Made available in DSpace on 2016-06-02T20:34:35Z (GMT). No. of bitstreams: 1 4393.pdf: 23560579 bytes, checksum: 0869152d96e7734e1dd6786fa23cb550 (MD5) Previous issue date: 2012-05-03 / Universidade Federal de Sao Carlos / The study of phytopathogenic fungi Alternaria alternata has improved the chemical of this micro-organism. The fungus was grown up in large scale incubated in rice medium for 26 days. After grown period was interrupted an extract of the microorganism was made and fractionated using several chromatographic techniques, obtaining 11 different classes of metabolites which are: alternariol monomethyl éter, alternariol, altertoxin, altenuen, altenusin, uridine, uracila, 5 -inosil, ergosterol, peróxido de ergosterol e triglycerides. Also, a study was made via LC-MS in order to identify some toxins produced by A. alternata like a pathogen of citrus, and it was possible to identify four of these. Other research via LC-MS was the study of co-cultivation of the fungus A. alternata, the causal agent of alternaria brown spot, and Guignardia citricarpa, the causal agent of citrus black spot. In this study was possible to observe the variation in concentration of alternariol and thenuazonic acid, mycotoxins produced by A. alternata when this fungus is in contact with G. citricarpa. The study identification of the volatiles produced by A. Alternaria in different ways contributed significantly to improve the knowledge of their chemistry. Once, the A. alternata is a pathogenic fungus of 'Murcott' a study was performed via scanning electron microscopy (ESEM). Thus it was possible to observe the penetration of this fungus by stomatal cavity of young leaves of citrus. In order to search for natural inhibitors to control the disease, were tested several classes of natural products against the A. alternata, and some alkaloids, a xxvi coumarin, a xanthyletin, and a flavonoid substances showed Higher inhibition of mycelial growth, spore germination and appressorium formation. With the aim to obtain information on natural inhibitors were tested metabolites produced by A. alternata opposite and G. citricarpa, the results indicated that alternariol monomethyl ether and alternariol were substances that had a potential fungistatic against the causal agent of citrus black spot. / O estudo do fungo fitopatógeno Alternaria alternata contribuiu com a química desse micro-organismo. O fungo foi cultivado em larga escala utilizando como meio de cultura arroz por um período de 26 dias. Após esse período o crescimento foi interrompido e foi feito um extrato do microorganismo o qual foi fracionado utilizando várias técnicas cromatográficas obtendo 11 metabólitos de distintas classes sendo eles: alternariol monometil éter, alternariol, altertoxina, altenueno, altenusina, uridina, uracila, 5 -inosil, ergosterol, peróxido de ergosterol e triglicerídeo. Além disso, foi feito um estudo via LC-MS buscando identificar algumas toxinas de citros produzidas por A. alternata, sendo que foi possível a identificação de quatro dessas. Outra pesquisa realizada via LC-MS foi o estudo de co-cultivo entre os fungos A. alternata, o agente causal da mancha marrom de alternaria, e Guignardia citricarpa, o agente causal da mancha preta de citros. Nesse estudo foi possível observar a variação de alternariol e ácido tenuazônico, micotoxinas produzidas por A. alternata, quando este fungo está em contato com outro fitopatógeno. O estudo de identificação dos voláteis produzidos por A. alternaria em diferentes meios contribuiu muito para um maior conhecimento de sua química. Sabendo que A. alternata é um fitopatógeno de tangor Murcott foi realizado um estudo via microscopia eletrônica de varredura (MEV) a qual foi possível observar a penetração desse fungo pela cavidade estomatal de folhas jovens desse citros. Visando a busca de inibidores naturais para o controle da doença, foram ensaiadas diversas classes de produtos naturais frente a A. alternata, sendo que alcalóides e uma cumarina, a xantiletina, foram as substâncias que apresentaram uma maior inibição no crescimento micelial, germinação de esporos e formação de apressórios. Pensando em inibidores naturais, foram ensaiados metabólitos produzidos pelo A. alternata frente ao G. citricarpa, e os resultados obtidos revelaram que alternariol monometil éter e alternariol foram as substâncias que apresentaram um potencial fungistático frente ao agente causal da mancha preta de citros.

Citrus is the main fruit crop in the world and Spain is the 6th producer and the major exporter for the fresh fruit market. Seedlessness is one of the most important fruit quality traits for this market since consumers do not accept seedy fruits. Recovery of triploid hybrids has become an important breeding strategy to develop new seedless citrus varieties and several of them have been already released from citrus breeding programs worldwide. Despite the undisputable importance of polyploidy in plant species, their genetics are much less well known than those of their diploid counterparts. Citrus triploid hybrids can be routinely recovered from sexual polyploidization (2x ¿ 2x) or interploid crosses (2x ¿ 4x and 4x ¿ 2x). In 2x ¿ 2x sexual crosses, spontaneous triploid hybrids arise from the union of an unreduced (2n) megagametophyte with haploid pollen. In the case of interploid sexual crosses (2x ¿ 4x and 4x ¿ 2x), triploid hybrids result from the fecundation of a diploid gamete arising from the tetraploid parent and a haploid gamete arising from the diploid parent. The genetic and phenotypic structures of triploid populations greatly depend on the parental heterozygosity restitution (HR) in the diploid gamete at each locus, which is mainly affected by the triploid recovery strategy. In 2x ¿ 2x crosses, HR depends on the underlying mechanism leading to the unreduced gamete formation, which are genetically equivalent to First Division Restitution (FDR) or Second Division Restitution (SDR) mechanisms. Moreover, under each restitution mechanism, HR also depends on the locus-centromere genetic distance. In the case of interploid crosses, parental heterozygosity restitution from tetraploid parents depends on the double reduction frequency. In citrus, the unreduced gamete formation mechanism is still controversial; FDR has been the mechanism proposed for sweet orange, whereas SDR has been proposed for clementine. On the other hand, inferring the allelic configurations of genetic markers is a main challenge in polyploidy crops to infer genotypic and gametic structures with the objective to analyze meiosis and inheritance mechanisms. According to this scientific context, the objectives of the thesis where: (i) to develop a new approach for allele dosis assignation when using co-dominant markers, (ii) to implement and apply methods for the analysis of 2n gametes origin and locate centromeres, and (iii) to take advantage of this knowledge to locate a major gene of resistance to Alternaria Brown Spot (ABS) which is a major constraint for triploid mandarin breeding. For microsatellite (SSR) markers, we have demonstrated that triploid progeny genotyping can be successfully performed using the microsatellite allele-counting peak ratio (MAC-PR) method. However, SSR analysis remains relatively costly and time consuming compared with actual SNP genotyping methods. Moreover, with the increasing availability of EST databases and whole genome sequences, SNPs have become the most abundant and powerful polymorphic markers that can be selected along the entire genome. In this thesis, a new method based on competitive allele-specific PCR has been developed to assign SNP allele dosage in an accurate, simple, and cost effective way. Combining the MAC-PR and the new developed SNP genotyping methods offers the possibility to utilize a broad range of molecular markers in genotyping triploid genotypes. Both methods have been used in further works included in this thesis. SDR has been demonstrated as the mechanism underlying unreduced gamete production in `Fortune¿ mandarin by genotyping triploid progenies with SSR markers. In addition, a new method to locate the centromere, based on the best fit between observed heterozygosity restitution within a linkage group and theoretical functions under either partial or no chiasma interference hypotheses has been developed and successfully applied. To expand the knowledge of the mechanism underlying unreduced gamete formation to other citrus genotypes besides clementines and `Fortune¿ mandarin, a maximum likelihood method based on parental heterozygosity restitution of centromeric loci was developed and successfully applied in sixteen mandarin cultivars. The new method developed in the study allows inferring the restitution mechanism both at population level and even at individual level. Maternal origin of 2n gametes was confirmed for all triploid hybrids and SDR was proposed as the restitution mechanism for all analyzed progenies. The information acquired from the mode of heterozygosity restitution in citrus was useful to determine the genetic and phenotypic structures of new triploid populations arising from different breeding strategies. We studied these structures for the resistance to Alternaria brown spot (ABS), a serious fungal disease producing necrotic lesions on fruits and young leaves in susceptible citrus genotypes. In the present work, different approaches were combined taking advantage of the particular genetic structures of 2n gametes resulting from SDR to map a genome region linked to ABS resistance in triploid citrus progeny. The monolocus dominant inheritance of the susceptibility, proposed on the basis of diploid population studies, was corroborated in triploid progeny. A 3.3 Mb genomic region linked to ABS resistance was located near the centromere on chromosome III, which includes clusters of resistance genes. SSR and SNP markers were developed for an efficient early selection of ABS resistant hybrids and they are currently used in our breeding program to perform marker assisted selection. The knowledge obtained in this thesis on the mechanism of sexual polyploidization and inheritance of concrete traits in citrus will allow implementing much more efficient triploid breeding programs on the basis of current and future needs. Indeed, applied outcomes of this PhD are already routinely used in the IVIA triploid breeding program. / Cuenca Ibáñez, J. (2013). Mechanisms of sexual polyploidization and inheritance in triploid citrus populations [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34671 / Premios Extraordinarios de tesis doctorales

Exposure to spores and hyphae of Alternaria alternata, an airborne ubiquitous fungus, is clinically associated with allergic airway disorders including allergic rhinitis, asthma, and chronic rhinosinusitis. Dendritic cells are known as the type of antigen presenting cells most often associated with allergic inflammation. In this study, we used mouse bone marrow-derived dendritic cells (BMDCs) as a model to study the ability of A. alternata spores and different components of the spore cell wall to stimulate innate immune responses. We found that BMDCs were highly sensitive to A. alternata spores, chitin and the major allergen Alt a 1. Following stimulation with these molecules, the expression of MHC II and other co-stimulators, like CD40, CD86, and OX40L, were highly up regulated. In order to determine how different cell wall components affect the T cells, we conducted co-culture experiments of BMDCs and lymphocytes in this study. Both spores and Alt a1 did not induce IL-4 in mixed lymphocytes reactions. Interestingly, we found that Alt a 1 induced the switching of the CD4+ T cell population to the Th17 type, with a major increase in IL-17A secretion. This study reveals that A. alternata components may balance the innate immune responses between Th2 and Th17 pathways, and/or contributes to the development and exacerbation of more serve neutrophilic forms of asthma. / Master of Science / Alternaria alternata, is an airborne ubiquitous fungus, which is associated with allergic airway disorders including allergic rhinitis, asthma, and chronic rhinosinusitis. While dendritic cells, which are known as the classical antigen presenting cells, play an important function in antigen recognition at the early stage of immune response and then pass the signal to other immune cells. In this study, we used mouse bone marrow-derived dendritic cells (BMDCs) as a model to study the ability of A. alternata spores and different components of the spore cell wall to stimulate innate immune responses. As the results in this study, we found that BMDCs were highly sensitive to A. alternata spores and their cell wall components. Following stimulation with these molecules, the expression of major histocompatibility complex (MHC) molecules and other co-stimulators, were highly up regulated. In order to determine how different cell wall components affect other immune cells through BMDCs, we conducted co-culture experiments of BMDCs and different T cells population in this study. Both spores and cell wall component did not induce IL-4, a featured Th2 cytokine, in those co-culture studies. Interestingly, we found that cell wall component induced the switching of the T helper cell population to the Th17 type. This study reveals that A. alternata components may balance the innate immune responses between Th2 and Th17 pathways.

Made available in DSpace on 2016-06-02T19:30:12Z (GMT). No. of bitstreams: 1 6721.pdf: 2764879 bytes, checksum: ca82bcb193d5ae0d2e21032afd6ed1dd (MD5) Previous issue date: 2015-03-30 / Financiadora de Estudos e Projetos / The Brazilian savanna holds the richest flora among the tropical savannas, being one of the hot spots for conservation of the biodiversity in the world. However, the chemical potential of its plant species is still underused while deforestation and loss of biodiversity are main issues. In the Brazilian savanna, plants are exposed to environmental pressures (competition, pathogens, drought, injurious UV radiation, etc) that may stimulate the production of secondary metabolites as a defense. These compounds can generally be explored due to their biological properties. The aim of this study was to assess the fungicidal and antioxidant properties of plants from the Brazilian savanna. First a screening was conducted with ethanolic leaf extracts of 28 plant species. There was no activity against Alternaria alternata, a filamentous fungi that causes one of the most severe diseases in tangerines (Alternaria brown spot disease). However, the same extracts presented a high antioxidant potential associated with the phenolic content. Other investigations were made using infusion extractions focusing on the Myrtaceae family, one of the most abundant families of plants in the Brazilian savanna. Most species also presented high antioxidant activity and phenolic content. At last, the extraction of piceid, a compound with high antioxidant activity, was optimized from roots of Psidium laruotteanum. A response surface methodology was applied to assess the influence of solvent, pH, amplitude, cycle, temperature and solvent-solid ratio using an ultrasound extraction. A high amount of piceid was extracted and Psidium laruotteanum root was found to be the most concentrated source of this compound reported up to date. These results point the plant species from the Brazilian savanna as an important source of bioactive compounds, emphasizing the need to preserve and study its biodiversity. / O cerrado possui a flora mais rica entre as savanas tropicais, sendo um dos pontos quentes para conservação da biodiversidade no mundo. No entanto, o potencial químico de suas espécies vegetais é ainda subutilizado, enquanto o desmatamento e a perda de biodiversidade são questões de grande relevância. No cerrado, as plantas estão expostas a pressões ambientais (competição, patógenos, seca, radiação UV nociva, etc) que podem estimular a produção de metabólitos secundários como defesa. Estes compostos geralmente podem ser explorados devido suas propriedades biológicas. O objetivo deste estudo foi avaliar as propriedades fungicidas e antioxidante de plantas do cerrado. Primeiro uma triagem foi realizada com extratos etanólicos de folhas de 28 espécies vegetais. Não houve nenhuma atividade contra Alternaria alternata, um fungo filamentoso que causa uma das doenças mais severas em tangerinas (mancha marrom de Alternaria). No entanto, os mesmos extratos apresentaram um potencial antioxidante elevado associado com o conteúdo fenólico. Outras investigações foram feitas usando extrações por infusão enfocando a família Myrtaceae, uma das famílias mais abundantes de plantas no cerrado. A maioria das espécies apresentou alta atividade antioxidante e alto conteúdo fenólico. Por fim, a extração de piceido, um composto com alta atividade antioxidante, foi otimizada de raízes de Psidium laruotteanum. Uma metodologia de superfície de resposta foi aplicada para avaliar a influência do solvente, pH, amplitude, ciclo, temperatura e razão solvente-sólido usando uma extração de ultrasonido. Uma alta quantidade de piceido foi extraída e a raiz de Psidium laruotteanum se mostrou como sendo a fonte mais concentrada deste composto relatada até hoje. Estes resultados apontam as espécies de plantas do cerrado como uma importante fonte de compostos bioativos, enfatizando a necessidade de preservar e estudar sua biodiversidade.