Plants and microorganisms interact with each other constantly, with some interactions being mutually beneficial and others being detrimental to the plants. The features of the organisms involved in such interactions will determine the characteristics of individual pathosystems. Plants respond readily to pathogen attacks, regardless of the pathosystem; furthermore, variation in the resistance to pathogens within species is common and well documented in many plant species. The variability in pathogen resistance is at the core of genetic improvement programs for disease resistance. True resistance to pathogens in plants is a genetically determined and complex trait that can involve both constitutive and induced mechanisms at different levels of organization. The complexity of this phenomenon makes the study of compatible plant - pathogen interactions challenging, and typically, disease resistance studies focus on specific aspects of a pathosystem, such as field resistance, anatomical or physiological features of resistant plants, or molecular mechanisms of resistance. The Thuja sp. - Didymascella thujina (E.J. Durand) Maire interaction is an important pathosystem in western North America, which has been studied for more than five decades. Western redcedar (Thuja plicata Donn ex D. Don) is very susceptible to cedar leaf blight (D. thujina), a biotroph that affects the tree at all stages, although seedlings are the most sensitive to the pathogen. The characteristics of the Thuja sp. - D. thujina interaction, the wealth of information on the pathosystem and the excellent Thuja sp. genetic resources available from the British Columbia Ministry of Forests, Lands, Natural Resource Operations and Rural Development make this interaction an ideal system to advance the study of disease resistance mechanisms in conifers. This Doctoral project presents a comprehensive investigation of the constitutive and induced resistance mechanisms against D. thujina in T. plicata, Thuja standishii (Gord.) Carrière and a Thuja standishii x plicata hybrid at the phenotypic and gene expression levels, undertaken with the objective of exploring the resistance mechanisms against the biotroph in these conifers. The project also aimed to establish base knowledge for the future development of markers for marker-assisted breeding of T. plicata. The investigations included a combination of histological, chemical and next generation sequencing (NGS) methodologies. NGS data were analyzed, in addition to the traditional clustering analyses, with cutting edge machine learning methods, including grade of membership analysis, dynamic topic modelling and stability selection analysis. The studies were progressively more controlled to narrow the focus on the resistance mechanisms to D. thujina in Thuja sp. Histological characteristics related to D. thujina resistance in Thuja sp. were studied first, along with the relationship between climate of origin and disease resistance. The virulence of D. thujina was also documented early in this project. Chemical and gene expression constitutive and induced responses to D. thujina infection in T. plicata seedlings were studied next. T. plicata clonal lines were then comprehensively studied to shed light on the mechanisms behind known physiologically determined resistance. A holistic investigation of the resistance mechanisms to D. thujina in T. standishii, T. plicata and a T. standishii x plicata hybrid explored the possibility of a gene-for-gene resistance model. Thirty-five T. plicata families were screened during the four field seasons carried out between 2012 and 2015, totalling more than 1,400 seedlings scored for D. thujina severity. Thirteen of those families were used in the five studies performed during the program, along with two T. plicata seedling lines self-pollinated for five generations and three T. plicata clonal lines. One T. standishii clonal line, and one T. standishii x plicata clone were also investigated during the program. A total of 16 histological and anatomical characteristics were studied in more than 750 samples, and more than 270 foliar samples were analyzed for 60 chemical and nutritional compounds. Almost one million transcriptomic sequences in four individually assembled reference transcriptomes were examined during the program. The results of the project support the variability in the resistance to D. thujina in T. plicata, as well as the higher resistance to the pathogen in plants originating from cooler and wetter environments. The data collected also depicted the existence of age-related resistance in T. plicata, and confirmed the full resistance to the disease in T. standishii. Western redcedar plants resistant and susceptible to D. thujina showed constitutive differences at the phenotypic and gene expression levels. Resistant T. plicata seedlings had thicker cuticles, constitutively higher concentrations of sabinene, alpha-thujene, and higher levels of expression of NBS-LRR disease resistance proteins. Resistant clones of T. plicata and T. standishii had higher expression levels of bark storage proteins and of dirigent proteins. Plants from all ages, species and resistance classes studied that were infected with D. thujina showed the accumulation of aluminum in the foliage, and increased levels of sequences involved in cell wall reinforcement. Additional responses to D. thujina infection in T. plicata seedlings included the downregulation of some secondary metabolic pathways, whereas pathogenesis-related proteins were upregulated in clonal lines of T. plicata. The comprehensive approach used here to study the Thuja sp. - D. thujina pathosystem could be applied to other compatible plant-pathogen interactions. / Graduate / 2020-08-31

Made available in DSpace on 2014-07-29T15:10:31Z (GMT). No. of bitstreams: 1 Dis Heleno Dias Ferreira - pre textuais.pdf: 666003 bytes, checksum: a67975494e3cb7ed970b88ad268aca1b (MD5) Previous issue date: 2010-04-29 / The genus Hyptis, with 280 species worldwide, belongs to the family Labiatae (Lamiaceae), subfamily Nepetoideae, tribe Ocimae, and subtribe Hyptidinae. The genus was divided in 27 sections, with 13 of them (Apodotes, Cephalohyptis, Cyanocephalanthus, Cyrta, Eriosphaeria, Gymneia, Induratae, Mesosphaeria, Polydesmia, Pusilae, Trichosphaeria, and Hylodontes) occur in the states of Goiás and Tocantins (Brazil). The collection of botanical material for morphological, taxonomical, anatomical, phytochemical studies, as well as geographical distribution and phylogenetic relationships, was realized with several field trips in the states of Goiás and Tocantins. For morphological studies, several loans of herbarium specimens of Hyptis were requested from Brazilian and international institutions. The genus Hyptis is mostly American, with Neotropical distribution, ranging from southern United States to Argentina. In South America, most species of Hyptis are associated with open areas, and occur from 300 m altitude in the valley of the Rio Araguaia, in Goiás, to 3300 m in the western slopes of the Peruvian Andes. In Goiás and Tocantins they can be found in several vegetation types: cerrado, rocky outcrops, margins of cow pastures, cultivated fields, wet fields, humid forests, and flooded areas with Mauritia palms. In these states they occur in a variety of soils: sandy, sandygravelly, with rocky outcrops, hydromorphic, and latossoils. Specie of the section Cyrta are exclusive of humid and flooded soils. The two main centers of diversity of Hyptis in Brazil are in the states of Minas Gerais and Goiás. Many herbarium specimens were analyzed and many species were studied and collected during field trips in Goiás and Tocantins. The geographic position of each collection was verified with the use of a GPS instrument. The specimens collected were deposited in the UFG Herbarium of the Federal University of Goiás. Thirty-two distribution maps of Hyptis in Goiás and Tocantins were elaborated from the specimens studied. A total of 89 species was catalogued, and 18 of them reported from Goiás and Tocantins were not found during the collecting trips. A key for the identification of the species was elaborated using morphological characters. A parsimony analysis using 35 morphological characters was realized, obtaining 1864 most parsimonious trees and a strict consensus tree. The parsimony analysis supports the monophyly of the sections Gymneya, Cyrta, Apodotes, and Cyanocephalus (except for the H. nitidula - H. peduncularis clade), and indicates that the sections Mesospheria, Xylodontes, Hyptis, Polydesmia, and Eriosphaeria are not monophyletic. Section Trichosphaeria is not well resolved. An anatomical study of 60 species, representing 11 sections of Hyptis, was undertaken. Some characters, as hypodermis, schlerenchimatic sheath extensions, trichomes, stomatal cripts, supply useful taxonomic for inferring phylogenetic relationships within the genus. For the analyses of chemical components, 29 species and two varieties of Hyptis were selected. With the chemical analises, 216 constituents of essential oils were identified, mostly monotherpenes and sesquiterpenes. Average percentage and pattern of each chemical component were calculated. The most common chemical components were arbitrarily selected, as present in nine or more species: sabinene (9 spp.), 1, alpha-tujene, alpha-cubebene, beta-selinene, and 14-hidroxy-(Z)-cariophyllene (10 spp.). alpha-muurolol and 8(13)-dien- 5beta-ol (11 spp.), mircene, cariophil-4 and germacrene B (12 spp.), gamma-cadinene, epialpha- caninol (13 spp.) and 14-hydroxy-9-epi-(E)-cariofilene (13 spp.), 1-epi-cubenol- and gamma-muurolene (14 spp.), humulene poxide II and bicyclogermacrene (16 spp.), betaelemene and alpha-cadinol (17 spp.), beta-pinene (18 spp.), limonene (19 spp.), espatulenol and germacrene D (24 spp.), alpha copaene and beta-bourbonene (25 spp.), delta-cadinene (26 spp.), alpha-humulene (27 spp.), cariofilene oxide and (E)-cariofilene (28 spp.). Two dendrograms were obtained with two multivariate analyses of chemical constituents, one with quantitative data and the other with qualitative data (presence/absence). Species of section Cyanocephalus were the only ones found in a consistent group in both dendrograms based on chemical constituents. The data obtained in the present study contribute important information for the taxonomy and phylogenetic relationships of the genus Hyptis and the subtribe Hyptidinae, and ultimately for the family Labiatae. / O gênero Hyptis, com 280 espécies, pertence à família Labiatae, subfamília Nepetoideae, tribo Ocimae e subtribo Hyptidinae. O gênero Hyptis foi dividido em 27 seções, sendo que 13 seções (Apodotes, Cephalohyptis, Cyanocephalus, Cyrta, Eriosphaeria, Pachyphyllae, Gymneia, Induratae, Mesosphaeria, Polydesmia, Pusilae, Trichosphaeria e Xylodontes) ocorrem em Goiás/Tocantins. Para coleta do material botânico, destinado aos estudos morfológicos, taxonômicos, anatômicos, fitoquímicos, distribuição geográficas e relações filogenéticas, foram realizadas excursões de campo em Goiás e algumas regiões do Tocantins.Para complementação dos dados morfológicos, foram solicitados exemplares de espécies de Hyptis dos diferentes herbários do Brasil e de outros países. O gênero Hyptis é essencialmente americano com distribuição na Região Neotropical, estendendo desde o sul dos Estados Unidos da América até a Argentina. As espécies de Hyptis estão associadas principalmente a áreas abertas. A altitude de ocorrência varia desde 300m, como no vale do Rio Araguaia em Goiás, até 3300m no oeste dos Andes do Peru. Habitat e solos em que ocorrem as espécies em Goiás/ Tocantins são variados. Podendo ser encontradas nos cerrados, campos rupestres, margens de lavouras, lotes baldios, brejos, campos úmidos e veredas. Podem ocorrer em solos arenosos, arenosopedregosos, solos com afloramento rochosos, latossolos e solos hidromórficos. As espécies da seção Cyrta são exclusivas de ambientes úmidos e pantanosos. Os dois principais centros de diversidade de Hyptis, no Brasil, são Minas Gerais e Goiás. Foram analisadas exsicatas de diversos herbários e indivíduos coletados de diferentes espécies nas excursões de campo em Goiás e Tocantins. Os espécimes coletados foram depositados no Herbário UFG, da Universidade Federal de Goiás. Foram elaborados 32 mapas de distribuição geográfica de Hyptis. A posição geográfica de cada espécime coletado no campo foi verificada com auxílio de GPS. Foram catalogadas 89 espécies, sendo que 18 espécies citadas para Goiás/Tocantins não foram coletadas nas excursões de campo. Baseado nos caracteres morfológicos foi elaborada uma chave de identificação das espécies. Desses caracteres foram selecionados 35 para análise de parcimônia, obtendo 1864 árvores igualmente parcimoniosas e a árvore consenso estrito. A análise de parcimônia suporta que as seções Gymneia, Pusillae, Apodotes e Cyanocephalus (exceto o clado formado por H. nitidula e H. peduncularis) são monofiléticas, enquanto que as seções Mesosphaeria, Xylodontes, Polydesmia e Eriosphaeria não são consideradas monofiléticas. Trichosphaeria é uma seção que não está bem resolvida. Foi realizado estudo anatômico de 60 espécies de Hyptis, distribuídas em 11 seções. Alguns caracteres como hipoderme, extensão de bainha parenquimática, tricomas, criptas estomáticas são úteis para a taxonomia e para inferir relações filogenéticas dentro do gênero. Para análise dos componentes químicos, dos óleos esssenciais foram selecionados 29 espécies e duas variedades de Hyptis. Através de análise química foram identificados 216 componente dos óleos essenciais, principalmente monoterpenos e sesquiterpenos. Foram feitos os cálculos das médias percentuais e desvios padrões de cada componente químico. Arbitrariamente foram considerados os componentes químicos mais comuns aqueles presentes em nove ou mais espécies: sabineno (9 spp), 1, α-tujeno, α-cubebeno (9 spp), β-selineno e 14-hidroxi-(Z)- cariofileno (10 spp), α-muurulol e 8(13)-dien-5 β-ol (11 spp), mircenno, cariofila-4 e germacreno β (12 spp), γ-cadineno, epi- α-cadinol e 14-hidroxi-9-epi-(E)-cariofileno (13 spp), 1-epi-cubenol e γ-muuroleno (14 spp), póxido de hemuleno II e biciclogermacreno (16 spp), β- elemeno e α-cadinol (17 spp), β-pineno (18 spp), limoneno (19 spp), espatulenol e germacreno D (24 spp), α-copaeno e β-bourboneno (25 spp), δ-cadineno (26 spp), α-humuleno (27 spp),óxido de cariofileno e (E)-cariofileno (28 spp). Atravé de análise multivariada dos constituintes químicos foram elaborados dois dendrogramas, um baseado em dados quantitativos e outro em dados qualitativos (presença/ausência). As espécies da seção Cyanocephalus foram as únicas que formaram grupos muito afim, em ambos os dendrogramas, baseados nos constituintes químicos. Os dados obtidos, no presente trabalho, contribuirá para a taxonomia, anatomia, distribuição geográfica e relações filogenéticas do gênero Hyptis e da subtribo Hyptidinae, bem como para melhor conhecimento da família Labiatae.