Made available in DSpace on 2014-12-17T14:03:28Z (GMT). No. of bitstreams: 1 LeonardoLPM.pdf: 466570 bytes, checksum: 827ec94d9844ad03ef852080e819058f (MD5) Previous issue date: 2008-09-21 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The fruit fly Ceratitis capitata is considered the most destructive pest of the world fruitculture. Many pest management practices, mainly based on agrochemicals, have been developed to allow the world-wide commerce of fruit. Solutions to decrease the use of synthetic insecticides in agriculture are based on the development of new target-specific compounds which cause less damage to the environment, especially vegetal proteins with insecticidal effects. The aim of this work was to evaluate the deleterious effect of a purified vicilin of E. velutina (EvV) seeds to C. capitata larvae and adult insects and to investigate the mechanisms involved in these effects. EvV was purified, characterized and its deleterious effect was tested in bioassay systems. EvV mechanism of action was determined by immunodetection techniques and fluorescence localization in chitin structures that are present in C. capitata digestory system. EvV is a glycoprotein with affinity to chitin. Its molecular weight, of 216,57 kDa, was determined by gel filtration chromatography in FPLC system. Using SDS-PAGE, it was possible to observe EvV dissociation in two main subunits of 54,8 and 50,8 kDa. When it was submitted to eletrophoresis in native conditions, EvV presented only one band of acid characteristic. The WD50 and LD50 values found in the bioassays were 0,13% and 0,14% (w/w), respectively for the larvae. EvV deleterious effects were related to the binding to chitin structures presented in peritrophic membrane and gut epithelial cells, associated with its low digestibility in C. capitata digestive tract. The results described herein are the first demonstration of the larvicidal effects of plant protein on C. capitata larvae. EvV may be part of the pest management programs, in the toxic bait composition, or an alternative in plant improvement program / A mosca-da-fruta Ceratitis capitata ? considerada a praga mais destrutiva da fruticultura mundial, para o seu controle v?rios programas de erradica??o baseados em agroqu?micos foram criados para permitir o com?rcio mundial de frutas. Solu??es para a diminui??o do uso de inseticidas sint?ticos na agricultura est?o baseadas no desenvolvimento de novos compostos alvos-espec?ficos com menos persist?ncia no meio ambiente, em especial prote?nas vegetais com efeitos inseticidas. Neste trabalho o principal objetivo foi avaliar o efeito delet?rio de uma vicilina purificada de sementes de E. velutina (EvV) para larvas de C. capitata e propor o mecanismo de a??o da prote?na. EvV foi purificada, caracterizada e o seu efeito delet?rio foi testado em sistemas de bioensaios. O mecanismo de a??o de EvV foi determinado por t?cnicas de imunodetec??o e localiza??o por fluoresc?ncia em estruturas quitinosas, presentes no sistema digest?rio de C. capitata. EvV ? uma glicoprote?na com afinidade a quitina cuja massa molecular foi de 216,57 kDa, determinado por cromatografia de gel filtra??o em sistema de FPLC. Por SDS-PAGE, EvV dissociouse em duas subunidades principais de 54,8 e 50,8 kDa, e quando foi submetida a eletroforese em condi??es nativas apresentou uma banda ?nica de caracter?stica eletrofor?tica ?cida. Nos bioensaios a WD50 e a LD50 para as larvas foram de 0,13% e 0,14% (p/p). Os efeitos delet?rios de EvV foram relacionados a sua liga??o a estruturas quitinosas presentes na membrana peritr?fica e no epit?lio intestinal, associado com a baixa capacidade de C. capitata em digeri-la. Com esses resultados ? poss?vel propor a EvV como candidata a fazer parte de programas de manejo integrado de pragas, na composi??o de iscas t?xicas, como um potencial bioinseticida de natureza prot?ica.

Este trabalho teve o objetivo de avaliar, em condições de laboratório, a influência da temperatura e do tempo de exposição na viabilidade e virulência dos fungos Beauveria bassiana e Metarhizium anisopliae em suspensões preparadas com produtos à base de Conídios puros, Arroz+fungo e nas formulações Pó molhável e Suspensão concentrada em óleo emulsionável. Inicialmente foi necessário o desenvolvimento de um método eficiente e econômico para avaliação da viabilidade, especialmente para produtos formulados em óleo emulsionável misturados em água (suspensão de aplicação). Durante o desenvolvimento do método de avaliação foram definidos parâmetros e condições adequadas para cada formulação, como o meio de cultura, e também a influência de antibióticos e fungistáticos. O protocolo estabelecido baseiase resumidamente no plaqueamento de 150 µL de uma suspensão contendo entre 0,7 e 1×106 conídios/mL em placas tipo Rodac® com 5 mL de meio de cultura Batata-Dextrose-Ágar + 5 mg/L de Pentabiótico® e 10 µL/L de Derosal® (Carbendazim), efetuando-se posteriormente a contagem dos conídios germinados. Este método foi comparado com outra técnica de viabilidade direta (VD) e com as técnicas de Unidade Formadoras de Colônia (UFC) e viabilidade por fluorescência, confrontando-se a precisão obtida pelo coeficiente de variação (CV) das análises de cada método. Os resultados demonstraram que além da facilidade de execução, o método desenvolvido apresentou maior precisão que as demais técnicas (com CV até 7 vezes menor em relação à VD, e até 32 vezes menor que UFC). Foi constatado que a técnica de UFC não é adequada para avaliações de produtos formulados, visto que em relação à quantificação inicial subestimou a concentração viável na maioria das formas dos fungos testadas, sendo que nos produtos em óleo emulsionável estes valores foram 54% menores para B. bassiana, e 73% menores para M. anisopliae. A técnica para avaliação da viabilidade da suspensão preparada com produtos formulados em óleo se mostrou eficaz, após a retirada do óleo da suspensão e outras adaptações. Suspensões de produtos e formulações a base de B. bassiana e M. anisopliae foram mantidas a 26°C, 36°C e 46°C por 1h, 4h e 6h e os efeito destes tratamentos foram medidos na viabilidade e virulência dos fungos contra lagartas de Diatraea saccharalis. Verificou-se que quanto maior o tempo de exposição e maior a temperatura, maior é a influência negativa nos parâmetros dos fungos. Entretanto, a formulação em óleo emulsionável foi a que apresentou melhor proteção aos efeitos detrimentais da temperatura. Mesmo após 6h de exposição a temperatura de 36°C a viabilidade se manteve acima dos 85% para ambos os fungos avaliados. Esta formulação também apresentou os melhores resultados de mortalidade dos insetos, e mesmo após 4h de exposição a 46°C atingiu 39% e 50% de eficiência para B. bassiana e M. anisopliae, respectivamente, sendo recomendável para o controle de D. saccharalis. O protocolo para avaliação da viabilidade de conídios de fungos entomopatogênicos proposto constitui-se como uma ferramenta importante para estudos da influência de fatores bióticos e abióticos durante o período de pré-aplicação na eficiência de micoinseticidas. / This study aimed to evaluate, under laboratory conditions, the influence of temperature and exposure time on the viability and virulence of Beauveria bassiana and Metarhizium anisopliae in suspensions prepared with pure conidia, rice plus fungus and in formulations of wettable powder and concentrated suspension in emulsifiable oil. Initially, an efficient and economical methodology was developed to evaluate conidia viability for each formulation, especially for products formulated in emulsifiable oil mixed in water (suspension of application). During the development of the methodology, the influence of culture media, antibiotics and fungistatics and appropriate conditions were defined for each formulation. Briefly, the established protocol was based on plating 150 L of suspensions containing between 0.7 and 1×106 conidia/mL on a plate type Rodac® with 5 ml of Potato Dextrose Agar culture medium plus 5 mg/L pentabiotic® and 10mg/L and 10 µL/L Derosal® (Carbendazim) and the subsequent counting of germinated conidia. This method was compared to a direct counting (DC) technique, the technique of colony-forming unit (CFU) and viability through fluorescence. The precision of each methodology was measured by their coefficient of variability (CV). Besides its easiness of execution, the new developed methodology was the most precise of all methods (with CV up to 7 times lower compared to the DC, and up to 32 times lower than the UFC). Our results showed that the technique of UFC is not appropriate for evaluation of formulated products, as compared to the conidial quantification by hemacytometer, this technique underestimated the viable concentration in most forms of fungi tested, and for the products in emulsifiable oil these values were 54% lower for B. bassiana, and up to 73% lower for M. anisopliae. The technique for assessing the viability of the suspension prepared with products formulated in emulsifiable oil was effective, after the removal of oil from the suspension and others adaptations. The effect of temperature and period of time on conidia viability of B. bassiana and M. anisopliae-based products and formulations and their virulence against larvae of Diatraea saccharalis was measured at 26°C, 36°C and 46°C for 1h, 4h and 6h. The longer the time of heat exposure and the higher the temperature, the greater the negative influence on vital parameters of the fungi. However, the emulsifiable oil formulation was the least affected by the detrimental effects of heat for both fungi. Even after 6 hours of exposure to temperature of 36°C, the viability remained above 85% for both species evaluated. This formulation also showed the best results of insect mortality, and even after 4h of exposure to 46°C the efficiency was 39% (for B. bassiana) and 50% (for M. anisopliae), and therefore can be useful for control of D. saccharalis. The proposed protocol of evaluation of entomopathogenic fungi viability is a valuable tool for studies of biotic and abiotic factors effect on efficiency of micoinsecticides during the pre-application period.

Os fungos entomopatogênicos do gênero Metarhizium e Beauveria compreendem um importante grupo de patógenos de artrópodes-praga. A seleção de isolados de fungos promissores é a primeira e uma das mais importantes etapas no desenvolvimento de um biopesticida, pois alguns isolados podem apresentar alta virulência e não necessariamente boa produção em substrato e vice-versa, sendo interessante a combinação desses dois parâmetros para a viabilidade comercial de um produto. A dificuldade de criação ou manutenção de algumas espécies de pragas em laboratório é um limitante para a condução de testes de virulência, tornando-se interessante a utilização de espécies modelo, de fácil criação, nas etapas preliminares de seleção. Nesse sentido, este estudo objetivou selecionar isolados com alta produção de conídios e virulência, comparando a eficiência de controle de M. anisopliae e B. bassiana às pragas alvo Mahanarva fimbriolata e Bemisia tabaci Biótipo B, respectivamente, com a mortalidade em Tenebrio molitor. Inicialmente, foram selecionados 50 isolados a partir de 100 isolados de cada gênero, baseado em avaliações visuais do crescimento e esporulação em meio de cultivo em placas de Petri. Estes isolados selecionados foram cultivados em arroz parboilizado para quantificação do rendimento produtivo de conídios. Os 25 isolados mais produtivos de cada espécie de fungo foram utilizados nos bioensaios com T. molitor. Posteriormente, os cinco isolados que causaram maior e menor mortalidade de cada gênero, foram utilizados nos bioensaios com as respectivas pragas-alvo. A variação no rendimento de conídios de Beauveria spp., foi de 0,3 a 7,7 x 109 conídios.grama de arroz-1 e de Metarhizium spp. foi de 0,1 a 2,5 x 109 conídios.grama de arroz-1. A mortalidade confirmada de larvas de T. molitor por Beauveria spp., variou de 5,5 a 96,4% e por M. anisopliae variou de 29,1 a 89,1%. Alguns isolados causaram mortalidade elevada tanto no inseto modelo quanto na praga-alvo, porém, não foi verificada uma relação entre a virulência para as duas espécies. Da mesma forma, não foi observada associação entre os parâmetros produção de conídios e virulência. O isolado ESALQ 4958 de B. bassiana se destacou nos dois bioensaios apresentando mortalidades elevadas de ninfas de B. tabaci Biótipo B. Nos bioensaios utilizando ninfas de M. fimbriolata, ESALQ 1641 foi o isolado que apresentou os maiores percentuais de mortalidade nos dois bioensaios. Analisando conjuntamente as variáveis produção de conídios e virulência a T. molitor e a espécie alvo, os isolados ESALQ 540 (B. bassiana) e ESALQ 1116 (M. anisopliae) se destacaram por apresentarem valores elevados para todas as variáveis de interesse. Os resultados reforçam a necessidade de uma análise conjunta destas variáveis com um peso diferenciado para cada variável na seleção de isolados para utilização em produtos microbianos para o controle de pragas. / The genus Metarhizium spp. and Beauveria spp. are important entomopathogenic fungi used to control arthropod pests. The selection of promising fungal isolates is the first and one of the most important steps on the development of a biopesticide, since some isolates may present high virulence and not necessarily good production in substrate and vice-versa, being the combination of these two parameters important for the commercial viability. Difficulties of rearing or maintaining some species of pests in laboratory are limitations for the conduction of virulence tests, justifying the use of easy to breed model species on the preliminary steps of selection. Therefore, this study aimed to select isolates with high conidia production and virulence, comparing the control efficiency of M. anisopliae and B. bassiana to the target pests, Mahanarva fimbriolata and Bemisia tabaci biotype B, respectively, with mortality in Tenebrio molitor. At first, 50 isolates were selected from 100 isolates of each genus, based on growth and sporulation in culture medium on Petri dishes. These isolates were grown in parboiled rice to quantify the yield of conidia. The 25 most productive isolates of each fungus species were used in the bioassays with T. molitor. After, the five isolates that caused higher and lower mortality of each genus were used in the bioassays with the respective target pests. Beauveria spp. conidia yield ranged from 0.3 to 7.7 x 109 conidia.grams of rice-1 and Metarhizium spp. from 0.1 to 2.5 x 109 conidia.gram of rice-1. The confirmed mortality of T. molitor larvae by Beauveria spp. varied from 5.5 to 96.4% and M. anisopliae varied from 29.1 to 89.1%. Some isolates caused high mortality in both, model insect and the target pest; however, no relationship between the virulence of both species was observed. Similarly, there was no association between the parameters conidia production and virulence. The B. bassiana isolate ESALQ 4958 in both bioassays presented high mortalities of B. tabaci Biotype B. In bioassays using M. fimbriolata nymphs, ESALQ 1641 was the isolate that presented the highest mortalities in both bioassays. Analyzing the variables, conidia production and virulence to T. molitor and the target species, the isolates ESALQ 540 (B. bassiana) and ESALQ 1116 (M. anisopliae) showed high values for all variables of interest. The results reinforce the necessity of a joint analysis of these variables with different weight for each one in the selection of isolates, aiming to use them in microbial products for pest control.

Orientadores: Susanne Rath, Sonia Claudia do Nascimento de Queiroz / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-27T01:49:14Z (GMT). No. of bitstreams: 1 Porto_RafaelSilveira_M.pdf: 1983268 bytes, checksum: f89fcb853911e559df9889c577be5a8e (MD5) Previous issue date: 2015 / Resumo: O Brasil é um dos maiores produtores de frutas do mundo, no entanto, estima-se que doenças pós-colheita possam gerar perdas de até 50% em sua produção. A forma mais comum de tratamento para essas doenças envolve a aplicação de fungicidas sintéticos. Contudo, nos últimos anos, a demanda por tratamentos alternativos tem crescido, com destaque para o uso de biopesticidas, produtos desenvolvidos a partir de plantas, microrganismos e insetos. Este trabalho teve como objetivo investigar a presença dos compostos bioativos (4Z)-lachnophyllum lactona, (4Z,8Z)-matricaria lactona e (2Z,8Z)-matricaria ester nos espécimes brasileiros da planta Conyza canadensis, bem como avaliar a atividade antifúngica dessas substâncias isoladas contra diversos fungos associados a doenças pós-colheita de frutas. Por cromatografia flash preparativa foi possível isolar a (4Z)-lachnophyllum lactona e a (4Z,8Z)-matricaria lactona a partir de extratos da planta obtidos com diclorometano. Os compostos foram caracterizados por GC-MS/MS, NMR 1H e 13C, 1H-1H COSY e 1H-13C HSQC. Foram realizados ensaios de difusão em disco com 10 fungos filamentosos causadores de doenças pós-colheita em frutas. Os fungos Aspergillus niger, Cladosporium spp. e Penicillium digitatum se mostraram susceptíveis ao tratamento e, para eles, a concentração mínima inibitória dos compostos variou de 32 a 64 µg mL-1. Também foi desenvolvido um método de extração empregando água quente pressurizada, no qual foram otimizados os parâmetros de temperatura (100 °C), tempo de ciclo (1 min) e número de ciclos (quatro). Com essa técnica foi possível obter um rendimento de 1,46 mg g-1 e 0,24 mg g-1 para a (4Z)-lachnophyllum lactona e a (4Z,8Z)-matricaria lactona, respectivamente. O extrato aquoso da Conyza canadensis pode ser aplicado diretamente nos frutos com a vantagem de não conter resíduos de solventes orgânicos tóxicos / Abstract: razil is one of the largest fruit producers in the world. Nevertheless, it is estimated that postharvest diseases can lead to losses of up to 50% in its production. The most common treatment for these diseases involves the application of synthetic fungicides. Nonetheless, in recent years, the demand for alternative treatments has increased, especially for the use of biopesticides, products developed from plants, microorganisms and insects. This study aimed to investigate the presence of the bioactive compounds (4Z)-lachnophyllum lactone, (4Z,8Z)-matricaria lactone and (2Z,8Z)-matricaria ester in Brazilian specimens of the weed Conyza canadensis, as well as to evaluate the antifungal activity of these isolated substances against several fungi associated with postharvest diseases of fruits. With the use of preparative flash chromatography it was possible to isolate (4Z)-lachnophyllum lactone and (4Z,8Z)-matricaria lactone from plant extracts obtained with dichloromethane. The compounds were characterized by GC-MS/MS, NMR 1H e 13C, 1H-1H COSY and 1H-13C HSQC. Disk diffusion assays were performed in order to investigate the activity of the isolated compounds against 10 filamentous fungi regarded as common postharvest pathogens of fruits. Aspergillus niger, Cladosporium spp. and Penicillium digitatum proved susceptible to the treatment and, for them, the minimum inhibitory concentration of the compounds varied from 32 to 64 µg mL-1. An extraction method using pressurized hot water was also developed, in which the parameters of temperature (100 ° C), cycle time (1 min) and number of cycles (four) were optimized. By using this technique, it was possible to obtain a yield of 1.46 mg g-1 and 0.24 mg g-1 for the (4Z)-lachnophyllum lactone and (4Z,8Z)-matricaria lactone, respectively. The aqueous extract of Conyza canadensis can be applied directly on fruits with the advantage of not containing residues of toxic organic solvents / Mestrado / Quimica Analitica / Mestre em Química

Este trabalho teve o objetivo de avaliar, em condições de laboratório, a influência da temperatura e do tempo de exposição na viabilidade e virulência dos fungos Beauveria bassiana e Metarhizium anisopliae em suspensões preparadas com produtos à base de Conídios puros, Arroz+fungo e nas formulações Pó molhável e Suspensão concentrada em óleo emulsionável. Inicialmente foi necessário o desenvolvimento de um método eficiente e econômico para avaliação da viabilidade, especialmente para produtos formulados em óleo emulsionável misturados em água (suspensão de aplicação). Durante o desenvolvimento do método de avaliação foram definidos parâmetros e condições adequadas para cada formulação, como o meio de cultura, e também a influência de antibióticos e fungistáticos. O protocolo estabelecido baseiase resumidamente no plaqueamento de 150 µL de uma suspensão contendo entre 0,7 e 1×106 conídios/mL em placas tipo Rodac® com 5 mL de meio de cultura Batata-Dextrose-Ágar + 5 mg/L de Pentabiótico® e 10 µL/L de Derosal® (Carbendazim), efetuando-se posteriormente a contagem dos conídios germinados. Este método foi comparado com outra técnica de viabilidade direta (VD) e com as técnicas de Unidade Formadoras de Colônia (UFC) e viabilidade por fluorescência, confrontando-se a precisão obtida pelo coeficiente de variação (CV) das análises de cada método. Os resultados demonstraram que além da facilidade de execução, o método desenvolvido apresentou maior precisão que as demais técnicas (com CV até 7 vezes menor em relação à VD, e até 32 vezes menor que UFC). Foi constatado que a técnica de UFC não é adequada para avaliações de produtos formulados, visto que em relação à quantificação inicial subestimou a concentração viável na maioria das formas dos fungos testadas, sendo que nos produtos em óleo emulsionável estes valores foram 54% menores para B. bassiana, e 73% menores para M. anisopliae. A técnica para avaliação da viabilidade da suspensão preparada com produtos formulados em óleo se mostrou eficaz, após a retirada do óleo da suspensão e outras adaptações. Suspensões de produtos e formulações a base de B. bassiana e M. anisopliae foram mantidas a 26°C, 36°C e 46°C por 1h, 4h e 6h e os efeito destes tratamentos foram medidos na viabilidade e virulência dos fungos contra lagartas de Diatraea saccharalis. Verificou-se que quanto maior o tempo de exposição e maior a temperatura, maior é a influência negativa nos parâmetros dos fungos. Entretanto, a formulação em óleo emulsionável foi a que apresentou melhor proteção aos efeitos detrimentais da temperatura. Mesmo após 6h de exposição a temperatura de 36°C a viabilidade se manteve acima dos 85% para ambos os fungos avaliados. Esta formulação também apresentou os melhores resultados de mortalidade dos insetos, e mesmo após 4h de exposição a 46°C atingiu 39% e 50% de eficiência para B. bassiana e M. anisopliae, respectivamente, sendo recomendável para o controle de D. saccharalis. O protocolo para avaliação da viabilidade de conídios de fungos entomopatogênicos proposto constitui-se como uma ferramenta importante para estudos da influência de fatores bióticos e abióticos durante o período de pré-aplicação na eficiência de micoinseticidas. / This study aimed to evaluate, under laboratory conditions, the influence of temperature and exposure time on the viability and virulence of Beauveria bassiana and Metarhizium anisopliae in suspensions prepared with pure conidia, rice plus fungus and in formulations of wettable powder and concentrated suspension in emulsifiable oil. Initially, an efficient and economical methodology was developed to evaluate conidia viability for each formulation, especially for products formulated in emulsifiable oil mixed in water (suspension of application). During the development of the methodology, the influence of culture media, antibiotics and fungistatics and appropriate conditions were defined for each formulation. Briefly, the established protocol was based on plating 150 L of suspensions containing between 0.7 and 1×106 conidia/mL on a plate type Rodac® with 5 ml of Potato Dextrose Agar culture medium plus 5 mg/L pentabiotic® and 10mg/L and 10 µL/L Derosal® (Carbendazim) and the subsequent counting of germinated conidia. This method was compared to a direct counting (DC) technique, the technique of colony-forming unit (CFU) and viability through fluorescence. The precision of each methodology was measured by their coefficient of variability (CV). Besides its easiness of execution, the new developed methodology was the most precise of all methods (with CV up to 7 times lower compared to the DC, and up to 32 times lower than the UFC). Our results showed that the technique of UFC is not appropriate for evaluation of formulated products, as compared to the conidial quantification by hemacytometer, this technique underestimated the viable concentration in most forms of fungi tested, and for the products in emulsifiable oil these values were 54% lower for B. bassiana, and up to 73% lower for M. anisopliae. The technique for assessing the viability of the suspension prepared with products formulated in emulsifiable oil was effective, after the removal of oil from the suspension and others adaptations. The effect of temperature and period of time on conidia viability of B. bassiana and M. anisopliae-based products and formulations and their virulence against larvae of Diatraea saccharalis was measured at 26°C, 36°C and 46°C for 1h, 4h and 6h. The longer the time of heat exposure and the higher the temperature, the greater the negative influence on vital parameters of the fungi. However, the emulsifiable oil formulation was the least affected by the detrimental effects of heat for both fungi. Even after 6 hours of exposure to temperature of 36°C, the viability remained above 85% for both species evaluated. This formulation also showed the best results of insect mortality, and even after 4h of exposure to 46°C the efficiency was 39% (for B. bassiana) and 50% (for M. anisopliae), and therefore can be useful for control of D. saccharalis. The proposed protocol of evaluation of entomopathogenic fungi viability is a valuable tool for studies of biotic and abiotic factors effect on efficiency of micoinsecticides during the pre-application period.

No description available.

Bacteria are ubiquitously present in every ecosystem on earth. While bacterial communities that reside in specific habitats, called the microbiota, have characteristic compositions, their constituents are exchanged between habitats. To understand the assembly processes and function of a microbial community in an ecosystem, it is thus important to identify its putative sources and sinks. The sources and sinks of the plant leaf microbiome, also called the phyllosphere microbiome, are still under debate. Here, I hypothesized that precipitation is a so far neglected source of the phyllosphere microbiome. Using 16S rRNA amplicon and metagenomic sequencing, I identified the genera Massilia, Sphingomonas, Methylobacterium, Pseudomonas, Acidiphilium, and Pantoea as members of the core rain microbiome in Blacksburg, VA. Further, I used rainwater as a bacterial inoculum to treat tomato plants. I showed that rain-borne bacteria of the genera Chryseobacterium, Enterobacter, Pantoea, Paenibacillus, Duganella, Streptomyces, Massilia, Shinella, Janthinobacterium, Erwinia, and Hyphomicrobium were significantly more abundant in the tomato phyllosphere 7 days post-inoculation, suggesting that these rain-borne bacteria successfully colonized the tomato phyllosphere and had a direct impact on the composition of its microbiome. These results were confirmed by comparing the phyllosphere microbiota of tomato plants grown under greenhouse conditions, and thus never exposed to rain, compared to plants grown outside under environmental conditions, including precipitation. Since a large diversity of bacteria is associated with rain, I also hypothesized that rain-borne bacteria are well adapted to environmental stresses, similar to the stressors microbial biopesticides are exposed to in the field. I thus explored rain as a source of resilient biopesticides to control fire blight, caused by the bacterial pathogen Erwinia amylovora, on apple. In an in-vitro dual culture assay, I identified rain-borne isolates displaying broad-range inhibition against E. amylovora and several other plant pathogens. Two rain-borne isolates, identified as Pantoea agglomerans and P. ananatis, showed the strongest inhibition of E. amylovora. Further experiments showed that these two Pantoea isolates survive under environmental conditions and have a strong protective effect against E. amylovora. However, protection from disease in an orchard was inconsistent, suggesting that the timing of application and formulations must be improved for field applications. Using a UV-mutagenesis screen and whole-genome sequencing, I found that a phenazine antibiotic produced by the P. agglomerans isolate was the likely active molecule that inhibited E. amylovora. Bacterial communities are constantly released as aerosols into the atmosphere from plant, soil, and aquatic sources. When in the atmosphere, bacteria may play crucial roles in geochemical processes, including the formation of precipitation. To understand the potential role of decaying vegetation as a source of atmospheric Ice Nucleation Particles (INPs), I analyzed a historic leaf litter sample collected in 1970 that had maintained Ice Nucleation Activity (INA) for 48 years. A culture-dependent analysis identified the bacterial species Pantoea ananatis and the fungal species Mortierella alpina to have INA and to be present in the leaf litter sample. Further, I determined that both P. ananatis and M. alpina produced heat-sensitive sub-micron INPs that may contribute to atmospheric INPs. The development of new sequencing technologies has facilitated our understanding of microbial community composition, assembly, and function. Most research in bacterial community composition is based on the sequencing of a single region of the 16S rRNA gene. Here, I tested the potential of culture-independent 16S rRNA sequencing of the phyllosphere microbiome for disease diagnosis. I compared the community composition of the microbiome of the aerial parts of cheddar pinks (Dianthus gratianopolitanus) that showed disease symptoms with the microbiome of healthy plants to identify the causative agent. However, I found that the pathogen is probably ubiquitous on cheddar pinks since it was present at similar abundance levels in symptomatic as well as healthy plants. Moreover, the low-resolution of 16S rRNA sequencing did not allow to identify the pathogen at the species or strain level. In summary, in this thesis, I found support for the hypothesis that rain is one of the sources of the phyllosphere microbiome, that rain is a promising source of biopesticides to control plant diseases in the field, that leaf litter is a source of atmospheric INPs, and that 16S rRNA sequencing is not well suited for pathogen identification in support of plant disease diagnosis. Finally, in additional research to which I contributed but that is not included in this thesis, I found that metagenomic sequencing can identify pathogens at the species and strain level and can overcome the limitations of 16S rRNA sequencing. / Doctor of Philosophy / Bacteria are present in nearly every ecosystem on earth. Bacterial communities that reside in a specific habitat are known as microbiota and have characteristic compositions and functions that directly impact the health of ecosystems. Microbiota associated with plants, the so-called plant microbiota, play a crucial role in plant fitness. Thus, it is important to study the assembly and diversity of plant microbiota and their impact on the ecosystem. The sources of leaf microbiota remain to be elucidated. Here, I have studied the contribution of rainfall to the bacteria that live on and in plant leaves. First, using DNA sequencing, I identified the bacteria present in rainfall in Blacksburg, VA. Then, using rain as bacterial inoculum, I found that some rain-borne bacteria, including members of the genera Pantoea, Massilia, Janthinobacterium, and Enterobacter, are efficient colonizers of tomato leaves. Either absence or low abundance of rain-borne bacteria from tomato leaves never exposed to rainfall confirmed further that bacteria in rain contribute to the assembly of plant leaf microbiota. The identification of all putative sources and sinks of leaf microbiota is important when trying to manipulate them to improve plant health and crop yield. Since I found that rainfall contains many different bacteria, I also studied the potential application of rain-borne bacteria in agriculture. The main limitations of commercial bio-pesticides are their poor survival and limited efficacy in the field. Here, I speculated that rain-borne bacteria are well adapted to environmental stressors and could represent efficient bio-pesticides under field conditions. In fact, I isolated two rain-borne bacteria from the genus Pantoea that strongly inhibited Erwinia amylovora, the causal agent of the fire blight disease of apple, in the laboratory under controlled conditions. However, I observed inconsistent results in a 2-year field trial in an orchard. Using mutagenesis and DNA sequencing, I found the active molecule that likely inhibited E. amylovora, in one of the rain-borne isolates. Finally, the access to newer and cheaper sequencing technologies has recently facilitated the study of bacteria at large scale. Most research of microbiota is based on the sequencing of a single region of one gene, the 16S rRNA gene. Here, I tested the potential of 16S rRNA sequencing of leaf microbiota for disease diagnosis. However, I identified the pathogen in healthy and diseased plants, suggesting its ubiquitous presence. Further, due to the low-resolution of 16S rRNA sequencing, it was impossible to identify the pathogen at the species level. In summary, I found that rain is a source that contributes to leaf microbiota, that rain is a promising source of bio-pesticides to control plant diseases, and that 16S rRNA sequencing is not recommended as a tool to diagnose plant diseases.

Jatropha curcas L. est une espèce trouvée dans presque toutes les régions tropicales. Ces graines contiennent de l’huile qui convient à la production de biodiesel et par conséquent, elle a reçu un intérêt mondial en tant que source de biocarburant. Cependant, à ce jour Jatropha curcas n’a pas atteint son plein potentiel. Outre son utilisation pour la production de biodiesel, le développement d’autres sous-produits de graines de Jatropha comme la cire s’ajoute à la valeur économique de cette plante. Cependant, aucune étude détaillée n’a été apportée sur le rendement en cire de graines de Jatropha et ses utilisations dans la production de biopesticide. La présente étude a révélé que le rendement en cire de graines de Jatropha par les méthodes d’extraction utilisant le n-hexane, l’eau bouillante et l’azote liquide, était de 0,11%, 0,03% et 0,02%, respectivement. L’analyse des thermo grammes DSC par calorimétrie différentielle à balayage montre que la cire extraite par l’hexane est plus complexe indiquant la présence de plusieurs composés différents, comparée aux cires obtenues par les deux autres méthodes. Ceci est en accord avec les résultats obtenus au microscope électronique par balayage qui montrent moins de cire sur la surface et l’intérieur de graines de Jatropha traitées à l’hexane par rapport aux deux autres solvants. Le test biologique exploratoire effectué a révélé que les extraits cireux devraient posséder une certaine propriété biocide. Ils induisaient 100 % de mortalité des larves de stade 4 de Choristoneura fumiferana à une concentration de 20%. / Jatropha curcas L. is a species found in almost all tropical regions. The seeds of Jatropha curcas contain oil that is suitable for biodiesel production and therefore has received global interest as a source of biofuel. However, so far Jatropha curcas has not reached its full potential. In addition to its use for biodiesel production, the development of other Jatropha seed by-products such as wax will add to the economic value of this plant. No detailed studies have been conducted on Jatropha seed wax performance and its uses in biopesticide production. The present study revealed that the wax yield of Jatropha seeds was 0.11%; 0.03% and 0.02% respectively for extraction methods using n-hexane, boiling water and liquid nitrogen. Differential scanning calorimetry analysis of the DSC thermograms shows that wax extracted with the hexane is more complex indicating the presence of several different compounds, compared to the waxes obtained by the other two methods. This is in agreement with the scanning electron microscope results which show less wax on the surface of hexane-treated Jatropha seeds compared to the other two solvents. The exploratory biological test carried out revealed that the waxy extracts should have some biocidal property. They induced 100% mortality of stage 4 Choristoneura fumiferana larvae at a concentration of 20%.

La mosca mediterránea de la fruta, Ceratitis capitata, es la principal plaga de la fruticultura en el mundo. El desarrollo de métodos ambientalmente seguros de control de plagas, como Bacillus thuringiensis (Bt), adquiere cada vez mayor importancia. Esta tesis doctoral evalúa la validez de Bt y sus delta-endotoxinas como agentes de control de C. capitata. El análisis de la biodiversidad de Bt ha reflejado la gran riqueza presente en el agroecosistema de los cítricos. Sin embargo, ninguna de las cepas ensayadas (905) muestra alta toxicidad sobre C. capitata cuando esporas/cristales o sobrenadantes de su cultivo son ensayados. En cambio, la solubilización de los cristales de una selección de 42 cepas de Bacillus sp. ha demostrado que, para las cepas de la subespecie israelensis (Bti), este tratamiento produce una ganancia de función biológica. Adicionalmente, la predigestión de dichas protoxinas con proteasas de otro díptero, Culex pipiens, aumenta su actividad larvicida (CL50 31.26 µg/cm2). Por otro lado, se ha demostrado que, entre las delta-endotoxinas producidas por Bti, la protoxina Cyt1Aa es el factor determinante, con una CL50 sobre larvas de 4.93 µg/cm2. Sobre adultos, Cyt1Aa produce efectos subletales. Complementariamente, esta tesis propone un nuevo método de control basado en el desarrollo de toxinas recombinantes de fusión Cry-anticuerpo. Se ha puesto en práctica un sistema modelo para evaluar esta estrategia: se han desarrollado 4 variantes proteicas por la fusión entre partes de la protoxina Cry1Ab y un anticuerpo específico contra GFP (VHH anti-GFP) y éstas se han ensayado sobre larvas transgénicas de Drosophila melanogaster que expresan GFP en su intestino. Deficiencias en la unión de las toxinas de fusión a GFP, han impedido demostrar, por el momento, la estrategia propuesta. Por último, se han detectado al menos 160 proteínas distintas en las membranas intestinales de C. capitata y se han identificado las siguientes: V-ATPasa subunidades A y B, y alfa-tubulina. / Vidal Quist, JC. (2010). Estrategias para la utilización de la bacteria entomopatógena Bacillus thuringiensis (Berliner) en el control de Ceratitis capitata (Wiedemann) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/8336 / Palancia

Parmi les méthodes alternatives respectueuses de l'environnement pour la lutte contre les ravageurs et la protection des cultures agricoles, les bioinsecticides à base de Bacillus thuringiensis sont les plus largement utilisés. L’activité insecticide de B.thuringiensis réside dans la production d’inclusions cristallines protéiques appelées δ-endotoxines qui sont formées au cours de la sporulation. En préalable à ces travaux, de nouvelles souches de B.thuringiensis ont été isolées du sol libanais et ont montré un potentiel insecticide élevé, supérieur à des souches classiquement utilisées. Dans le cadre du développement d'une production industrielle, au Liban, de biopesticides à base de ces souches de B.thuringiensis, ce travail a visé à optimiser la production des δ-endotoxines afin d'augmenter les rendements et de réduire les coûts de production. A l'échelle industrielle, le milieu de culture des bactéries représente une part importante du coût de production. Ainsi, l'effet, sur la concentration des δ-endotoxines, de substitution des sources synthétiques coûteuses d'azote, de carbone et de minéraux, par un sous-produit de la minoterie de blé (le son de blé) a été étudié. L’efficacité de ce sous-produit agro-industriel comme source complète de nutriments, disponible localement à faible prix, a été prouvée pour des fermentations submergées de deux souches différentes toxiques contre des diptères ou des lépidoptères. La comparaison du milieu son de blé à d’autres milieux (synthétiques ou non), classiquement utilisés en bibliographie, a montré que ce milieu présente de nombreux avantages en termes de productivité, de rendement relatif des δ-endotoxines et de coût. Parallèlement à ce travail, une méthode simple et rapide de purification des cristaux a été développée de façon à obtenir des cristaux protéiques purs à 99 % nécessaires comme standards pour le dosage des cristaux dans le milieu de fermentation. Un autre objectif du travail a été d’analyser l'effet de l'aération sur la cinétique de croissance et de sporulation d'une nouvelle souche de B.thuringiensis var kurstaki. Les différentes conditions d’aération testées en fermenteurs de 2 L, caractérisées par le coefficient volumétrique de transfert de dioxygène (KLa), ont permis de montrer l’importance de ce paramètre et de l’optimiser pour obtenir la meilleure productivité en biopesticides. Le meilleur rendement en protéines de toxines a été obtenu dans un milieu son de blé 6 % (masse/volume) dans des conditions d’aération correspondant à un KLa de 65,5 h-1 alors que la meilleure productivité des protéines de toxines a été atteinte dans le milieu 9 % pour un KLa de 102 h-1. Sur la base de cette étude, le dimensionnement de la production de B.thuringiensis à l’échelle 1000 L a été réalisé. L’unité de production a été construite et les premiers essais réalisés ont permis de produire de façon satisfaisante des lots de biopesticides. L’ajustement des protocoles industriels reste à faire. / Among the safe and environment friendly methods for the fight against pests and agricultural crops protection, Bacillus thuringiensis based biopesticides are the most widely used. The insecticidal activity of B. thuringiensis resides in the production of protein crystal inclusions called δ-endotoxins, which are formed during sporulation. Preliminary to this work, new B. thuringiensis strains were isolated from Lebanese soil and showed higher insecticidal potential than the conventionally used strains. As part of the development of an industrial production, in Lebanon, of biopesticides based on these strains of B.thuringiensis, this work aimed to optimize the production of δ-endotoxins to increase yields and reduce the cost of mass production. At industrial scale, the bacteria culture medium represents a significant part of the production cost. Thus, the effect on the concentration of the δ-endotoxins, of substituting expensive synthetic sources of nitrogen, carbon, and minerals, with a byproduct of wheat mill (wheat bran) was studied. The effectiveness of this agro-industrial by-product available locally at low prices, as complete source of nutrients, in submerged fermentations of two different strains of B.thuringiensis toxic against Lepidoptera and Diptera, has been proven. The comparison of the wheat bran medium to other mediums (synthetic or not), conventionally used in the bibliography, has shown that this medium this medium has many advantages in terms of productivity, relative yield of δ-endotoxins and cost. In parallel to this work, a simple and quick method of crystal purification was developed to obtain pure protein crystals to 99 % required as standards for the determination of crystals concentration in the fermentation medium. Another objective of the study was to analyze the effect of aeration on growth and sporulation kinetics of a new strain of B.thuringiensis var kurstaki. The different aeration conditions tested in fermenters of 2 L, characterized by the volumetric oxygen transfer coefficient (KLa), permitted to show the importance of this parameter and to optimize it to get the best biopesticides productivity. The best yield of toxin proteins relative to the wheat bran ratio in the culture medium was obtained in the 6 % (weight/volume) medium in aeration conditions corresponding to a KLa of 65.5 h-1, while the highest productivity of toxin proteins has been reached in the 9 % medium for KLa of 102 h-1. On this study basis, the scale up of B. thuringiensis production was performed in a bioreactor of 1000 L. The production unit was built and the first tests carried out have produced satisfactory batches of biopesticides. Industrial protocols are still to be adjusted.