Fall-sown barley will be increasingly important in the era of climate change due to higher yield potential and efficient use of water resources. Resistance/tolerance to biotic and abiotic stresses will be critical. Low temperature is an abiotic stress of great importance. Resistance to barley stripe rust (incited by Puccinia striifomis f. sp. hordei) and scald (incited by Rhynchosporium secalis) will be important in higher rainfall areas. Simultaneous gene discovery and breeding will accelerate the development of agronomically relevant germplasm. The role of FR-H1 and FR-H2 in low temperature tolerance (LTT) has been well documented. However the question still remains: is LTT due only to FR-H1 and FR-H2 or are there other, undiscovered, determinants of this critical trait? We developed two doubled haploid mapping populations using two lines from the University of Nebraska (NE) with superior cold tolerance and one line from Oregon State University (OR) with good malting quality and disease resistance: NB3437f/OR71 (facultative x facultative) and NB713/OR71 (winter x facultative). Both were genotyped with a custom 384 oligonucleotide pool assay (OPA). QTL analyses were performed for LTT, vernalization sensitivity (VS), and resistance to barley stripe rust and scald. Disease resistance QTL were identified with favorable alleles from both NE and OR germplasm. The role of VRN-H2 in VS was confirmed and a novel alternative winter allele at VRN-H3 was discovered in the Nebraska germplasm. FR-H2 was identified as a determinant of LTT and a new QTL, FR-H3, was discovered on chromosome 1H that accounted for up to 48% of the phenotypic variation in field survival at St. Paul, Minnesota, USA. The discovery of FR-H3 is a significant advancement in barley LTT genetics and will assist in developing the next generation of fall-sown varieties. / Graduation date: 2012

Made available in DSpace on 2016-12-06T17:42:06Z (GMT). No. of bitstreams: 1 PGPV15DA017.pdf: 2167142 bytes, checksum: f46dc26724e5902a13e50a01c53ada5b (MD5) Previous issue date: 2015-04-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Four experiments were conducted to evaluate the effect of 1-MCP in CS and CA, and the ULO storage on the sensory quality and incidence of physiological disorders in function of the maturation at harvest in pears Conference and Alexander Lucas harvested at three production location. The fruit harvest was carried out in the cities of Ravensburg (orchard 1) for both cultivars and Langenargen (Orchard 2) and Öhringen (orchard 3) only for Alexander Lucas . All locations are in the state of Baden-Württemberg, southwestern Germany. For the experiments 1, 2 and 3 the design was completely randomized in a factorial scheme five (storage conditions) for two (harvest maturity). Treatments were: cold storage (CS-21.0 kPaO2+0.03 kPa CO2), controlled atmosphere (CA-2 kPa O2 / <0.7 kPa CO2), CS with 1-MCP application (CS+1-MCP-300 nL L-1), CA with application of 1-MCP (CA+1-MCP) and ultra low oxygen (ULO, 0.7 kPa O2 /<0.7 kPa CO2) versus harvest 1 (05/09/2012; Streif index of 0.15, 0.12, 0.09 to 1, 2 and 3 orchards, respectively) and harvest 2 (18/09/2012; Streif index of 0.08; 0.06, 0.08 for orchard 1, 2 and 3, respectively). All treatments were stored at 0±0.1°C and 94±2% relative humidity. In the experiment 4 was added high CO2 treatment (CO2-2 kPa O2/CO2 3 kPa). After seven months of storage plus seven days at ambient conditions (20±2°C/60±5% RH) fruits were evaluated for sensory attributes of firmness, color of skin (h°), soluble solids (SS), titratable acidity (TA) and aromatic compounds aldehydes, alcohols and esters. In addition, it were measured production of ethylene, &#945;-farnesene, 6-methyl-5-heptene-2-one and respiratory rate, phenolics, vitamin C of epidermis, scald and internal browning , percentage of fruit with cavities and rot, and mineral content. In general the best storage condition to Conference would be in CA+1-MCP in fruit picked with IS 0.08. This condition provides larger fruits with reduced scald index without internal browning, besides development of aromatic compounds. The storage of the fruits of Alexander Lucas for seven months plus seven days at room temperature is too long. All the storage conditions caused internal disorder. Nevertheless, there are indications of the best storage condition in CS+1-MCP in fruits picked with IS 0.08. In this condition beyond the larger return occurs in the production of volatile reduction in surface escaldaura and browning occurs and no appearance of cavities / Foram conduzidos quatro experimentos com o objetivo de avaliar o efeito do 1-MCP, em AR e AC, e do ULO sobre a qualidade sensorial e incidência de distúrbios fisiológicos, em função do estádio de maturação em peras Conference e Alexander Lucas colhidas em três locais de produção. A colheita dos frutos foi realizada nos municípios de Ravensburg (pomar 1), para ambas as cultivares e Langenargen (pomar 2) e Öhringen (pomar 3) somente para Alexander Lucas . Todos os municípios localizam-se no estado de Baden-Württemberg, sudoeste da Alemanha. Para os experimentos 1, 2 e 3 o delineamento utilizado foi o inteiramente casualizado em esquema bifatorial cinco (condições de armazenamento) por dois (pontos de maturação). Os tratamentos foram: atmosfera refrigerada (AR-21,0 kPa O2+0,03 kPa CO2), atmosfera controlada (AC-2 kPa O2/<0,7 kPa CO2), AR com aplicação de 1-MCP (AR*-300 nL L-1), AC com aplicação de 1-MCP (AC*) e ultra baixo oxigênio (ULO-0,7 kPa O2/<0,7 kPa CO2) combinados com colheita 1 (05/09/2012; índice de Streif (IS) de 0,15; 0,12; 0,09 para pomares 1, 2 e 3, respectivamente) e colheita 2 (18/09/2012; IS de 0,08; 0,06; 0,08 para pomares 1, 2 e 3, respectivamente). Todos os tratamentos foram armazenados a 0±0,1ºC e 94±2% de umidade relativa. Ao experimento 4 foi acrescida a condição de armazenamento alto CO2 (2 kPa O2/3 kPa CO2). Após sete meses de armazenamento mais sete dias em condições ambiente (20±2ºC / 60±5% de UR) os frutos foram avaliados no experimento 1 e 2 quanto às taxas de produção de etileno e taxa respiratória, acidez titulável (AT), sólidos solúveis (SS), firmeza de polpa, cor da epiderme (h°) e compostos aromáticos. No experimento 3 foi avaliado o índice de frutos com escaldadura superficial, produção de &#945;-farneseno e 6-metil-5-hepteno-2-ona, compostos fenólicos, vitamina C da epiderme e no experimento 4 o índice de escurecimento da polpa, porcentagem de frutos com cavidades e com podridão e teor de minerais. De maneira geral, apesar da perda na eficácia do 1-MCP no retardo do amadurecimento para ambas cultivares com o atraso na colheita a melhor condição de armazenamento para cultivar Conference seria em AC* em frutos colhidos com IS 0,08 (colheita 2). Está condição proporciona frutos maiores, com menor índice de escaldadura, ausência de distúrbios internos e com desenvolvimento de compostos aromáticos. O armazenamento dos frutos de Alexander Lucas por sete meses mais sete dias em temperatura ambiente é muito longo, pois todas as condições de armazenamento causaram distúrbios internos. Apesar disso, há indícios de que a melhor condição para o armazenamento desta cultivar é AR* em frutos colhidos com IS 0,08 (colheita 2), já que, ocorre retorno na produção de aroma, redução na escaldaura superficial e do escurecimento da polpa, sem o aparecimento de cavidades

A Escaldadura das Folhas da Ameixeira (EFA) é a principal doença da cultura no Brasil, sendo causada pela bactéria Xylella fastidiosa e transmitida entre plantas pela ação de insetos vetores, mas há carência de informações sobre a identidade dos vetores e plantas hospedeiras para estirpes de X. fastidiosa causando EFA. Objetivando subsidiar uma proposta de manejo da EFA, foram realizados estudos sobre a transmissão de X. fastidiosa por vetores em ameixeira, identificação de plantas hospedeiras da bactéria em vegetação de cobertura dos pomares que possam servir como fontes de inóculo, capacidade de colonização de estirpes de ameixeira, cafeeiro e citros em inoculações cruzadas e validação da técnica de inoculação mecânica como método de avaliação de resistência de cultivars a X. fastidiosa em programas de melhoramento de ameixeira. Inicialmente, por meio de testes de colonização por X. fastidiosa foram identificadas Ocimum basilicum, Vernonia condensata e Pentas lanceolata como plantas não hospedeiras da bactéria, permitindo a criação de cigarrinhas sadias que foram utilizadas nos ensaios de transmissão. As cigarrinhas Macugonalia cavifrons, M. leucomelas e Sibovia sagata (Hemiptera: Cicadellidae: Cicadellinae) foram identificadas como vetoras de X. fastidiosa em ameixeira com eficiência de transmissão por indivíduo variando de 12 a 21%. Para identificação de hospedeiros alternativos do patógeno, avaliaram-se 12 espécies herbáceas (folhas e raízes) predominantes na vegetação de cobertura de pomares de ameixeira com elevada incidência de EFA, sendo dois no município de Videira, SC e três no município de Jarinú, SP. Amostras de nove delas (Bidens pilosa, Lepidium ruderale, Lolium multiflorum, Plantago major, Parthenium hysterosphorus, Raphanus sativus, Rumex sp., Solanum americanum e Vernonia sp. foram positivas para X. fastidiosa em folhas e/ou raízes, pelo teste de reação de polimerase em cadeia (PCR), Testes de inoculação mecânica com um isolado de X. fastidiosa de ameixeira comprovaram a capacidade dessa estirpe bacteriana em colonizar plantas de B. pilosa, L. ruderale, R. sativus e S. americanum. Estudos de inoculação cruzada demonstraram que isolados de X. fastidiosa de citros são capazes de colonizar cafeeiro, embora em menor proporção que isolados de cafeeiro. Um isolado de ameixeira foi capaz de colonizar citros e cafeeiro, sendo que em citros a proporção de plantas infectadas foi maior. Isolados de X. fastidiosa de citros e cafeeiro não colonizam plantas de ameixeira, sendo estas colonizadas apenas pelos isolados homólogos. Estudos de inoculação mecânica de dois isolados de X. fastidiosa de ameixeira em diferentes cultivares dessa planta hospedeira, expressaram resultados corroborativos com a pré-caracterização da resistência das cultivares, validando a técnica para uso em programas de melhoramento, com melhor precisão nos resultados e ganho de tempo na avaliação de resistência à EFA. / The Plum Leaf Scald (PLS) is the main disease of plum crop in Brazil being caused by the bacterium X. fastidiosa, which is transmitted among plants by insect vectors. However, additional information is needed about the identity of vectors and host plants of X. fastidiosa isolates that cause PLS. In order to subsidize PLS management proposal, studies on X. fastidiosa vector transmission in plum, identification of host plants from orchard vegetation cover that can be source of inoculum, bacterial colonization of strains isolated from plum, coffee and citrus in cross inoculations, and validation of mechanical inoculation technique as method for assessing resistance of plum varieties against X. fastidiosa in breeding programs, were carried out. Firstly, studies of colonization by X. fastidiosa revealed that three plants are non-hosts: Ocimum basilicum, Vernonia condensata and Pentas lanceolata, allowing the maintenance of healthy leafhoppers colonies. The sharpshooters Macugonalia cavifrons, M. leucomelas and Sibovia sagata were confirmed as vectors of X. fastidiosa in plum with transmission efficiency per individual ranging from 12 to 21%. In order to identify alternative hosts of the bacterium in plum orchards, 12 plant species (leaves and roots), which are predominant in the vegetation from orchards where PLS incidence is high, two of them being located at Videira - SC and three at Jarinu - SP, were chosen to be tested. Samples of leaves and/or roots from nine plant species (Bidens pilosa, Lepidium ruderale, Lolium multiflorum, Plantago major, Parthenium hysterosphorus, Raphanus sativus, Rumex sp., Solanum americanum and Vernonia sp.) were positive for X. fastidiosa by Polymerase Chain Reaction (PCR). Mechanical inoculation tests using a plum isolate of X. fastidiosa showed the capacity of this strain in colonizing the plants B. pilosa, L. ruderale, R. sativus and S. americanum. Studies on cross inoculation demonstrated that citrus X. fastidiosa isolates are able to colonize coffee plants, although in lower intensity than strains isolated from coffee. A plum isolate was able to colonize citrus and coffee plants, but the proportion of infected plants was higher in citrus. On the other hand, isolates of X. fastidiosa from citrus and coffee do not colonize plum plants and these were only colonized by homologous isolates. Studies on mechanical inoculation of two plum strains of X. fastidiosa in different plant cultivars revealed a pre-characterization on the resistance of these cultivars, validating the technique for use in breeding programs with a better precision in results as well as saving time on the assessment of resistance against PLS.

A escaldadura das folhas, causada por Xanthomonas albilineans, é uma importante doença da cana-de-açúcar, a qual interfere no rendimento e na longevidade de plantas infectadas. A intensidade de doença é variável, especialmente em relação a fatores climáticos e variedades cultivadas. A quantificação de danos é difícil de ser feita através de observações de plantas no campo. O uso de variedades resistentes tem sido eficiente para controle da doença, mas a diversidade do patógeno pode promover a quebra desta resistência. Em campos comerciais localizados no Estado de São Paulo, tem sido observado que a mesma variedade de cana é resistente à escaldadura em uma região, mas suscetível em outra. Este tipo de comportamento tem sugerido a ocorrência de variantes de X. albilineans que podem ser responsáveis pela quebra da resistência das variedades cultivadas. Seria relevante determinar a ocorrência de variabilidade para este patógeno dentro do estado de São Paulo, pois esta informação seria útil para indicação de variedades para as diferentes regiões e para orientar programas de melhoramento visando a obtenção de variedades resistentes. Com base nestas considerações, o objetivo do presente estudo foi investigar a diversidade genética de isolados coletados em áreas comerciais, usando técnicas moleculares como PCR, rep-PCR, RFLP e testes de patogenicidade. Os resultados mostraram que todos os isolados de plantas que exibiam sintomas de escaldadura eram pertencentes à espécie X. albilineans. Ainda, foi possível determinar a ocorrência de diversidade genética e variabilidade patogênica entre os isolados amostrados nas diferentes regiões. Os resultados obtidos no presente trabalho podem contribuir para novas investigações visando confirmar a associação da quebra da resistência de variedades com a variabilidade do patógeno. / Leaf scald, caused by Xanthomonas albilineans, is an important disease of sugarcane, That can interfere on the yield and longevity of infected plants. The disease intensity is variable, especially according to climate factors and cultivated varieties, and the damage quantification is very difficult through observation of plants in the field. Resistant varieties has been efficient to disease control, but the pathogen diversity can promote the breakdow of resistance. In commercial fields located in São Paulo State has been observed that the same sugarcane variety is resistant to leaf scald in a region, but susceptible in another one. This kind of behavior has suggested the occurrence of strains of X. albilineans that can be responsible for the breakdow of resistance of the cultivated varieties. It would be relevant to determinate pothogen variability in São Paulo State, because that information would be useful to indicate varieties for different regions and to guide breeding programs to obtain resistant varieties. Based upon the above points, the objective of the present study was to investigate the genetic diversity of bacterial isolates collected in commercial areas, using molecular techniques as PCR, rep-PCR, RFLP, and pathogenicity assays. The results showed that all isolates from plants exhibiting typical leaf scald symptoms belonged to species X. albilineans. It was also possible to determinate the occurrence of genetic diversity and pathogenic variability among the isolates sampled in the distinct regions. Thus, the results obtained in the present study can contribute to new investigations to confirm the association of the breakdow of resistance with the variability of the pathogen.

Xanthomonas albilineans est la bactérie responsable de l'échaudure des feuilles, une maladie vasculaire et létale de la canne à sucre. Cette plante, domestiquée depuis plus de 5000 ans, est aujourd'hui cultivée sur près de 26 millions d'hectares. Depuis le début du 20ème siècle, les variétés naturelles de canne à sucre ont été remplacées par des variétés hybrides issues de croisements artificiels et présentant des caractères agronomiques plus intéressants. La transmission de X. albilineans est essentiellement liée au mode de multiplication par bouturage de la canne à sucre, les boutures produites à partir d'un plant infecté ayant de fortes chances d'être contaminées. L'utilisation d'outils de coupe favorise également la transmission de la bactérie à partir d'un plant infecté. Une transmission aérienne a toutefois été décrite pour au moins un groupe génétique de souches de X. albilineans appelé PFGE-B et caractérisé à l'aide de la technique d'électrophorèse en champ pulsé. Mon sujet de thèse visait, par une étude de génomique comparative et évolutive sur plusieurs souches de X. albilineans, à, d'une part, comprendre l'histoire de l'association de cette bactérie avec la canne à sucre, et, d'autre part, identifier des gènes candidats qui pourraient expliquer la pathogénie de X. albilineans et la capacité des souches du groupe PFGE-B à être transmises par voie aérienne.En comparant 12 souches de X. albilineans appartenant à neuf groupes PFGE différents, j’ai construit une phylogénie solide proposant l'existence de huit lignées regroupées en deux clades. Cette phylogénie a été confirmée en utilisant trois méthodes de phylogénomique différentes. De façon intéressante, cette phylogénie permet de proposer des hypothèses sur l'évolution de X. albilineans et sur l'introduction de cette bactérie chez la canne à sucre. L'analyse des gènes présents dans le génome de ces 12 souches appartenant à neuf groupes PFGE différents m'a également permis d'identifier un système RM (restriction/modification) qui n'est présent que chez les souches PFGE-B et qui pourrait expliquer pourquoi ces souches sont plus résistantes aux phages que celles appartenant aux autres groupes PFGE. En facilitant la survie des bactéries dans l'environnement, cette résistance aux phages pourrait jouer un rôle important dans la transmission aérienne.La comparaison de 11 souches de X. albilineans appartenant au groupe PFGE-B m'a permis de construire, à partir de 67 SNP (Single Nucleotide Polymorphism), une phylogénie qui indique que les Caraïbes, où la canne à sucre n'a été introduite qu'à la fin du 15ème siècle, pourraient être un centre de dispersion des souches PFGE-B. Cette étude a également mis en évidence un polymorphisme au niveau des spacers d'un locus CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats system). L'étude de ce polymorphisme sur 18 souches de X. albilineans non séquencées a permis de démontrer que les souches de Floride sont originaires des Caraïbes et de proposer des hypothèses concernant l'origine des souches PFGE-B d’autres régions du monde, même si le séquençage d'un plus grand nombre de souches sera nécessaire pour confirmer ces hypothèses.En analysant la séquence du génome de deux souches qui avaient été initialement classées dans l'espèce X. albilineans mais qui n'avaient pas été isolées à partir de tiges de canne à sucre, j’ai mis en évidence une nouvelle espèce que nous avons appelée 'Xanthomonas pseudalbilineans'. Enfin, la comparaison du génome fini et annoté de la souche PFGE-B GPE PC73 avec le génome d'autres espèces bactériennes phytopathogènes m'a permis de proposer des hypothèses pour expliquer la pathogénie de X. albilineans. / Xanthomonas albilineans is the bacterium causing leaf scald, a vascular disease being lethal for sugarcane. This plant, domesticated for over 5,000 years, is nowadays grown across almost 26 million hectares. Since the beginning of 20th century, natural varieties of sugarcane have been replaced by hybrid varieties resulting from artificial crossings. The transmission of X. albilineans is mainly linked to the fact that sugarcane is propagated by taking cuttings, with the cuttings from an infected plant likely to be contaminated. The use of cutting instruments also facilitates transmission of the bacteria from an infected plant. An aerial transmission has nevertheless been described, at least for one genetic group of strains from X. albilineans, namely PFGE-B, characterized using the pulsed-field gel electrophoresis technique. My thesis topic consisted in performing a comparative and evolutive genomic study of several strains of X. albilineans in order, on one hand, to better understand the history of association of this bacterium with the sugarcane, and, on the other hand, to identify candidate genes which may explain the pathogenicity of X. albilineans as well as the ability of PFGE-B strains to be aerially transmitted. Comparing 12 strains of X. albilineans belonging to nine different PFGE groups, I built a robust phylogeny which proposes the existence of eight lineages clustered in two clades. This phylogeny has been confirmed by three different phylogenomic methods. Interestingly, this phylogeny allows to propose hypotheses about the evolution of X. albilineans and the introduction of this bacterium in sugarcane. The analysis of genes from the genomes of these 12 strains belonging to nine different PFGE groups also allowed me to identify a RM (Restriction/Modification) system which is present only in PFGE-B strains and which may explain why these strains are more resistant to phages that those belonging to other PFGE groups. By facilitating the survival of bacteria in the environment, this resistance to phages may play an important role in aerial transmission. The comparison of 11 strains of X. albilineans belonging to the PFGE-B group allowed me to build, based on 67 SNPs (Single Nucleotide Polymorphism), a phylogeny which indicates that the Caribbean area, where sugarcane was introduced not before the end of the 15th century, might be a center for dispersion of PFGE-B strains. This study also revealed a polymorphism of spacers of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats system) locus. The study of this polymorphism in 18 non-sequenced strains of X. albilineans allowed to demonstrate that strains from Florida originated in the Caribbean region, and to propose hypotheses regarding the origin of PFGE-B strains from other regions of the world, even if the sequencing of a higher number of strains will be required to confirm these hypotheses.Analyzing the sequence of the genome of two strains, initially thought to belong to the X. albilineans species but which weren’t isolated from sugarcane stalks, I revealed a new species that we called ‘Xanthomonas pseudalbilineans’. Finally, the comparison of the finished and annotated genome of the PFGE-B strain GPE PC73 with the genome of other phytopathogenic bacteria allowed me to propose hypotheses to explain X. albilineans’ pathogenicity.

Large doses of recombinant growth factors formulated in solution form directly injected into the body is usual clinical practice in treating second-degree scald injuries, with promising results, but this approach creates side effects; furthermore, it may not allow appropriate levels of the factor to be sensed by the target injured tissue/organ in the specific time frame, owing to complications arising from regeneration. In this research, two delivery methods (infusion pumping and local topical application) were applied to deliver recombinant human erythropoietin (rHuEPO) for skin regeneration. First, rHuEPO was given in deep second-degree scald injury sites in mice by infusion pump. Vascularization was remarkably higher in the rHuEPO pumping group than in controls. Second, local topical application of rHuEPO gel was given in deep second-degree scald injury sites in rats. Histological analysis showed that epithelialization rate was significantly higher in the rHuEPO gel-treated group than in controls. Immunohistochemical studies showed that the rHuEPO gel-treated group showed remarkably higher expression of skin regeneration makers than the control group. An accurate method for visualization and quantification of blood vessel networks in target areas has still not been developed up to this point, because of technical difficulties in detecting such thin blood vessels. A method which utilizes a series of steps to enhance the image, removes noise from image background, and tracks the vessels edges for vessel segmentation and quantification has been used in this study. Using image analysis methods, we were able to detect the microvascular networks of newly formed blood vessels (less than 500 μm thickness), which participate in the healing process, providing not only nutrition and oxygen to grow tissues but also necessary growth factors to grow tissue cells for complete skin regeneration. The rHuEPO-treated group showed higher expression of stem cell markers (CD 31, CD 90, CD 71, and nestin), which actively contribute to in-wound-healing processes for new hair follicle generation as well as skin regeneration. Collectively, both rHuEPO group pumping into the systemic circulation system, and injection into the local injury area, prompted mice and rats to form new blood vessel networks in scald injury sites, which significantly participate in the scald healing process. These results may lead to the development of novel treatments for scald wounds.