Made available in DSpace on 2014-08-20T14:33:01Z (GMT). No. of bitstreams: 1 tese_luciane_soares_ribeiro.pdf: 649496 bytes, checksum: 2b9568824b4ab41d46496a2499f734b5 (MD5) Previous issue date: 2012-12-21 / Currently, the use of alternative products in disease control is a promising field mainly when associated with low cost technologies. This work comprises experiments designed to evaluate the reaction of common bean (Phaseolus vulgaris L.) seedlings under Biochemical Oxygen Demand (BOD) incubator conditions and the reaction of adult plants under field conditions to anthracnose (Colletotrichum lindemuthianum), when subjected to fermentation products treatment. At BOD were tested the effects of the fermented products vinegar, white wine and beer in concentrations of 0, 0.1, 1 and 10%, using the cultivars Carioca and BR-1 Ipagro Macanudo, according to the methodology described for the use of BOD incubator for anthracnose research. Fermented products were evaluated as anthracnose resistance inductors in seeds and seedlings, as well as a curative treatment for seedlings. Results obtained from BOD, revealed that only vinegar showed a linear significant response for concentrations tested and only for the experiment of induced resistance in seedlings. For field experiments, which followed those conducted under BOD conditions, it has been used five research-derived genotypes (TB 02-02, BRS Expedito, TB 98-20, BR IPAGRO 1 - Macanudo and TB 02-21) and five landrace genotypes (Guabiju, TB 02-26, 02-23, TB 02-25 and Amarelinho Iolanda) at the same vinegar concentrations for the BOD experiment, verifying the action of vinegar as anthracnose resistance induction agent. Regarding the field experiment, there were no interactions between vinegar and bean genotypes. There was no effect of inoculation with anthracnose, resulting in a similarity of response between control and inoculated plots. Significant vinegar concentration effects for number of plants per plot and grain yield were detected, with a cubic response, being negative the effects on grain yield. The observed trend for low anthracnose incidence on leaves and pods displayed by more susceptible genotypes when treated with vinegar at the higher concentration, may suggest that the vinegar treatment for susceptible cultivars under high anthracnose incidence might display more pronounced effects. The genotypes showed significant differences for all variables suggested by their distinct phenotypic characteristics, the availability of different market alternatives to farmers producing beans. / Atualmente, a utilização de produtos alternativos no controle de doenças é um campo promissor que vem sendo utilizado, principalmente quando associada a tecnologias de baixo custo. O presente trabalho compreende experimentos de avaliação da reação à antracnose (Colletotrichum lindemuthianum) de plântulas de feijão (Phaseolus vulgaris) sob condições de estufas incubadoras tipo BOD com fotoperíodo (BOD) e de plantas adultas em campo, objetivando o estudo da eficiência de produtos fermentados no controle desta doença. Em BOD foram testadas concentrações de 0; 0,1; 1 e 10% dos produtos vinagre, vinho e cerveja branca, utilizando as cultivares Carioca e BR-Ipagro 1 Macanudo, conforme metodologia para estufas incubadoras tipo BOD com fotoperíodo (BOD). Foi verificada a ação dos produtos como agentes indutores de resistência em sementes e em plântulas, e também como tratamento curativo em plântulas. Resultados obtidos em câmara BOD, revelaram que apenas vinagre apresentou resposta significativa linear para as concentrações testadas e apenas no experimento de indução de resistência em plântulas. No experimento em campo, que se seguiu àqueles conduzidos em BOD, foram utilizados cinco genótipos desenvolvidos em centros de pesquisa (TB 02-02, BRS Expedito, TB 98-20, BR IPAGRO 1 Macanudo e TB 02-21) e cinco genótipos de origem crioula (Guabiju, TB 02-26, TB 02-23, TB 02-25 e Amarelinho Iolanda) submetidos às mesmas concentrações do experimento em BOD verificando-se a ação do vinagre, que foi o produto fermentado que apresentou melhor desempenho nos testes em BOD, como agente indutor de resistência à antracnose. Em relação ao experimento de campo, não foram detectadas interações entre vinagre e genótipos de feijão. Não houve efeito da inoculação com antracnose, resultando em similaridade de resposta entre parcelas inoculadas e não inoculadas. Foi detectado efeito significativo das diferentes concentrações de vinagre sobre o número de plantas da parcela e sobre a produtividade de grãos, encontrando-se uma equação cúbica na explicação destes efeitos, sendo que a aplicação de vinagre apresentou efeitos negativos sobre a produtividade do feijão. A tendência de uma menor incidência de antracnose na folha e na vagem em genótipos mais suscetíveis para o tratamento com vinagre na sua maior concentração, pode sugerir que a ação do vinagre em cultivares suscetíveis sob condições de alta incidência de antracnose possa apresentar efeitos positivos mais pronunciados. Os genótipos apresentaram diferenças estatisticamente significativas para todas as variáveis sugerindo, pela suas distintas características fenotípicas, a disponibilidade de diferentes alternativas de mercado aos agricultores produtores de feijão.

La santé des cheptels et la maîtrise des maladies est un enjeu majeur de compétitivité des élevages et de durabilité des filières. Les maladies d'origine bactérienne sont responsables de pertes économiques importantes en pisciculture. La bactérie Flavobacterium psychrophilum, qui touche l’ensemble des salmonidés et plus particulièrement la truite arc-en-ciel (Oncorhynchus mykiss),est très largement distribuée dans le monde et en France. En l'absence de vaccin commercial efficace, la maladie est jusqu’à présent systématiquement combattue à l'aide de traitements antibiotiques. Dans un contexte d’antibiorésistance croissante, il est nécessaire de trouver d’autres moyens de lutte et la sélection d'animaux naturellement plus résistants constitue une priorité pour la filière trutticole française. Si le caractère héritable de la résistance à la maladie est bien démontré chez la truite, une meilleure connaissance de ses différentes composantes et des déterminismes génétiques sous-jacents est nécessaire pour optimiser les modalités d’introduction de la résistance dans les objectifs des schémas de sélection conduits par les entreprises françaises.Dans ce contexte, l’objectif de cette thèse est d’étudier le déterminisme génétique de différentes composantes de la réponse à l’infection par F. psychrophilum par une approche de cartographie de QTL (quantitative trait locus) ; puis d’évaluer dans quelle mesure ces déterminismes dépendent des protocoles infectieux utilisés pour tester la résistance.Pour ce faire, nous avons combiné les résultats obtenus avec des infections expérimentales par injection et balnéation et une infection naturelleEn utilisant, d’une part des croisements expérimentaux entre lignées isogéniques à la résistance contrastée et d’autre part, avec une lignée commerciale élevée dans une entreprise de sélection française.Nous mettons en évidence que la réponse de la truite arc-en-ciel à l’infection par F. psychrophilum est un caractère complexe, contrôlé par un grand nombre de QTL d’effet modéré et en interaction. Nos résultats suggèrent également que les différentes composantes de la réponse à l’infection (résistance, endurance, résilience, portage) sont en partie contrôlées par des déterminants génétiques différents, et que certains mécanismes de défense contre l’infection par F. psychrophilum dépendent de la voie d’infection (infection par balnéation ou par injection). Cette étude ouvre la voie à une meilleure compréhension des mécanismes immunitaires sous-jacents à la réponse de la truite arc-en-ciel à l’infection par F. psychrophilum, et constitue une première étape vers la mise en place de la sélection génomique pour la résistance à F. psychrophilum dans les populations de truites françaises. / Health management and disease control are of both major economic and environmental concerns for animal production systems. Bacterial diseases are responsible for important economic losses in fish farming. Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD), affects particularly rainbow trout (Oncorhynchus mykiss) and is found worldwide where salmonids are raised. As no commercial vaccine is available at the moment, the only way to fight BCWD remains through the use of antibiotic treatments. In a context of growing antibiotic resistance threat, additional methods to tackle fish disease are highly needed. Improving the natural host defense mechanisms against infectious pathogens through selective breeding is one of the priorities for French trout farmers. Moderate heritabilities were estimated for resistance against BCWD, indicating that selective breeding is a promising approach to improve trout defense mechanisms against F. psychrophilum. In order to implement disease resistance in French breeding schemes, we need to better understand the genetic determinism of this trait.In this context, the objective of this PhD is to study genetic determinism of different components of trout response to different infection protocols with F. psychrophilum using a QTL (Quantitative Trait Loci) mapping approach.To do so we used three experimental crosses between isogenic lines of trout with contrasting susceptibility. Finally, a study on a commercial line under selection in a French breeding company was performed after a natural disease outbreak to validate the interest of QTL previously detected.In this study, we showed that an important number of interacting QTL controls rainbow trout response to infection with F. psychrophilum. Our results suggest that different components of response to infection (resistance, endurance, resilience, bacterial load) might be controlled by different genetic determinisms. We also showed that different infection protocols trigger different immune mechanisms that may be specific to the route of entry of the pathogen. This study paves the way toward a better understanding of underlying immune mechanisms of rainbow trout response to F. psychrophilum. Finally, this study is the first step toward implementing genomic selection of resistance to BCWD in French rainbow trout population.

Since the 1970s, loss of herbivores, coral bleaching, pollution, and disease epidemics have reshaped the ecological framework of coral reefs. Staghorn coral, Acropora cervicornis, was a major reef-building scleractinian coral found throughout Florida and the Caribbean that experienced unprecedented population declines primarily due to disease and coral bleaching. These two stressors are coupled; the highest coral disease prevalence occurs after periods of thermal stress caused by increased sea surface temperature. Previous research documented three disease-resistant A. cervicornis genotypes in Panama, but it is unknown if disease-resistant genotypes exist in the Florida Keys. Thermal tolerance has been found to be variable among different species of corals and is relatively unknown in A. cervicornis. To investigate disease resistance and thermal tolerance in corals collected from the Florida Keys, pathogen transmission, thermal tolerance experiments, and coral outplanting studies were conducted, along with histological work to assess the condition of coral tissues. Corals were challenged in situ with exposure to rapid tissue loss (RTL) and bleaching resistance was evaluated ex situ in temperature-controlled seawater tanks, using 39 A. cervicornis genotypes. Disease and bleaching were further characterized in the wild using outplanted colonies. In a pathogen transmission pilot study, 7 out of 39 genotypes developed signs of rapid tissue loss transmission. An expanded transmission experiment that used 12 potentially disease resistant genotypes (based on anecdotal information and results from the pilot study), all genotypes developed signs of RTL transmission. However, susceptibility was variable but not statistically different among genotypes (p>0.05), ranging from 40-100% transmission. Histological analyses revealed significant (p0.05) related to photosynthetic efficiency and tissue condition metrics. No significant differences in mortality, disease, or predation were found between disease resistant and disease susceptible genotypes in outplanting experiments (p>0.05). This study reports the first evidence that disease resistance is present in Florida A. cervicornis genotypes. The variability of disease resistance found within genotypes suggests that genotype is not the only factor influencing disease transmission. Short-term exposure to thermal stress revealed heat tolerant A. cervicornis genotypes, which corroborates with recent published studies. Taken together, these results provide insights into how Caribbean Acropora and other scleractinian species persist through multiple disease and coral bleaching events.

Melon (Cucumis melo L.) and watermelon (Citrullus lanatus (Thunb.) Matsum and Nakai) belong to the family Cucurbitaceae. Bacterial fruit blotch (BFB) disease of cucurbits is an economically devastating plant disease that has caused an estimated loss of up to $450M on watermelon crops and $75M (worldwide) to the seed and transplant industries since 1996. Disease symptoms include water-soaked cotyledons, leaf necrosis, and internal fruit rot. Current commercial management strategies are very limited and include: seed production field sanitation, greenhouse transplant sanitation, copper-based bactericide sprays, crop rotation, disease-free healthy seeds, isolating diseased plants, and peroxyacetic acid seed treatments. The seedborne disease is usually spread by contaminated seeds, and there is a zero-tolerance policy in the seed industry for infected seeds. No nondestructive assays are commercially available to detect BFB in seeds. This research investigated several different aspects of BFB disease such as non-destructive seed detection, green tea seed treatment, candidate NB-LRR genes for disease resistance, and optimization of virus induced gene silencing for melon and watermelon crops. The potential application of attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (ATR-FTIR) and high-resolution X-ray analysis methods for detection of BFB on seeds were evaluated. It was possible to detect BFB in seeds that were pistil inoculated via x-ray imaging and pericarp inoculated via ATR FT-IR. In vitro and in vivo experiments evaluated the potential of tea (Camellia sinensis) and tea polyphenols as seed treatments to sanitize seeds infected with A. citrulli. Green tea unlike black tea inhibited growth of A. citrulli because of polyphenols. Eighty one melon and forty four watermelon NB-LRR genes were reidentified, and genes that have potential resistance against A. citrulli on melon plants were screened based on host selectivity of the pathogen. Finally, the virus-induced, gene-silencing method was optimized for melon and watermelon for further analysis of potential disease resistance genes. BFB can be nondestructively identified in seeds and green tea may be an effective seed treatment with further development. Promising candidate R genes were identified that might confer stable resistance in the right genetic background. / Doctor of Philosophy / Melon and watermelon crops both belong to the gourd family. Bacterial fruit blotch (BFB) disease of cucurbits is an economically devastating plant disease that has caused an estimated loss of up to $450M on watermelon crops and $75M (worldwide) to the seed and transplant industries since 1996. Disease symptoms include water-soaked cotyledons, leaf necrosis, and internal fruit rot. Current commercial management strategies and detection methods are very limited. The seedborne disease is usually spread by contaminated seeds, and there is a zero-tolerance policy in the seed industry for infected seeds. This research investigated several different aspects of BFB disease such as non-destructive seed detection, green tea seed treatment, candidate disease resistance genes, and optimization of virus induced gene silencing methodology for melon and watermelon crops. There are currently no nondestructive assays available to detect BFB in seeds. We evaluated the potential application of attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (ATR-FTIR) and high-resolution X-ray analysis methods for detection of BFB on seeds. It was possible to detect BFB inside layers of seeds that were naturally inoculated through the flowers via x-ray imaging and seedcoat inoculated via ATR FT-IR. In vitro and in vivo experiments evaluated the potential of tea and tea constituents as seed treatments to sanitize seeds infected with BFB. Green tea unlike black tea inhibited growth of BFB. Eighty one melon and forty four watermelon disease resistance genes were reidentified and genes that have potential resistance against BFB on melon plants were screened based on host selectivity of the pathogen. Finally, the virus induced gene silencing method was optimized for melon and watermelon plants for further analysis of potential disease resistance genes. BFB can be nondestructively identified in seeds and green tea may be an effective seed treatment with further development. Promising candidate resistance genes were identified that might confer stable resistance in the right genetic background.

No description available.

No description available.

Switchgrass is a C4 perennial grass that is currently being developed for use as a second generation lignocellulosic biofuel crop. For switchgrass to be fully utilized as a bioenergy crop, large-scale plantings of elite switchgrass germplasm, possibly in monoculture, are likely to occur. This practice may increase the selection pressure on plant pathogens, such as switchgrass rust, which could result in devastating disease epidemics. The identification and deployment of quantitative trait loci (QTLs) and major plant disease resistance genes (R) in switchgrass breeding programs could offer broad spectrum and durable disease resistance in commercial switchgrass cultivars. 'Alamo', a lowland cultivar, is generally resistant to switchgrass rust whereas 'Dacotah', an upland cultivar, is highly susceptible. I hypothesized that major R genes and/or QTLs were contributing to the differences in disease phenotypes of these two cultivars. In this dissertation, bioinformatics and molecular biology approaches were employed to dissect the genetic mechanisms underlying switchgrass rust disease resistance. Novel pseudo-F2 mapping populations were created from a cross derived from 'Alamo' and 'Dacotah'. RNA-sequencing of the pseudo-F2 progenies of 'Alamo' x 'Dacotah' was used to construct a genetic linkage map and to identify potential QTLs correlating with disease resistance. In addition, a homology-based computational method was used to identify 1,011 potential NB-LRR R genes in the switchgrass genome (v 1.1). These potential R genes were characterized for polymorphism and expression differences between 'Alamo' and 'Dacotah'. Moreover, I found that some NB-LRR genes are developmentally regulated in switchgrass. One of the major objectives of switchgrass breeding programs is to develop cultivars with improved feedstock quality; however, changes in the components of the plant cell wall may affect disease resistance. I hypothesized that genetically modified switchgrass plants with altered cell wall components will respond differently than the wild-type to switchgrass rust. Transgenic switchgrass plants overexpressing AtSHN3, a transcription factor with known functions in epicuticular wax accumulation and cell wall deposition, were created. I found that AtSHN3-overexpressing transgenic switchgrass lines were more susceptible than wild-type plants in their response to switchgrass rust. Overall, the results of this dissertation provide a platform for elucidating the molecular mechanisms underlying resistance of switchgrass to switchgrass rust. These findings will help breeders create switchgrass cultivars with improved disease resistance, and will ultimately allow switchgrass to be used for sustainable biomass production. / Ph. D.

Thesis (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Agriculture worldwide is under great pressure to produce enough food in order to sustain the ever-growing world population. Among the many challenges faced by food producers, crop losses and damage caused by fungal plant pathogens is a major problem. The study of fungal pathogens and the interaction between plants and fungi is therefore essential, and has been carried out for many years. Much has been learned in this time, but the full mechanisms of the various modes of fungal attack and plant defence have still not been elucidated. Many fungi rely on the action of cell-wall degrading enzymes (CWDEs) to breach the plant cell wall and facilitate access to the nutrients within. CWDEs are among the very first enzymes to be secreted at the start of fungal attack, and many of them are considered to be essential pathogenesis factors. Endopolygalacturonases (ePGs) are CWDEs that cleave the homogalacturonan stretches of the plant cell wall and are vital virulence factors for a number of fungi, including Botrytis cinerea. An important defence mechanism of plants involves the inhibition of CWDEs in order to halt or slow down the fungal attack. Plant polygalacturonaseinhibiting proteins (PGIPs) are cell wall associated CWDE-inhibiting proteins that specifically act on fungal ePGs. Many different PGIPs from a number of diverse plant species have been described to date. They are known to have differential inhibition capabilities that often result from only a few key amino acid changes within the leucine-rich repeat (LRR) active domains. Previously, the first grapevine PGIP was isolated and characterised from Vitis vinifera cultivar Pinotage (Vvpgip1). This Vvpgip1 gene was overexpressed in the tobacco species Nicotiana tabacum, and was shown to be very effective in reducing the susceptibility of tobacco towards B. cinerea. The combined results confirmed transgene overexpression, increased PGIP activity and a strong resistance response against Botrytis, leading to the characterisation of these lines as having PGIP-specific resistance phenotypes. In a subsequent transcriptomic analysis of these lines it was found that they display differential expression of cell wall metabolism genes and biochemical characteristics that might indicate possible cell wall strengthening compared to wild-type tobacco under uninfecting conditions. The V. vinifera cultivars are all very susceptible to fungal attack, whereas other grapevine species, specifically the North American Vitis species, are known for their strong resistance and even immunity against many fungal pathogens. Thirty seven PGIPs have previously been isolated from these more resistant species. The amino acid sequences of the active domains of these PGIPs were previously aligned with that of VvPGIP1, and the proteins were found to be highly homologous with each other and with VvPGIP1. The different nonvinifera PGIPs separated into 14 subgroups based on their active domain sequences. For this study, one PGIP from each group was selected for functional analysis in tobacco. The selected PGIP-encoding genes were transformed into tobacco by means of Agrobacterium tumefaciens. Analyses of the putatively transformed plantlets were performed to test for transgene presence, transgene expression, and PGIP activity: final transgenic tobacco populations consisting of three to twelve individually transformed lines of nine different nonvinifera PGIPs were obtained. A subset of the resultant transgenic lines was infected with B. cinerea in two independent whole plant infections over 11-14 days in order to investigate the disease resistance afforded by the various PGIPs towards this fungus. A line from the previously characterised VvPGIP1 population was included as reference; all the infections were contrasted to the WT tobacco. All the infected lines overexpressing the non-vinifera PGIPs displayed very strong disease reduction in comparison to the WT control: after initial primary lesion formation, the spread of fungal infection was contained and halted in these lines, while wild-type tobacco plants were severely affected. Although the VvPGIP1 line displayed the characteristic PGIP-defense response, the non-vinifera PGIP plants displayed smaller lesions, indicating very strong resistance phenotypes. The characterised non-vinifera PGIP overexpressing lines, together with the VvPGIP1 line and the WT control were also used to further evaluate the previous observation that overexpression might lead to changes in expression of cell wall genes. Analysis of the expression of a xyloglucan endotransglycosylase (xth) gene in the transgenic population showed that this gene was down-regulated in healthy uninfected tissue from all the transgenic lines tested. This confirmed previous results and have confirmed in all grapevine PGIP overexpressing lines tested so far that this gene is downregulated. XTH is typically involved in cell wall metabolism and specifically in controlling the strength and elasticity of the plant cell wall. From previous work it is known that downregulation of this gene leads to strengthening of the wall. The results obtained in this study showed that the PGIP-specific resistance phenotype seen for VvPGIP1-overexpressing tobacco could be confirmed in transgenic tobacco overexpressing non-vinifera PGIPs from more resistant grapevine species as well. The fact that these PGIPs lines all performed even better than the VvPGIP1 lines in conferring resistance towards B. cinerea provides an interesting angle for further investigation into the structural differences between the non-vinifera PGIPs and VvPGIP1. The transgenic lines are also excellent material to study the in vivo functions of PGIPs further in the context of plant-pathogen interactions. / AFRIKAANSE OPSOMMING: Die landboubedryf is wêreldwyd onder groot druk om genoeg voedsel te produseer vir die groeiende wêreldbevolking. Een van die grootste probleme wat die bedryf ondervind, is die groot skade wat aan gewasse aangerig word deur patogeniese swamme. Dit is dus noodsaaklik om swamme en die interaksie tussen plante en swamme te bestudeer, en dit word al vir jare gedoen. Hoewel daar al baie geleer is in hierdie tydperk, is die volle meganismes van die verskeie maniere hoe swamme aanval en hoe plante hulleself verdedig, nog nie bekend nie. Verskeie swamme maak staat op die aktiwiteit van selwand-afbrekende ensieme (SWAEe) om deur die plantselwand te breek en sodoende toegang tot voedingstowwe in die plantsel te fasiliteer. SWAEe is van die eerste ensieme wat tydens die begin van patogeniese aanval deur swamme afgeskei word en verskeie SWAEe word as noodsaaklike patogeniese faktore beskou. Endopoligalakturonases (ePGs) is SWAEe wat die homogalakturoniese dele van die plantselwand verteer en is noodsaaklike virulensie faktore vir ‘n aantal swamme, onder andere Botrytis cinerea. ‘n Belangrike weerstandsmeganisme van plante behels die inhibering van swam SWAEe om sodoende die patogeen-aanval te stop of te vertraag. Die poligalakturonase-inhiberende proteïne (PGIPs) van plante is selwand-geassosieerde SWAEinhiberende proteïne wat spesifiek teen swam ePGs optree. Verskeie verskillende PGIPs vanuit verskillende plantspesies is tot dusver beskryf. Dit is bekend dat hulle differensiële inhiberende vermoëns het wat dikwels toegeskryf kan word aan slegs ‘n paar belangrike aminosuurvolgordeverskille in die leusien-ryke herhalende (LRH) aktiewe areas. Die eerste wingerd PGIP is vantevore geïsoleer vanuit Vitis vinifera kultivar Pinotage (Vvpgip1) en gekarakteriseer. Hierdie Vvpgip1 geen is ooruitgedruk in die tabakspesie Nicotiana tabacum en was baie effektief om die weerstand van tabak teen die swam Botrytis cinerea te verhoog. Die ooruitdrukking van die transgeen, verhoogde PGIP aktiwiteit en goeie weerstand teen Botrytis cinerea is bevestig, en het gelei daartoe dat die transgeniese VvPGIP1 plantlyne geklassifiseer is as lyne met PGIP-spesifieke weerstandsfenotipes. ‘n Daaropvolgende transkriptomiese analise van die plantlyne het gewys dat hulle differensiële uitdrukking van selwand-geassosieerde gene het, asook biochemiese eienskappe, wat ‘n moontlike selwandversterking aandui in vergelyking met wilde-tipe tabak in die afwesigheid van infeksie. Die V. vinifera kultivars is hoogs vatbaar vir swamme, terwyl ander wingerdspesies, spesifiek die Noord-Amerikaanse spesies, bekend is vir hoë weerstand en selfs immuniteit teenoor verskeie patogeniese swamme. Sewe-en-dertig PGIPs is vantevore geïsoleer vanuit hierdie meer weerstandbiedende spesies. Die aminosuurvolgordes van die aktiewe areas van hierdie PGIPs is vantevore vergelyk met die van VvPGIP1 en dit is gevind dat hierdie proteïne hoogs homoloog is aan mekaar, sowel as aan VvPGIP1. Die verskillende nie-vinifera PGIPs het in 14 groepe verdeel na aanleiding van die homologie van hulle aktiewe areas. Vir hierdie studie is een PGIP vanuit elkeen van hierdie groepe gekies vir verdere funksionele analise in tabak. Die 14 nie-vinifera PGIP-koderende gene is stabiel oorgedra na tabak deur middel van Agrobacterium tumefaciens. Die vermeende transgeniese plante is geanaliseer vir die teenwoordigheid van die transgeen, die uitdrukking daarvan en PGIP aktiwiteit: bevestigde transgeniese tabak populasies wat wissel van drie tot 12 individuele getransformeerde lyne kon verkry word vir nege van die verskillende nie-vinifera PGIPs. ‘n Aantal van die transgeniese lyne is geïnfekteer met B. cinerea in twee onafhanklike heelplantinfeksies vir 11-14 dae om die siekteweerstand van hierdie PGIPs teenoor die swam te evalueer. ‘n Plantlyn van die VvPGIP1-populasie is as ‘n verwysing ingesluit en al die infeksies is vergelyk met die wilde-tipe tabak. Al die geïnfekteerde lyne wat die nie-vinifera PGIPs ooruitdruk het ‘n baie sterk afname in siektesimptome getoon in vergelyking met die wilde-tipe kontrole: na aanvanklikle primêre lesies gevorm het, is die verspreiding van die infeksie ingeperk en gestop in hierdie lyne, terwyl die wilde-tipe plante baie erg geaffekteer is. Terwyl die VvPGIP1 lyn ook die tipiese PGIPweerstandsrespons getoon het, het die nie-vinifera PGIPe kleiner lesies ontwikkel, wat dui op baie sterk weerstandsfenotipes. Die gekarakteriseerde nie-vinifera PGIP ooruitdrukkende lyne, asook die VvPGIP1 lyn en die wilde-tipe kontrole, is gebruik om die vorige waarneming dat die ooruitdrukking kan lei tot veranderinge in selwandgeen-uitdrukking verder te ondersoek. Analise van die uitdrukking van ‘n xiloglukaan-endotransglikosilase (xth) geen in die transgeniese populasie het getoon dat hierdie geen afgereguleer is in gesonde, oninfekteerde weefsel van al die transgeniese lyne wat getoets is. Dit het vorige resultate bevestig en het ook bevestig dat hierdie geen afgereguleer is in alle wingerd PGIP-ooruitdrukkende lyne wat tot dusver getoets is. XTH is tipies betrokke by selwandmetabolisme, spesifiek by die beheer van selwandsterkte en selwandelastisiteit. Dit is uit vorige werk bekend dat die afregulering van hierdie geen lei tot versterking van die plantselwand. Die resultate verkry tydens hierdie studie het gewys dat die PGIP-spesifieke weerstand fenotipe van VvPGIP1-ooruitdrukkende tabak ook bevestig kon word in transgeniese tabak wat nie-vinifera PGIPs vanuit meer weerstandbiedende wingerdspesies ooruitdruk. Die feit dat hierdie PGIP lyne almal selfs beter weerstand teen B. cinerea bied as VvPGIP1 lyne is ‘n interessante invalshoek vir opvolgende ondersoeke na die belang van strukturele verskille tussen die nie-vinifera PGIPs en VvPGIP1. Hierdie transgeniese lyne is ook uitstekende hulpbronne om die in vivo funksies van PGIPs verder te bestudeer in die konteks van plantpatogeen interaksies.

Este trabalho objetivou investigar a existência de polimorfismos de seqüência nucleotídica (SNPs "single nucleotide polymorphisms") em fragmentos genômicos de cana-de-açúcar homólogos a genes de resistência. Para tal, iniciadores sintéticos homólogos às extremidades de uma seqüência expressa identificada (ESTs "expressed sequence tag") como homóloga ao gene Xa1 de arroz (EST SCCCCL3080E03.g) e outra ao gene Rp1-D de milho (EST SCCCCL3080B03.g) foram utilizados para amplificar, em duplicata, fragmentos genômicos de variedades de cana-de-açúcar. Em seguida, os fragmentos foram clonados e seqüenciados. No caso do primeiro EST, foram obtidas seqüências de 119 insertos de uma variedade resistente (SP804966) e 156 de uma variedade suscetível (SP80180) a Xanthomonas albilineans. No segundo caso, foram obtidas seqüências de 167 insertos de uma terceira variedade resistente (R570) e 135 de outra suscetível (SP811763) a Puccinia melanocephala. Para cada EST considerado, as seqüências foram comparadas por meio do programa DNA Sequencher 3.0 (Gene Codes Corporation, Ann Arbor, MI). Nesta análise, somente foram consideradas seqüências reproduzíveis, isto é, que ocorreram nas duas repetições. No caso de ambos ESTs, a comparação de seqüências entre variedades possibilitou a identificação de quatro a seis fragmentos distintos. A comparação entre variedades, por sua vez, revelou a existência de até duas seqüências comuns. Presume-se que estas seqüências reproduzíveis e confirmadas por meio de digestões de clones representativos de cada uma com enzimas de restrição, correspondam a seqüências alélicas. Iniciadores foram sintetizados com base em polimorfismos encontrados entre os diferentes alelos. No entanto, tentativas de amplificar alelos específicos por meio destes iniciadores se revelaram infrutíferas. Tal insucesso deve-se possivelmente à condição polialélica da cana-de-açúcar, que faz com que polimorfismos entre dois alelos sejam compensados por monomorfismos entre estes e os demais alelos. Assim, a utilização de SNPs como marcadores moleculares baseados em PCR em cana-de- açúcar mostra-se mais complexa quando comparada a plantas diplóides. / The objective of this work was to investigate the existence of single nucleotide polymorphisms (SNPs) in genomic fragments homologous to resistance genes in sugarcane. For this purpose, primers were designed based on the sequence of the extremities of an identified expressed sequence tag (ESTs) similar to the Xa1 gene of rice (EST SCCCCL3080E03.g) and another to the maize Rp1-D gene (EST SCCCCL3080B03.g). These primers were used to amplify genomic fragments of sugarcane varieties in duplicate. The fragments were then cloned and sequenced. In the case of first EST, sequences of 119 inserts from a resistant variety (SP804966) and 156 from a susceptible variety (SP80180) to Xanthomonas albilineans were analyzed. In the case of the second EST, sequences of 167 inserts from a third variety (R570) and 135 of a fourth one (SP811763), resistant and susceptible respectively to Puccinia melanocephala, were analyzed. Sequences were compared using the program DNA Sequencher 3.0 (Gene Codes Corporation, Ann Arbor, MI). In this analysis, only reproducible sequences were considered, that is, sequences that occurred in both replicates. Four to six different sequences were identified within varieties whereas comparisons among varieties revealed the existence of up to two sequences in common. These reproducible sequences, which were further confirmed through digestion of representative clones with appropriate restriction enzymes, could correspond to allelic sequences. Primers were designed based on the SNPs detected among these so-called alleles. However, attempts to amplify specific alleles using these primers were unsuccessful. This could be due to the polyallelic condition of sugarcane in which polymorphisms between two alleles could be compensated by monomorphisms between these and other alleles. Thus the use of SNPs as PCR-based molecular markers is not as straightforward in sugarcane as in diploid species.

A mancha preta dos citros (MPC) é uma doença que limita a exportação de laranja brasileira para o Japão e países da Europa. Exceto para Citrus aurantium e seus híbridos, todas as outras variedades são susceptíveis ao patógeno. O fungo Guignardia citricarpa, descoberto por Kiely em 1948 em New South Wales na Austrália, é o estádio sexual do agente causal da MPC e a sua fase imperfeita é Phyllosticta citricarpa. Uma importante característica da MPC é seu longo período de latência. A infecção pode ser efetuada por ascósporos e picnidiósporos. A lesão nos frutos fica restrita ao flavedo (epicarpo), sendo que o fungo não infecta o albedo (mesocarpo). O albedo é rico em celulose, carboidratos solúveis, pectinas, compostos fenólicos (flavonóides), aminoácidos e vitaminas. Os compostos fenólicos presentes nas plantas são produtos do metabolismo secundário, e podem ser resultantes da interação planta-ambiente e podem sintetizados como resposta ao ataque de fitopatógenos. Os fenólicos presentes nos citros incluem flavonóides, antocianinas, coumarinas e psorolenos entre outros. Estes compostos podem exibir ação antimicrobiana e antiviral e podem contribuir controle da MPC. A aplicação de fungicidas é o método de controle da MPC. Uma outra possibilidade de controle seria a ativação de fatores de resistência através do uso de indutores bióticos (Saccharomyces cerevisiae) e abióticos (Bion e ácido salicílico). Deste modo, este trabalho teve como objetivos estudar os efeitos do uso de extratos aquosos, metanólicos e etanólicos de albedo de laranja na germinação formação de apressório e crescimento micelial de P. citricarpa in vitro, bem como avaliar o uso da indução de resistência para o controle da MPC através dos indutores S. cerevisiae, Bion e ácido salicílico em pós e pré-colheita em frutos de laranja ‘Pêra-Rio’ e folhas de limão ‘Siciliano’. Os resultados experimentais mostraram que os extratos de albedo, nas concentrações de 10 e 100 mg / mL de água, foram capazes de inibir em 100% a germinação, formação de apressório e o crescimento micelial de P. citricarpa. Foi observado que os extratos, dependendo da concentração, podem ter ação fungicida ou fungistática. No tocante a utilização de S. cerevisiae, Bion e ácido salicílico, aplicados em pós-colheita, foi observado que estes agentes não foram capazes de impedir o desenvolvimento de novas lesões em frutos de laranja ‘Pêra-Rio’. Também foi constatado que S. cerevisiae e Bion, aplicados em pré-colheita, não foram capazes de induzir resistência contra P. citricarpa em folhas de limão ‘Siciliano’ inoculadas previamente a campo com G. citricarpa. Nesse sentido, sugere-se que outros estudos sejam conduzidos, principalmente no que se refere ao uso potencial de extratos do albedo de laranja como medida alternativa de controle da MPC. / Black spot of citrus (CBS) has been a limiting factor in the export of brazilian oranges to Japan and European countries and Japan. Except for Citrus aurantium and its hybrids, all commercially growing Citrus spp. are susceptible to the pathogen. The fungus Guignardia citricarpa, discovered by Kiely in 1948 in New South Wales, is the sexual stage of the causal agent of CBS and Phyllosticta citricarpa is the imperfect stage. An important characteristic of CBS is the long latent period after infection. The infection is carried out by ascospores and pycnidiospores. The fungicidal application is the most important method of control of CBS. The CBS lesion in citrus fruits is limited to the flavedo, since P. citricarpa does not infect the albedo. The albedo is rich in cellulose, soluble carbohydrates, pectin, phenolic compounds, amino acids and vitamins. The phenolics present in the plants are secondary metabolic products and are believed to be produced as a result of the plant interaction with the enviromment and synthesized as a response to attempted phytopathogen attacks. The phenolics that occur in Citrus include flavonoids, anthocyanins, coumarins and psorolens. These compounds may exhibit antiviral and antimicrobial activities, and may contribute to the control of CBS disease. An another possibility to the CBS control is the activation of factors resistance by the use of abiotics (Bion and salicylic acid) and biotics (Saccharomyces cerevisiae) "plant defence activator" (inducers). Therefore, the objectives of this paper were to study the in vitro effects of aqueous, ethanolic and methanolic albedo orange extracts on the germination, appressorium formation and mycelial growth of P. citricarpa as well as to evaluate the use of the S. cerevisiae, Bion and salicylic acid as "plant defence activator" at post and preharvest conditions in fruit of "Pêra-Rio" and leaves of ’Siciliano’ lemon. The results showed that the use of albedo extracts, 10 and 100 mg per mL of water, inhibited 100 % the germination, appressorium formation and mycelial growth of P. citricarpa. It was also observed that the extracts of albedo, depending upon the concentration, exhibited fungicidal or fungistatic activity. The use of S. cerevisiae, Bion and salicylic acid at postharvest conditions did not affect the development of new lesions of CBS in ‘Pêra-Rio’ orange fruit. It was also observed that the use of S. cerevisiae and Bion at preharvest conditions, did not induce resistance against P. citricarpa in leaves of ‘Siciliano’ lemon naturally infected with G. citricarpa under field conditions. Thus, it is suggested that other studies be carried out, mainly regarding the potential of orange albedo’s extracts as a alternative method for CBS control.