistance in certain pest species to chemical applications and a growing public awareness of the safety implications of residual insecticides. Several entomopathogenic fungi were tested against four insect pests found in the Republic of South Africa (RSA), the greenhouse whitefly, Trialeurodes vaporariorum, the rice weevil, Sitophilus oryzae, the grapevine mealybug, Planococcusficus and the sugarcane stem borer, Eldana saccharina. Further concentration, temperature and humidity studies were conducted with selected isolates on the rice weevil, S. oryzae. Sitophilus oryzae is considered one ofthe most important pests of stored grain. Several fungal isolates were tested against the rice weevil, four of which, B1, PPRI 6690, PPRI 6864 and PPRI 7067, were selected for further testing based on the mortality results over a 21 d period. Varying conidial concentrations were applied and at high doses of 1x10 -6 conidia ml -1 with mortality rates of to 84% achieved. LT 50 values ranged from 6 - 68d. Increased spore concentration resulted in an increase in overall mortality. Temperature and humidity was found to affect the infection potential of the four isolates tested. Four temperatures ranging from 15 - 30°C were tested. The highest mortality rates were obtained at 25°C where mortality ranged from 46 - 65% in 14d. Mortality rates decreased with decreasing temperature, and no mortality was recorded at 30°C. Temperature was found to significantly alter the LT 50 values, increasing the LT 50 with decreasing temperatures. Decreasing the humidity resulted in an increased LT 50 and a reduction in the overall mortality rates. The mortality of S. oryzae ranged according to the RH and isolate. Isolates Bland PPRI 6690 resulted in the highest mortalities of 80 and 83% at 92.5% RH, with LT 50's of 6.3d and 6.4d, respectively. Several entomopathogenic fungi were tested against T vaporariorum, P. ficus and E. saccharina, three key pests of South African crops. Nine fungal isolates were tested against the greenhouse whitefly, T vaporariorum, with mortalities ranging from 26.7 - 74.7% over 14d. Beauveria bassiana Isolates Bl and PPRl 6690 produced the highest mortality rates and were recommended for further pathogenicity testing against T. vaporariorum. Planococcus ficus is a common pest ofvineyards in the Western Cape Province, South Africa. Nine entomopathogenic fungi were screened against P.ficus, only two of which produced mortality. Eldana saccharina is a stalk borer, which infests sugarcane in large areas of Southern Africa. Five isolates were tested against second and third instar larvae, three of which, B1, PPRl 6864 and PPRl 6690 resulted in mortalities. Mean percentage mortality was low for all three isolates. From the study it was evident that two of the isolates tested, Bland PPRI 6690 (B. bassiana), showed potential against three of the four pests, and two isolates of Lecanicillium lecanii caused mortality in P. ficus. Further research and understanding of the effect of environmental conditions, spore concentration and epizootic potential would result in the further development of these isolates as future biological control agents. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.