Upon exposure to heat or cold, Arabidopsis thaliana seedlings undergo rapid transcriptional reprogramming of several hundreds of genes that promote stress tolerance. Despite extensive characterisation of the transcriptional responses to these stimuli, however, relatively little is known about the mechanisms by which temperature signals are perceived and transduced in plant cells. High or low seasonal temperatures have large impacts on crop productivity and are expected to intensify given current global climatic projections. It is therefore of agricultural importance to better understand temperature signalling pathways in plants in order to find solutions to this problem. In this thesis, a chemical genomics screen for molecules activating or repressing heat-inducible genes in A. thaliana was performed in collaboration with Syngenta and the biological targets of these chemicals were predicted based on structural similarities to compounds with known modes of action. Many molecules that affect the function of chloroplasts or mitochondria either activate or repress heat-responsive genes, thus implicating these organelles in the regulation of plant temperature responses. In addition, the translation inhibitor cycloheximide was identified as a repressor of heat-inducible genes and an activator of early cold-inducible genes. Diverse translation inhibitors trigger a cytosolic influx of calcium ions and several inhibitors of translation elongation were found to strongly activate cold-inducible gene expression in a calcium-dependent manner. Furthermore, it was demonstrated that cold shock causes rapid translation repression in A. thaliana seedlings and that the elongation factor LOS1 is involved in cold- or cycloheximide-induced gene expression, thus implicating translational machinery in the regulation of temperature signalling in plants. Finally, one of the chemicals identified in the screen, S01A463859Y, was found to improve heat resilience in A. thaliana and may therefore be of potential use in enhancing crop productivity during thermal stress.

Various genes have evolved to protect the cell against stressor-induced damage or death including the heat shock proteins (HSPs). Stressor-induced HSP gene expression involves the activation of heat shock factor (HSF), which binds to the heat shock element (HSE) found in the promoter region of <em>hsp</em> genes. Previously, our laboratory has examined the expression and function of <em>hsp</em> genes in the South African clawed frog, <em>Xenopus laevis</em>. Amphibians are particularly susceptible to adverse environmental conditions, including high temperatures and toxicants. In contrast to the many known inducers of HSF activation in poikilothermic vertebrates, few inhibitors have been either discovered or described in the literature. The present study has compared for the first time the effect of two heat shock response (HSR) inhibitors, quercetin and KNK437, on <em>hsp</em> gene expression in <em>Xenopus</em> A6 cells, demonstrating their efficacy in poikilotherms. Northern blot and densitometric analysis showed that cells treated with either quercetin or KNK437 decreased the heat shock-induced accumulation of <em>hsp70</em>, <em>hsp47</em>, and <em>hsp30</em> mRNAs. Additionally, constitutive levels of <em>hsp47</em> and <em>hsc70</em> mRNAs were reduced. In comparison, neither quercetin nor KNK437 affected the levels of constitutively expressed <em>ef1&alpha;</em> mRNAs under control or heat shock conditions. Western blot and densitometric analysis in this study showed that under heat shock conditions, exposure to quercetin or KNK437 significantly decreased the accumulation of HSP30, and that KNK437 was more effective in doing so than quercetin. In comparison, levels of actin were not significantly affected by either heat shock or exposure to DMSO, quercetin, or KNK437. These findings suggest that one mechanism by which quercetin and KNK437 inhibits the HSR in <em>Xenopus</em> is through the inhibition of HSF activity. <br /><br /> Results of this study also suggest that KNK437 inhibits the acquisition of thermotolerance in poikilotherms, similar to observations in mammalian systems. In the presence of KNK437, cells given a 2 h heat pretreatment at 33ºC followed by a thermal challenge for 1 h at 37ºC, showed numerous ruffled membrane edges and some aggregates of disrupted stress fibers. In comparison, cells directly challenged for 1 h at 37ºC, showed a marked decrease in HSP30, which was located predominantly at the cellular periphery in conjunction with actin aggregates. These cells showed virtually no intact stress fibers spanning cells and no coherent cell-cell connections. A 3-D analysis of cells given a 1 h thermal challenge at 37ºC (after a prior 2 h heat shock at 33ºC) in the absence of KNK437, showed numerous linear actin bundles transversing the entire cell, even extending into areas of cell-cell contact, and abundant HSP30 concentrated in the perinuclear region surrounding an intact nucleus. However, in the presence of KNK437, there was a significant emergence of membrane ruffles indicating global instability of cellular adhesion. This study has demonstrated that KNK437, which is the more specific and efficient HSR inhibitor, will be an important inhibitor to compare with the well-documented quercetin for future investigations.

Various genes have evolved to protect the cell against stressor-induced damage or death including the heat shock proteins (HSPs). Stressor-induced HSP gene expression involves the activation of heat shock factor (HSF), which binds to the heat shock element (HSE) found in the promoter region of <em>hsp</em> genes. Previously, our laboratory has examined the expression and function of <em>hsp</em> genes in the South African clawed frog, <em>Xenopus laevis</em>. Amphibians are particularly susceptible to adverse environmental conditions, including high temperatures and toxicants. In contrast to the many known inducers of HSF activation in poikilothermic vertebrates, few inhibitors have been either discovered or described in the literature. The present study has compared for the first time the effect of two heat shock response (HSR) inhibitors, quercetin and KNK437, on <em>hsp</em> gene expression in <em>Xenopus</em> A6 cells, demonstrating their efficacy in poikilotherms. Northern blot and densitometric analysis showed that cells treated with either quercetin or KNK437 decreased the heat shock-induced accumulation of <em>hsp70</em>, <em>hsp47</em>, and <em>hsp30</em> mRNAs. Additionally, constitutive levels of <em>hsp47</em> and <em>hsc70</em> mRNAs were reduced. In comparison, neither quercetin nor KNK437 affected the levels of constitutively expressed <em>ef1&alpha;</em> mRNAs under control or heat shock conditions. Western blot and densitometric analysis in this study showed that under heat shock conditions, exposure to quercetin or KNK437 significantly decreased the accumulation of HSP30, and that KNK437 was more effective in doing so than quercetin. In comparison, levels of actin were not significantly affected by either heat shock or exposure to DMSO, quercetin, or KNK437. These findings suggest that one mechanism by which quercetin and KNK437 inhibits the HSR in <em>Xenopus</em> is through the inhibition of HSF activity. <br /><br /> Results of this study also suggest that KNK437 inhibits the acquisition of thermotolerance in poikilotherms, similar to observations in mammalian systems. In the presence of KNK437, cells given a 2 h heat pretreatment at 33ºC followed by a thermal challenge for 1 h at 37ºC, showed numerous ruffled membrane edges and some aggregates of disrupted stress fibers. In comparison, cells directly challenged for 1 h at 37ºC, showed a marked decrease in HSP30, which was located predominantly at the cellular periphery in conjunction with actin aggregates. These cells showed virtually no intact stress fibers spanning cells and no coherent cell-cell connections. A 3-D analysis of cells given a 1 h thermal challenge at 37ºC (after a prior 2 h heat shock at 33ºC) in the absence of KNK437, showed numerous linear actin bundles transversing the entire cell, even extending into areas of cell-cell contact, and abundant HSP30 concentrated in the perinuclear region surrounding an intact nucleus. However, in the presence of KNK437, there was a significant emergence of membrane ruffles indicating global instability of cellular adhesion. This study has demonstrated that KNK437, which is the more specific and efficient HSR inhibitor, will be an important inhibitor to compare with the well-documented quercetin for future investigations.

One unique feature of cancer cells that can be exploited for anticancer drug discovery is their dependence on their own cellular stress responses to survive the stressful acidotic, hypoxic and nutrient-deprived conditions within the tumor. Reasoning that desert organisms surviving under stressful conditions may have evolved to produce small molecule metabolites capable of modulating heat shock protein 90 (Hsp90) function, and/or other cell stress responses, we employed the cellular heat shock response in a moderate-throughput phenotypic assay. This strategy has resulted in the isolation and characterization of a number of small molecule natural products with heat shock induction activity from these organisms. Three such natural products are the subject of this study.In a limited structure-activity relationship (SAR) study, a previously known Hsp90 inhibitor radicicol (RAD), and several structurally related molecules including the fungal metabolite monocillin 1 (MON) were found to interact with Hsp90. In addition, RAD and MON were shown to lead to the degradation of Hsp90 client proteins involved in the cancer cell survival the estrogen receptor (ER) and the insulin-like growth factor receptor 1 (IGF-1R).We further characterized MON and showed that by targeting the molecular chaperone Hsp90, this compound induces components of the heat shock response at the transcriptional and translational levels, and leads to the acquisition of a thermotolerant phenotype in seedlings of the plant Arabidopsis thaliana. These findings support our hypothesis that there is ecological significance to the elaboration of small molecules that target stress responses.A number of extracts active in our phenotypic assay contained small molecules with no apparent Hsp90 activity. One such extract afforded terrecyclic acid A (TCA) with significant anti-tumor activity against a panel of human cancer cell lines. To characterize the biological activities of TCA we examined three key stress responsesthe heat shock, oxidative, and inflammatory responsesand show that TCA destabilizes these pathways associated with cancer cell survival through induction of oxidative stress (ROS), and inhibition of NF-kappaB transactivation.The isolation of RAD, MON and TCA from Sonoran desert organisms provides proof of principle that we have developed an effective strategy for the discovery of small molecule modulators of cellular stress responses that can serve as leads for the development of new anticancer drugs with novel mechanisms of action.

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / A alface à uma das mais importantes hortaliÃas folhosas cultivadas no mundo. Considerada cultura de clima ameno, sua produÃÃo em regiÃes de baixa altitude e latitude à prejudicada, principalmente, devido Ãs elevadas temperaturas e à predominÃncia de dias longos encontrados nestes locais. Nessas condiÃÃes, ocorre baixa germinaÃÃo e desuniformidade na emergÃncia, por consequÃncia, verifica-se baixa eficiÃncia na produÃÃo de mudas. Tais acontecimentos sÃo importante indÃcio da manifestaÃÃo de um acontecimento fisiolÃgico desencadeado dentro da semente, a dormÃncia. Na alface, tal fenÃmeno parece ser controlado pela interaÃÃo genÃtipo (cultivar) x fatores ambientais, sendo que neste Ãltimo a temperatura tem sido considerada o fator mais relevante. AlÃm dos problemas relacionados ao estabelecimento do stand inicial, seja em campo ou viveiro de produÃÃo de mudas, plantas de alface submetidas a elevadas temperaturas tambÃm podem apresentar pendoamento precoce, o que resulta na necessidade de antecipaÃÃo da colheita provocando, assim, reduÃÃo na produÃÃo. Baseado no exposto, objetivou-se com este trabalho, avaliar o comportamento germinativo de cultivares de alface submetidas a diferentes nÃveis de temperatura e de espaÃamentos sob condiÃÃo de baixa altitude e latitude. No primeiro experimento foram utilizadas sementes de alface das cultivares Red Salad Bowl, Salad Bowl Green, Marianne, Elba, Mimosa Green Salad Bowl, Crespa Lollo Bionda e Crespa para VerÃo, sob diferentes condiÃÃes de temperatura diurna (15; 20; 25; 30; 35 e 40 ÂC). As âRed Salad Bowlâ e âSalad Bowl Greenâ foram as que apresentaram os maiores percentuais de germinaÃÃo, acima de 81%, atà a temperatura de 35 ÂC, sendo assim consideradas termotolerantes. No segundo experimento, estudou se o comportamento produtivo das cultivares que apresentaram os melhores percentuais de germinaÃÃo e emergÃncia no primeiro experimento (Red Salad Bowl, Salad Bowl Green, Mimosa Green Salad Bowl e Crespa Lollo Bionda) em diferentes espaÃamentos. As âSalad Bowl Greenâ e âMimosa Green Salad Bowlâ foram as que se destacaram, jà que apresentaram tolerÃncia mÃdia ao pendoamneto, 63 dias apÃs semeadura (DAS), e florescimento tardio, 95 DAS. A âSalad Bowl Greenâ, no espaÃamento 0,25 x 0,30 m, produziu plantas com maior massa fresca e seca total. O espaÃamento de 0,20 x 0,20 m foi o que possibilitou a maior produtividade para todos os cultivares avaliados, podendo ser considerado como o mais indicado para o cultivo nas condiÃÃes climÃticas de baixa altitude e latitude. / Lettuce is one of the most important leafy vegetables grown in the world. Considered a mild climate culture, its production in low-lying regions and latitude is impaired mainly due to high temperatures and the predominance of long days found in these locations. Under these conditions, germination and low unevenness occurs in the emergency, therefore, there is low efficiency in the production of seedlings. Such events are an indication of the manifestation of a physiological event triggered within the seed dormancy. In lettuce, this phenomenon appears to be controlled by the genotype (cultivar) x environmental factors, and in the latter the temperature has been considered the most important factor. Besides the problems related to the initial stand establishment, either in the field or production nursery seedlings, lettuce subjected to elevated temperatures may also have bolting, resulting in the need for earlier harvesting thus causing a reduction in puduction. Based on the above, the aim of this work was to evaluate the germination behavior of lettuce cultivars under different levels of temperature and spacing on condition of low altitude and latitude. In the first experiment were used lettuce seeds of the cultivars Red Salad Bowl, Salad Bowl Green, Marianne, Elba, Mimosa Green Salad Bowl, Crespa Lollo Bionda and Crespa para VerÃo, under different conditions of day temperature (15; 20; 25; 30; 35 and 40 ÂC). The 'Red Salad Bowl' and 'Green Salad Bowl' were the ones that showed the highest germination rates above 81%, until the temperature of 35 ÂC and therefore were deemed thermotolerant. The second experiment studied the behavior of production of the cultivars that showed the higher resultos of germination percentage and emergence in the first experiment (Red Salad Bowl, Salad Bowl Green, Mimosa Green Salad Bowl and Crespa Lollo Bionda) at different spacings. The ' Salad Bowl Green ' and 'Mimosa Green Salad Bowl' were the ones that stood out, paralleling the average tolerance of bolting, 63 days after sowing (DAS), and late flowering 95 DAS. The 'Green Salad Bowl' cv., spaced 0,25 x 0,30 m, produced plants with increase in weight and total dry. The spacing of 0,20 x 0,20 m was the one that allowed the highest yield for all evaluated cultivars and may be considered as the most suitable for lettuce cultivation in the climatic conditions of low altitude and latitude.

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No. of bitstreams: 2 Dissertação - Lucas Prado Barreto - 2014.pdf: 3040237 bytes, checksum: 9b5b111ad7079b39159b4f2f8968b129 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-10-08 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Resistence of the tick Rhipicephalus sanguineus to chemicals is commonly reported. Entomopathogenic fungi emerge as an alternative to the exclusive use of chemical acaricides for tick control; however, heat and other environmental factors may limit the efficacy of fungi. Thus, the current study evaluated the effect of heat on conidial germination of Metarhizium anisopliae IP 119 when suspended in pure mineral oil (Impex®) or in water (Tween 80, 0.01%) and applied to the cuticle of R. sanguineus (in vivo) or in artificial medium PDAY (in vitro). Then, the results obtained with tests in vivo were compared to the results obtained with tests conducted in vitro, and finally, experiments were conducted to verify the virulence of M. anisopliae IP 119 for R. sanguineus engorged females. Conidial suspensions of M. anisopliae in water or oil, adjusted to 108 conidia ml-1, were exposed to 0 (control) or 4 h to 45 ± 0.5 °C in a water bath, and then, individually inoculated onto the dorsal surface of R. sanguineus engorged females. The samples were incubated at 27 ± 1 °C and RH > 80% for 0, 12, 18, 24, 36, 48 or 72 h. In parallel, aliquots of the aqueous conidial suspensions (control or 45 °C) were inoculated onto PDAY medium in Petri plates. The samples were incubated at the same temperature and for the same time periods described above. After each incubation time, ticks were fixed and dissected, i.e., a fragment of the dorsal cuticle was removed. Cuticles that received conidia suspended in water, exposed or not to heat, were processed for fluorescence microscopy for evaluation of conidial germination. Cuticles that received conidia suspended in water or oil, exposed or not to heat, were processed for scanning electron microscopy for evaluation of conidial germination. Conidial germination on PDAY plates was assessed using a phasecontrast microscope. A minimum of 300 conidia per cuticle or plate was evaluated for calculation of percent germination. When conidia were exposed to heat, a high percentage of germination was observed in PDAY medium (61.5%) in comparison to the tick cuticle (13%), 72 h after inoculation. Appressoria were observed 36 h after inoculation, only in conidia that were not exposed to heat. On tick cuticle, the percentage of germination of conidia suspended in mineral oil and exposed to heat was high in comparison to the percentage of germination of conidia suspended in water, at all incubation times investigated. At 36 h of incubation, the mean percentage of germination of conidia suspended in oil reached 16.3%, whereas conidia suspended in water reached only 2.2%. Conversely, in the control groups (ticks treated with conidia not exposed to heat), the percentage of germination of conidia suspended in water or oil differed at 48 and 72 h of incubation, where germination was also high for conidia suspended in oil and inoculated on the tick cuticle. M. anisopliae conidia suspended in oil exposed or not to heat showed control percentage greater than the observed with aqueous suspensions. Thus, the results showed a significant delay in germination for conidia suspended in water and exposed to 45 °C in comparison to conidia suspended in mineral oil; in this sense, the oil formulation of conidia when exposed or not to heat proved to be more effective for controlling R. sanguineus engorged females. Moreover, conidia suspended in water germinated faster when inoculated in PDAY than when they were applied to the tick cuticle. Therefore, the results indicated that the negative effect of heat on conidial germination was more expressive when conidia were suspended in water and applied to the arthropod cuticle than it would be predicted by the in vitro thermotolerance tests. Also, conidia suffered less interference from heat exposures when suspended in oil. In conclusion, mineral oil protects conidia against the heat effects and enhances the fungal germination on the cuticle of R. sanguineus. / Relatos da resistência do carrapato Rhipicephalus sanguineus a bases químicas utilizadas para controle tem sido comuns. Neste contexto, os fungos entomopatogênicos surgem como uma alternativa ao uso exclusivo do controle químico; entretanto, calor e outros fatores ambientais podem limitar a eficácia dos fungos. Sendo assim, o presente estudo avaliou o efeito do calor sobre a germinação de conídios de Metarhizium anisopliae IP 119 quando suspensos em puro óleo mineral (Impex®) ou em água (Tween 80, 0,01%) e aplicados sobre a cutícula de R. sanguineus (in vivo) ou em meio de cultivo BDAY (in vitro). Em seguida, a resposta dos testes in vivo foi comparada a dos testes in vitro, e bioensaios foram realizados para verificar a virulência do isolado IP 119 para fêmeas ingurgitadas de R. sanguineus. Suspensões conidiais de M. anisopliae em água ou óleo, ajustadas a 108 conídios ml-1, foram expostas a 0 h (controle) ou 4 h a 45 ± 0,5 ºC em banho-maria e, em seguida, inoculadas individualmente na superfície dorsal de fêmeas ingurgitadas de R. sanguineus. Os indivíduos foram incubados a 27 ± 1°C e UR > 80% por 0, 12, 18, 24, 36, 48 ou 72 h. Em paralelo, alíquotas das suspensões conidiais aquosas (controle ou 45ºC) foram inoculadas em placas de Petri contendo meio BDAY. As amostras foram incubadas na mesma temperatura e pelos mesmos períodos de tempo descritos acima. Após cada período de incubação, os carrapatos correspondentes foram fixados e dissecados, retirando-se um fragmento da cutícula dorsal. Cutículas que receberam conídios suspensos em água, e expostos ou não ao calor, foram processadas para microscopia de fluorescência para avaliação da germinação. Cutículas que receberam conídios suspensos em água ou óleo, e expostos ou não ao calor, foram processadas para microscopia eletrônica de varredura para avaliação da germinação. A germinação de conídios em BDAY foi avaliada por microscopia de contraste de fase. Um mínimo de 300 conídios por cutícula ou por placa foi avaliado para calcular o percentual de germinação. Verificou-se que quando conídios foram expostos ao calor, um maior percentual de germinação foi observado em BDAY (61,5%) em comparação com a cutícula do carrapato (13%), 72 h após a inoculação. Apressórios foram observados 36 h após a inoculação, apenas em conídios que não foram expostos ao calor. Na cutícula de R. sanguineus, o percentual de germinação de conídios suspensos em óleo mineral e expostos ao calor foi maior em comparação ao percentual de germinação de conídios suspensos em água, em todos os tempos de incubação investigados. Com 36 h de incubação, a germinação média de conídios suspensos em óleo chegou a 16,3%, enquanto conídios suspensos em água apresentavam 2,2% de germinação. Em contrapartida, nos controles, o percentual de germinação de conídios suspensos em água ou óleo diferiu nos tempos de 48 e 72 h de incubação, onde a germinação sobre a cutícula também foi maior em conídios suspensos em óleo. M. anisopliae quando suspenso em óleo e exposto ou não ao calor apresentou percentual de controle de fêmeas maior que o observado nas suspensões aquosas. Sendo assim, os resultados demonstraram: marcante atraso na germinação dos conídios suspensos em água e expostos a 45 ºC em comparação aos conídios suspensos em óleo mineral; a formulação oleosa de conídios quando exposta ou não ao calor mostrou-se mais efetiva para controle de R. sanguineus. Ainda, conídios suspensos em água germinaram mais rapidamente quando inoculados em BDAY do que quando foram aplicados sobre a cutícula do carrapato. Neste sentido, permite-se inferir que o efeito negativo do calor sobre a germinação de conídios suspensos em água foi maior quando os conídios foram aplicados sobre a cutícula do artrópode do que seria previsto nos testes in vitro, e os conídios sofreram menor interferência do calor quando suspensos em óleo. Portanto, conclui-se que o óleo mineral protege os conídios contra a ação do calor e potencializa a germinação do fungo na cutícula de R. sanguineus.

Fundação de Apoio a Pesquisa e à Inovação Tecnológica do Estado de Sergipe - FAPITEC/SE / The objective of this study was to evaluate the physiology and biochemistry of lettuce seed through germination and expression of the enzymes alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), catalase (CAT), Esterase (EST), pyruvate decarboxylase (PDC) and glutamate oxalacetate transferase (GOT) at different temperatures. Seeds of four lettuce cultivares were submitted to germination and the first germination was tested under the temperatures of 20, 25, 30, 35, 40 and 42°C. The germination speed index (IVG), the percentage of germination were estimated, and the expression of the enzyme alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), catalase (CAT), esterase (EST), pyruvate decarboxylase (PDC) and glutamate oxalacetate transferase (GOT) evaluated. The experimental design was completely randomized, in a 4x6 factorial scheme, with four cultivars and six different temperatures, with four replications. The highest values of germination and vigor were observed for cv. Everglades at 35ºC, proving the thermotolerance. The catalase may be considered a marker for identification of thermotolerant lettuce cultivars. Cultivar Everglades has potential for use in lettuce breeding programs aiming at temperature tolerance during germination. / O objetivo neste trabalho foi avaliar a fisiologia e bioquímica de sementes de alface por meio da germinação e expressão das enzimas álcool desidrogenase (ADH), malato desidrogenase (MDH), catalase (CAT), esterase (EST), piruvato descarboxilase (PDC) e glutamato oxalacetato transferase (GOT) em diferentes temperaturas. Sementes de quatro cultivares de alface foram submetidas aos testes de germinação e primeira contagem nas temperaturas de 20, 25, 30, 35, 40 e 42ºC. Foram calculados o índice de velocidade de germinação (IVG), a porcentagem de germinação, e avaliadas a expressão das enzimas álcool desidrogenase (ADH), malato desidrogenase (MDH), catalase (CAT), esterase (EST), piruvato descarboxilase (PDC) e glutamato oxalacetato transferase (GOT). Utilizou-se o delineamento experimental inteiramente casualizado, em arranjo fatorial 4x6, testando quatro cultivares e seis temperaturas, com quatro repetições. Os maiores valores de germinação e vigor foram observados para a cv. Everglades, à temperatura de 35ºC, comprovando a termotolerância. A enzima catalase pode ser considerada marcadora para identificação de cultivares de alface termotolerantes. A cultivar Everglades tem potencial para utilização em programas de melhoramento de alface com vistas à tolerância a altas temperaturas durante a germinação.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20439号 / 農博第2224号 / 新制||農||1049(附属図書館) / 学位論文||H29||N5060(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 間藤 徹, 教授 喜多 恵子 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The rise of atmospheric CO2 is a threat to many ecosystems, including coral reefs. Rising sea surface temperatures are known to interfere on coral health and cause extensive worldwide mortality. In the Red Sea, coral reefs are spread across 18 degrees of latitude, resulting in a temperature gradient with maximum monthly mean sea surface temperatures ranging from around 33 °C in the south to 27°C in the north. Such gradients can lead to adaptations to local conditions, with coral thermal performance expected to increase along temperature/latitude gradients. In this research, I used the corals Porites lobata and Platygyra daedalea collected in various locations in the Red Sea to resolve fine-scale populational differences in thermotolerance along the Red Sea, and assess if this variability can be explored for potential assisted gene flow projects (i.e. assisted migration and selective breeding) within the Red Sea region. In chapter two, a heat stress assay indicated that P. lobata colonies from the Southern Red Sea have a higher thermal bleaching threshold than conspecifics from Northern latitudes, highlighting their potential as source of temperature resilient colonies. In chapter three, a common garden experimental design showed that thermotolerance of southern P. lobata colonies is maintained when transplanted to a foreign environment. However, despite lack of bleaching in Southern colonies, mortality rates of approximately 20% suggested that other environmental constraints besides temperature might impact coral health and survival. In chapter four, purebred Arabian Gulf larvae generated by cross-breeding colonies of Platygyra daedalea collected along the Red Sea and the Arabian Gulf showed higher survival under heat stress. In addition, paternal survival index increased in crosses with fathers from warmer locations. These results combined indicate that assisted gene flow via translocation alone may have limited success due to a lack of local adaptations to environmental conditions other than temperature. Nonetheless, inter-populational breeding may overcome these limitations as they could generate offspring with both increased thermal tolerance and local adaptations.

O fungo entomopatogênico Isaria fumosorosea (Ascomycota: Hypocreales: Cordycipitaceae) é encontrado comumente nos solos e infectando diversas espécies de artrópodes. Bioprodutos à base de I. fumosorosea são utilizados principalmente na América do Norte e Europa para o controle de mosca-branca, pulgões e trips. Entretanto, no Brasil ainda não existe nenhum produto registrado à base deste patógeno. O objetivo deste trabalho foi estabelecer parâmetros para a seleção de isolados promissores para o desenvolvimento de um biopesticida à base de I. fumosorosea. Os isolados foram avaliados em três etapas sequênciais: esporulação em meio de cultura artificial, fermentação em grão de arroz e tolerância à radiação ultravioleta B e temperatura alta. A partir de uma coleção de 172 isolados de I. fumosorosea provenientes do solo e insetos de diferentes biomas do Brasil, foram escolhidos 72 isolados por apresentar visualmente maior esporulação das colônias em SDYA (Sabouraud-dextrose-extrato de levedura-ágar). Posteriomente, esses isolados foram utilizados para a fermentação sólida em garrafas de vidro contendo 50 gramas de arroz parboilizado. Destes, 14 isolados foram avaliados quanto à produção de conídios em sacos de polipropileno contendo arroz parboilizado e quanto aos efeitos da exposição à radiação ultravioleta B (UV-B) por 0, 2, 4, 6 e 8 horas e temperatura de 45°C por 0, 30, 60 e 90 minutos. Os isolados ESALQ-4556 e ESALQ-4778 apresentaram os maiores rendimentos de conídios em garrafas de vidro (4,65 e 4,40 ×109 conídios/g arroz seco) e em sacos de polipropileno (3,96 e 3,25 ×109 conídios/g arroz seco). Os isolados ESALQ-1296 e ESALQ- 3415 apresentaram maior tolerância aos efeitos da radiação ultravioleta-B e aquecimento. Para a elaboração de uma formulação do tipo dispersão oleosa foi ajustado o ponto de secagem dos conídios para incrementar o tempo de prateleira e avaliadas diferentes misturas do adjuvante KBRAdj e óleos vegetais. Foram utilizados conídios do isolado ESALQ-1296 produzidos em arroz com atividade de água (aw) de 0,527 e conídios secos com aw= 0,410; 0,248 e 0,182 adicionados ao adjuvante KBRAdj puro. A sobrevivência dos conídios mais secos foi maior do que de conídios úmidos. O adjuvante promoveu proteção aos conídios umidos, apresentando viabilidades significativamente maiores do que conídios umidos sem adjuvante em todos os períodos de armazenamento. Conídios com aw= 0,182 sem adjuvante apresentaram as maiores viabilidades (70,3%) após 60 dias de armazenamento. Para desenvolver uma formulação do tipo dispersão oleosa foram testadas misturas de 25% de óleo de soja ou canola + 75% do adjuvante KBRAdj com ou sem sílica gel, em conídios úmidos (aw=0,546 e UR= 13,18%) ou secos (aw=0,175 e UR= 4,42%). A adição de 25% de óleo vegetal ou sílica gel na formulação não contribuiu para o incremento do período de prateleira da formulação. A viabilidade dos conídios nas diferentes formulações após 90 dias de armazenamento variou de 42,3 ± 0,03% a 56,9 ± 0,01%. Estudos futuros deverão ser conduzidos para incrementar o período de prateleira e avaliar a virulência destas formulações para o controle de pragas, especialmente mosca-branca e psilídeo-dos-citros. / The entomopathogenic fungus Isaria fumosorosea (Ascomycota: Hypocreales: Cordycipitaceae) is commonly found in soil and infecting several species of arthropods. I. fumosorosea based bioproducts are mainly used in North America and Europe for the control of whitefly, aphids and thrips. However, in Brazil there is still no registered product based on this pathogen. The objective of this study was to establish parameters for the selection of promising isolates for developing I. fumosorosea based biopesticides. The isolates were evaluated in three sequential steps: sporulation on artificial medium, sporulation on rice grains, tolerance to ultraviolet-B radiation and high temperature. From a collection of 172 I. fumosorosea isolates from soil and insects of different Brazilian biomes, 72 isolates were chosen based on visual observations of higher sporulation of colonies in SDYA (Sabouraud-dextrose- yeast extract-agar). Later, these isolates were used for the solid fermentation in glass bottles containing 50 g of parboiled rice. Of these, 14 strains were evaluated for conidia production in polypropylene bags containing parboiled rice and the effects of exposure to ultraviolet-B (UV-B) for 0, 2, 4, 6 and 8 hours and 45°C for 0, 30, 60 and 90 minutes. The ESALQ-4556 and ESALQ-4778 isolates showed the highest spore yields in glass bottles (4.65 and 4.40 × 109 spores/g dry rice) and in polypropylene bags (3.96 and 3.25 × 109 spores/g dry rice). The ESALQ-1296 and ESALQ-3415 isolates showed a higher tolerance to the UV-B radiation and heating. For the development of oil dispersion formulations, the drying point of the conidia was adjusted to increase the shelf-life in different mixtures of the KBRAdj adjuvant and vegetable oils. Conidia produced on rice from the isolate ESALQ-1296 without drying with water activity (aw) = 0.527 and dried conidia with aw of 0.410; 0.248 and 0.182 added to the pure adjuvant. Survival of the dryer conidia was greater than humid conidia. The adjuvant provided protection to humid conidia, with significantly higher viability than conidia without drying and without adjuvant at all storage periods. Conidia with aw = 0.182 without adjuvant showed the highest viability (70.3%) after 60 days of storage. To develop an oil dispersion type formulation it were tested mixtures with 25% of soy or canola oil + 75% of KBRAdj adjuvant with or without silica gel using humid (aw = 0.546 and RH = 13.18%) or dried conidia (aw = 0.175 and RH = 4.42%). Addition of 25% of vegetable oil or silica gel in the formulation did not contribute to the increase of the shelf-life. The conidia viability in the different formulations after 90 days of storage ranged from 42.3% ± 0.03% to 56.9 ± 0.01. Future studies must be conducted to increase the shelf- life and to assess the virulence of these formulations for pest control, particularly whitefly and the citrus psyllid.