Stem rust and Yellow rust diseases are the two major wheat fungal diseases causing considerable yield losses in Turkey and all around the world. There are studies which are carried out to identify and utilize resistance sources in order to obtain resistant lines of wheat. However, virulent pathotypes are continuously being important threats to wheat production and yield. For that reason, new approaches for rapid identification are needed. The aim of this study was to investigate and to understand the structural and functional differences between the resistant and sensitive durum wheat cultivars to the plant fungal diseases of stem and yellow (stripe) rusts. To aim this, forty durum wheat recombinant inbred lines (RILs), which were previously determined to be resistant or sensitive to stem and yellow rust diseases, were investigated by the noninvasive Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy. Also, classification of the resistant and sensitive lines depending on the structural and functional differences has been attempted. The FTIR spectra for stem rust disease showed that, resistant durum wheat lines had a significant increase in the population of unsaturation in acyl chains of lipid molecules, an increase in lipid and in total protein content and also an increase in carboxylic acids and alcohols. For yellow rust disease, resistant lines had a significant increase in hydrogen bonding and they had also a more ordered membrane structure. In Principal Component Analysis for stem rust disease, according to 3700-650 cm-1 region, amide III band (1213-1273 cm-1 region) and C-H stretching region (3020- 2800 cm-1), the resistant and sensitive groups were separated successfully. For yellow rust disease, according to 3700-650 cm-1 region, Amide A and Amide III bands, the resistant and sensitive lines were grouped distinctly. FTIR spectroscopy provides a useful approach to determine the differences in molecular structure of durum wheat RILs regarding resistance of lines to fungal diseases. However, further research is still needed to ensure if the structural and functional differences in biomolecules of the samples could be used as molecular markers for discrimination of rust resistant materials from rust sensitive ones.

The barley scald pathogen (Rhynchosporium secalis) causes extensive economic losses, not only through lost product and quality, but also due to costs associated with chemical control. Economic and environmental impacts and the emerging resistance to fungicides and dominant resistance genes are reasons to understand molecular defence responses in order to develop new strategies to increase resistance of barley to this pathogen. In most pathosystems, defence gene expression in susceptible or resistant genotypes commonly differs quantitatively. Thus, differentially expressed genes between genotypes contrasting for response to infection by pathogens are considered candidate genes that have a role in resistance. This thesis presents functional analysis of a subset of genes isolated from a Suppression Subtractive Hybridisation library. The library was previously established and enriched for differentially expressed genes in epidermis of resistant and susceptible near-isogenic barley cultivars inoculated with R. secalis. Functional characterisation involved both investigating their putitative biochemical function as well as the genes‟ role(s) in biotic and abiotic stress responses. Three cDNA clones from the library were selected based on the putative function of the encoded proteins and the full length of the clones and their homologues were isolated from cDNA and genomic DNA. One of the clones represented a member of the pathogenesis-related protein family 17 (PR-17). Southern hybridisation showed that a small multigene family encodes the barley PR-17 proteins. Three members were cloned with two of them being novel. The second clone was homologous to galactinol synthases (GolS) and Southern blot analysis indicated existence of two GolS genes in the barley genome and subsequently two HvGolS members were isolated. The last clone (a single gene) showed similarity to very long chain fatty acid elongases, which indicates its involvement in synthesis of cuticular waxes. A characterised Arabidopsis mutant named fiddlehead (Atfdh) was highly similar to this gene and it was named HvFdh. Detailed expression analysis using Q-PCR, Northern blot analysis and publically available microarray data revealed that the isolated genes are regulated in response to a variety of abiotic and biotic stresses as well as different tissues during barley development. Under some treatments expression patterns were consistent with their putative roles and in agreement with results of other studies. Nevertheless, in other treatments expression profiles were not in agreement with previous findings in other plants indicating potentially different stress adaptation mechanisms between species. Further insight into the function of the encoded proteins was gained by their subcellular localisation using transient expression as GFP fusion proteins followed by confocal laser scanning microscopy. The results were in agreement with in silico predictions and their putative cellular function. In addition, a comprehensive list of homologous genes from other species was compiled for each gene by using public EST databases. Analyses of phylogenetic relationship and multiple sequence alignment of the homologues provided further clues to their function and conserved regions of the proteins. HvPR-17 anti-fungal properties were investigated by heterologous protein expression in E. coli and subsequent in vitro bioassays using purified protein under different conditions against a number of phytopathogenic fungi. However, no anti-fungal activity was observed. A construct with the AtFdh promoter driving the coding region of barley Fiddlehead was used for complementation of the Arabidopsis fiddlehead mutant to investigate functional orthology between these genes from dicots and monocots. The Arabidopsis fiddlehead mutant phenotype that shows contact-mediated organ fusion, germination of spore on epidermis and reduced number of trichomes was completely reverted by HvFdh. Finally, more than fifty transgenic barley lines were regenerated over-expressing or suppressing one of the three genes. The analyses of the transgenic progeny exhibited some interesting developmental phenotypes and resistance to scald and drought tolerance. These lines are awaiting further experiments to investigate the effect of altered expression in conferring resistance to other pathogens and abiotic stress tolerance as well as biochemical analysis. Collectively, in this work six barley genes were cloned and characterised by a variety of in silico techniques, temporal and transient expression analyses, subcellular localisation, in vitro bioassays and mutant complementation in Arabidopsis and loss- and gain-of-function transgenic barley plants. This work has provided insight into the function of these gene families in barley. Furthermore, the data suggest that they are regulated by the defence response to pathogenic fungi as well as drought, salinity and frost in barley. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1375755 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2009

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2010. / Härtill 4 uppsatser.

Thesis (MSc (Genetics))--Stellenbosch University, 2008.

Made available in DSpace on 2014-06-11T19:35:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-11-25Bitstream added on 2014-06-13T19:05:03Z : No. of bitstreams: 1 theodoro_gf_dr_botfca.pdf: 486962 bytes, checksum: 133c964b71961b171a0d897af6f2f21e (MD5) / Universidade Estadual Paulista (UNESP) / Este trabalho visou avaliar a ocorrência da bactéria Curtobacterium flaccumfaciens pv. flaccumfaciens em lavouras de feijoeiro localizadas em alguns municípios do Estado de Santa Catarina nas safras 2002/03 e 2003/04; a reação de genótipos de feijoeiro à murcha-de-curtobacterium; e o efeito de doses de nitrogênio (N) e potássio (K) sobre a severidade da doença, peso da matéria seca e o conteúdo de diversos nutrientes na parte aérea das cultivares IAC Carioca Pyatã, IPR 88 - Uirapuru e SCS 202 - Guará. Foram coletadas plantas com sintoma de murcha em lavouras de feijoeiro em 12 municípios. Posteriormente, procedeu-se ao isolamento, à purificação e à caracterização da bactéria e aos testes de patogenicidade. Aos 10 dias após a semeadura, plântulas de 24 genótipos de feijoeiro, conduzidas em casa-de-vegetação, foram inoculadas com o isolado FJ 36. A severidade da murcha-de-curtobacterium foi avaliada, a cada cinco dias, até aos 25 dias após a inoculação. Tanto no experimento que avaliou o efeito do N (uréia) quanto do K (cloreto de potássio) na severidade da doença, empregou-se a dose recomendada pela análise de solo e variações de 25 e 50 % abaixo e acima da mesma. Foi coletada a parte aérea das plantas antes e após as adubações, aos 25 DAS, para se aferir o peso da matéria seca e o conteúdo de N, fósforo (P), K, cálcio (Ca) e magnésio (Mg). Constatou-se que C. f. pv. flaccumfaciens esteve presente em plantas cultivadas nos municípios de Campos Novos, Faxinal dos Guedes, Guatambu, Ipuaçu, Ponte Serrada e Tigrinhos. Dos genótipos avaliados, somente as cultivares IAC Carioca Akytã, IAC Carioca Aruã e IAC Carioca Pyatã, considerados como padrões de resistência à doença, apresentaram-se com as menores notas de severidade média e valores de área abaixo da curva do progresso da murcha-de-curtobacterium (AACPMC)... . / This work aimed evaluate the occurrence of the bacteria Curtobacterium flaccumfaciens pv. flaccumfaciens in common bean fields of Santa Catarina State, during the harvest of 2002/03 and 2003/04; the reaction of bean genotypes to the bacterial wilt; and the effect of nitrogen (N) and potassium (K) levels on the severity of the disease, the weight of the dry mass and the content of nutrients I nthe aerial part of the cultivars IAC Carioca Pyatã, IPR 88 - Uirapuru and SCS 202 - Guará cultivars. Plants with symptoms of wilt were collected in bean fields of 12 cities. It was made the isolation, purification and cultural characterization of the bacteria and the procedures to fulfill the Kochþs postulates. The inoculation with the strain FJ 36 was done in the 10th day after the sow of 24 common bean genotypes, under greenhouse conditions. The bacterial wilt severity was evaluated, in each five days, until the 25th day after inoculation. It was adopted as treatments the recommended level of N (urea) and K (potassium chloride), by the soil analysis, as well levels 25% and 50% under and below it. The aerial part of the plants was collected before and after the fertilizations, to determine the weight of the dry mass and the content of N, phosphorus (P), K, calcium (Ca) and magnesium (Mg). Curtobacterium flaccumfaciens pv. flaccumfaciens was detected in plants cultivated in Campos Novos, Faxinal dos Guedes, Guatambu, Ipuaçu, Ponte Serrada and Tigrinhos. Regarding the evaluated genotypes, only the cultivars IAC Carioca Akytã, IAC Carioca Aruã e IAC Carioca Pyatã, considered as patterns of resistance, had the lower values of average severity and area under the bacterial wilt progress curve (AUBWPC). However, although to have been considered susceptible, the cultivars SCS 202 - Guará and IPR Graúna showed relatively low values of AUBWPC... (Complete abstract, click electronic address below).

A doença Huanglongbing (HLB) associada a bactéria Candidatus Liberibacter spp., que coloniza os vasos do floema, é considerada uma das mais graves doenças de citros. Uma importante estratégia para o controle desta doença consiste na produção de plantas transgênicas, expressando genes que codificam peptídeos antibacterianos especificamente no local de colonização do patógeno. O objetivo deste trabalho foi obter plantas transgênicas de laranja doce, expressando o gene que codifica o peptídeo antibacteriano atacina A (attA) dirigido por promotores específicos para a expressão gênica no floema. O trabalho foi iniciado com a elaboração de construções gênicas contendo o gene attA (associado ou não ao peptídeo sinal), sob o controle dos promotores AtSuc2 (transportador de sacarose), AtPP2 (proteína de floema 2), clonados de Arabidopsis thaliana, ou CsPP2 (proteína de floema 2), clonado de Citrus sinensis. Os experimentos de transformação genética foram realizados com C. sinensis cv. \'Hamlin\', \'Valência\', \'Pêra\' e \'Natal\', via Agrobacterium tumefaciens, utilizando-se segmento de epicótilo como explante. A identificação de plantas transgênicas foi realizada por meio da análise de PCR. Plantas PCR+ foram aclimatizadas e transferidas para casa-de-vegetação específica para o cultivo de plantas transgênicas. Análises de Southern e Northern blot foram realizadas em plantas aclimatizadas, confirmando-se a integração e transcrição do gene attA, respectivamente. A expressão do gene attA também foi confirmada pela análise de RT-qPCR. Plantas de laranja \'Hamlin\' contendo o gene attA (associado ou não ao peptídeo sinal), sob o controle dos promotores AtSuc2 ou AtPP2 foram propagadas por enxertia, para futura avaliação da resistência a Candidatus Liberibacter asiaticus / Huanglongbing (HLB) associated to Candidatus Liberibacter spp., which colonizes the phloem, is considered one of the most serious diseases of citrus. One important strategy to control this disease consists of producing transgenic plants expressing, in the bacteria colonization tissue, genes encoding antibacterial peptides. The objective of this work was to produce transgenic sweet orange plants expressing genes encoding the antibacterial peptide attacin A (attA) driven by phoem-specific promoters. The work started with the development of the gene constructs, containing the attacin A gene (with or without signal peptide) controlled by either sucrose transporter gene (AtSuc2) or phloem protein 2 gene promoters (AtPP2) from Arabidopsis thaliana, or phloem protein 2 gene promotor (CsPP2) from Citrus sinensis. The genetic transformation of C. sinensis \'Hamlin\', \'Valencia\', \'Pera\' and \'Natal\' cultivars was done via Agrobacterium tumefaciens. Epicotyls segments collected from in vitro germinated seedlings were used as explants. Transgenic plants were identified by PCR analyses. PCR positive plants were acclimatized and transferred to specific greenhouse. Integration and transcription of the attA gene was confirmed in acclimatized transgenic plants by Southern and Northern blot analysis, respectively. The attA gene expression was validated by RT-qPCR analysis. \'Hamlin\' transgenic cultivars containing the AtSuc2 or AtPP2 promoters controlling the expression of attA (with or without signal peptide) were propagated by grafting, for future evaluation of Candidatus Liberibacter asiaticus resistance

Orientador: Antonio Carlos Maringoni / Banca: Antonio Carlos Maringoni / Banca: Roberto Lyra Villas Boas / Banca: Julio Rodrigues Neto / Banca: Silvania Furlan de Oliveira / Banca: Ivan Paulo Bedendo / Resumo: Este trabalho visou avaliar a ocorrência da bactéria Curtobacterium flaccumfaciens pv. flaccumfaciens em lavouras de feijoeiro localizadas em alguns municípios do Estado de Santa Catarina nas safras 2002/03 e 2003/04; a reação de genótipos de feijoeiro à murcha-de-curtobacterium; e o efeito de doses de nitrogênio (N) e potássio (K) sobre a severidade da doença, peso da matéria seca e o conteúdo de diversos nutrientes na parte aérea das cultivares IAC Carioca Pyatã, IPR 88 - Uirapuru e SCS 202 - Guará. Foram coletadas plantas com sintoma de murcha em lavouras de feijoeiro em 12 municípios. Posteriormente, procedeu-se ao isolamento, à purificação e à caracterização da bactéria e aos testes de patogenicidade. Aos 10 dias após a semeadura, plântulas de 24 genótipos de feijoeiro, conduzidas em casa-de-vegetação, foram inoculadas com o isolado FJ 36. A severidade da murcha-de-curtobacterium foi avaliada, a cada cinco dias, até aos 25 dias após a inoculação. Tanto no experimento que avaliou o efeito do N (uréia) quanto do K (cloreto de potássio) na severidade da doença, empregou-se a dose recomendada pela análise de solo e variações de 25 e 50 % abaixo e acima da mesma. Foi coletada a parte aérea das plantas antes e após as adubações, aos 25 DAS, para se aferir o peso da matéria seca e o conteúdo de N, fósforo (P), K, cálcio (Ca) e magnésio (Mg). Constatou-se que C. f. pv. flaccumfaciens esteve presente em plantas cultivadas nos municípios de Campos Novos, Faxinal dos Guedes, Guatambu, Ipuaçu, Ponte Serrada e Tigrinhos. Dos genótipos avaliados, somente as cultivares IAC Carioca Akytã, IAC Carioca Aruã e IAC Carioca Pyatã, considerados como padrões de resistência à doença, apresentaram-se com as menores notas de severidade média e valores de área abaixo da curva do progresso da murcha-de-curtobacterium (AACPMC)... (Resumo completo, clicar acesso eletrônico abaixo). / Abstract: This work aimed evaluate the occurrence of the bacteria Curtobacterium flaccumfaciens pv. flaccumfaciens in common bean fields of Santa Catarina State, during the harvest of 2002/03 and 2003/04; the reaction of bean genotypes to the bacterial wilt; and the effect of nitrogen (N) and potassium (K) levels on the severity of the disease, the weight of the dry mass and the content of nutrients I nthe aerial part of the cultivars IAC Carioca Pyatã, IPR 88 - Uirapuru and SCS 202 - Guará cultivars. Plants with symptoms of wilt were collected in bean fields of 12 cities. It was made the isolation, purification and cultural characterization of the bacteria and the procedures to fulfill the Kochþs postulates. The inoculation with the strain FJ 36 was done in the 10th day after the sow of 24 common bean genotypes, under greenhouse conditions. The bacterial wilt severity was evaluated, in each five days, until the 25th day after inoculation. It was adopted as treatments the recommended level of N (urea) and K (potassium chloride), by the soil analysis, as well levels 25% and 50% under and below it. The aerial part of the plants was collected before and after the fertilizations, to determine the weight of the dry mass and the content of N, phosphorus (P), K, calcium (Ca) and magnesium (Mg). Curtobacterium flaccumfaciens pv. flaccumfaciens was detected in plants cultivated in Campos Novos, Faxinal dos Guedes, Guatambu, Ipuaçu, Ponte Serrada and Tigrinhos. Regarding the evaluated genotypes, only the cultivars IAC Carioca Akytã, IAC Carioca Aruã e IAC Carioca Pyatã, considered as patterns of resistance, had the lower values of average severity and area under the bacterial wilt progress curve (AUBWPC). However, although to have been considered susceptible, the cultivars SCS 202 - Guará and IPR Graúna showed relatively low values of AUBWPC... (Complete abstract, click electronic address below). / Doutor

A produção brasileira de laranjas destina-se à indústria de suco concentrado, principal produto citrícola, sendo o estado de São Paulo o maior produtor e exportador do país. Devido à diminuição da variabilidade genética causada pela multiplicação de plantas cítricas por enxertia, esta cultura se tornou alvo constante de inúmeras pragas e doenças. Dentre estas doenças que vem causando crescentes prejuízos para a citricultura brasileira destaca-se a pinta preta causada pelo fungo Guignardia citricarpa Kiely. O combate desta doença é feito por inúmeras pulverizações de fungicidas que aumenta significativamente o custo para os produtores. Uma possível alternativa para o combate a este patógeno seria a produção de plantas cítricas resistentes a doença, através de transformação gênica. Mas para isto, é necessário conhecer os mecanismos de respostas de defesa da planta e identificar quais genes podem estar relacionados com a defesa. Na tentativa de elucidar as respostas de defesa deste hospedeiro foram feitas análises anatômicas de frutos de laranja doce (Citrus sinensis Osbeck cv. Pêra) inoculados com o patógeno e análise transcricional de duas bibliotecas de cDNA, uma de albedo de frutos sadios(BAFS) e outra de albedo de frutos inoculados com o patógeno (BAFC). As análises anatômicas mostraram que os danos causados pelo patógeno na planta iniciam-se 24 horas após a inoculação com as lesões das células do epicarpo, e continuam gradativamente até 72 horas apresentando a diminuição na quantidade de amido e acúmulo de compostos fenólicos. Foi obtido 184 ESTs válidas da BAFS e 370 da BAF. A anotação e categorização destas sequencias apresentaram que o perfil transcricional encontrado nas duas bibliotecas foi semelhante, porém, na BAFC, foram encontrados transcritos pertencentes à defesa da planta como a catalase, a glutationa e monooxygenases. Está analise mostrou que a resposta de defesa da planta inicia-se com lesões nas células, que a interação é custo-intensiva (redução da reserva de amido) para o hospedeiro, e que a defesa da planta está relacionada a muitos genes de defesa. / The Brazilian production of oranges is mainly for the industry of concentrated juice, it is the most important citrus product, and the state of São Paulo is the largest producer and exporter of the country. Due to the low genetic variability caused by the multiplication of citrus plants by grafting, this culture became constant target of many pests and diseases. Among all the diseases that lead to elevated damage to the Brazilian citrus plantation, one is very relevant, the Black Spot disease caused by the fungus Guignardia citricarpa Kiely. The control of this disease is done by many sprays of fungicides that significantly increase the production cost to the producers. A possible alternative to control this pathogen is the development of resistant citrus plants to disease through genetic transformation. To that, it is necessary to understand the mechanisms of plant defense response and identify the genes related to the defense. In an attempt to elucidate the responses of this host both,anatomical analysis of sweet orange fruits (Citrus sinensis Osbeck cv. Pêra) inoculated with the pathogen and transcriptional analysis of two cDNA libraries of albedo section were done. One was from health fruits (BAFS) and another from albedo of fruits inoculated with the pathogen (BAFC). The analysis showed that the anatomical damage caused by the pathogen in the plant shall begin 24 hours after inoculation showing lesions on epicarp cells, and continue gradually until 72 hours also showing a decrease in the amount of starch and accumulation of phenolic compounds. It was obtained 184 valid ESTs of the BAFS and 370 from BAFC. The annotation and categorization of these sequences showed that the transcriptional profile in the two libraries were similar, however, in BAFC, were found transcripts belonging to plant defense such as catalase, the glutathione and monooxygenases. These analyses showed that the plant defense response begins with lesions in the cells, being it cost-intensive interaction for the host, (with reduction of starch reserves) and the plant defense to resistance is related to many genes.