The use of biological control agents (BCAs) in the past has shown limited success as its application has often been done incorrectly, and in addition, management practices are rarely altered to incorporate BCAs. Criteria for the correct application of BCAs have been devised as part of the research, and companies selling these products may use the said criteria. Such application will ensure the correct BCAs are used and, more specifically, used under the correct conditions. The powdery mildew (PM) fungus is often seen to develop resistance to synthetic fungicides and, therefore, alternative control measures are required. BCAs as an alternative pose less risk to the environment, workers and the consumer. A pre-trial has been conducted with a range of BCAs to see if they can control powdery mildew (PM) in a greenhouse environment on hydroponically grown cucumber (Cucumis sativus L.) plants using the variety Baccara that has only a moderate tolerance to PM. The BCAs have been compared to the control (synthetic fungicide: Bravo). Comparative work includes Coyier's model, which has been modified and adapted for these trials to determine the percentage of leaf area covered by the PM infection. Furthermore, the number of fruit harvested per treatment, kilogram yield, total mass of yield and average fruit mass is also used to determine the efficacy of the BCAs as these factors have economic significance to commercial growers. The pre-trial showed promise until the fertigation computer failed, resulting in a nutrient shortage and imbalance, confirming that BCAs alone cannot control PM. Synthetic fungicides were applied until control of PM and plant nutrition was regained. BCAs were re-introduced and used until the end of crop production. The confirmation from the pre-trial has led to the inclusion of silicon in conjunction with the BCAs in the two subsequent trials (Trials 1 & 2). Silicon was applied with the BCAs as a foliar spray on a weekly basis. In trials 1 and 2, the cucumber variety, Palladium, with a high genetic tolerance to PM is used, as this variety is suited to form part of the holistic approach used for trials 1 and 2. Trial 1 showed that treatment A, containing Streptomyces griseovirdis and Streptomyces aureofaciens, had the highest yield. Both of these are bacterial BCAs and demonstrated their adaptability to varied climatic conditions, notably when low humidity was experienced. In treatment B, Trichoderma harzianum strains, Rifai and Uppington, show the slowest rate of PM development. In trials 1 and 2, the best actual PM control was obtained by two fungal based BCAs (Trial 1, treatment C was Ampelomyces quisqualis) and (Trial 2, treatment B was Trichoderma harzianum strains, Rifai and Uppington), showing that fungal BCAs have a place for this application, but the growth-enhancing properties of bacterial based BCAs make economic sense and would make them attractive to growers. Treatment A (Streptomyces spp.) had the most number of fruit for the entire growing period and the best overall yield (kg yield) again. Two of the BCA / silicon treatments have marginally better PM control compared to that of the control (E) treatment, although not statistically significant. Treatment E (control) has the highest average fruit mass in this instance but does not have the highest yield (kg yield) when compared to treatments A and B, possibly due to the growth-enhancing properties of most of these BCAs. Therefore, most of these BCA treatments give fairly inconsistent results that vary possibly according to season, humidity and temperature, making it difficult to predict their efficacy. Using combinations or weekly alternations of these BCAs with extremes of climatic adaptation will probably be the most reliable method of obtaining consistent results. Bacterial BCAs are shown to have lower humidity requirements and produce the most consistent results in terms of fruit number, yield and fruit mass and a combination of bacterial and fungal based BCAs would possibly be the best as this would control PM and yet still have the growth enhancing properties from the bacterial based BCAs. From the research, it can be said that some BCAs in trials 1 and 2 produce results similar to that of the control in terms of percentage leaf area covered by PM and some are shown to have improved yields. Results produced from certain BCA treatments are thus equal to the control; yet provide an environmentally friendly alternative to synthetic fungicides. Silicon is listed as a beneficial element rather than an essential element; however, literature claims it to be highly effective in treating PM in cucurbits. Results from trials 1 and 2 show that control of PM is possible in most cases, when a holistic approach is used. This approach includes a cucumber variety with a high PM tolerance, optimum nutrition, cultural practices and silicon in combination with the BCAs. A complete change of management practices is necessary to implement such a BCA program. / Agriculture, Animal Health & Human Ecology / M. Tech. (Nature Conservation)

The use of biological control agents (BCAs) in the past has shown limited success as its application has often been done incorrectly, and in addition, management practices are rarely altered to incorporate BCAs. Criteria for the correct application of BCAs have been devised as part of the research, and companies selling these products may use the said criteria. Such application will ensure the correct BCAs are used and, more specifically, used under the correct conditions. The powdery mildew (PM) fungus is often seen to develop resistance to synthetic fungicides and, therefore, alternative control measures are required. BCAs as an alternative pose less risk to the environment, workers and the consumer. A pre-trial has been conducted with a range of BCAs to see if they can control powdery mildew (PM) in a greenhouse environment on hydroponically grown cucumber (Cucumis sativus L.) plants using the variety Baccara that has only a moderate tolerance to PM. The BCAs have been compared to the control (synthetic fungicide: Bravo). Comparative work includes Coyier's model, which has been modified and adapted for these trials to determine the percentage of leaf area covered by the PM infection. Furthermore, the number of fruit harvested per treatment, kilogram yield, total mass of yield and average fruit mass is also used to determine the efficacy of the BCAs as these factors have economic significance to commercial growers. The pre-trial showed promise until the fertigation computer failed, resulting in a nutrient shortage and imbalance, confirming that BCAs alone cannot control PM. Synthetic fungicides were applied until control of PM and plant nutrition was regained. BCAs were re-introduced and used until the end of crop production. The confirmation from the pre-trial has led to the inclusion of silicon in conjunction with the BCAs in the two subsequent trials (Trials 1 & 2). Silicon was applied with the BCAs as a foliar spray on a weekly basis. In trials 1 and 2, the cucumber variety, Palladium, with a high genetic tolerance to PM is used, as this variety is suited to form part of the holistic approach used for trials 1 and 2. Trial 1 showed that treatment A, containing Streptomyces griseovirdis and Streptomyces aureofaciens, had the highest yield. Both of these are bacterial BCAs and demonstrated their adaptability to varied climatic conditions, notably when low humidity was experienced. In treatment B, Trichoderma harzianum strains, Rifai and Uppington, show the slowest rate of PM development. In trials 1 and 2, the best actual PM control was obtained by two fungal based BCAs (Trial 1, treatment C was Ampelomyces quisqualis) and (Trial 2, treatment B was Trichoderma harzianum strains, Rifai and Uppington), showing that fungal BCAs have a place for this application, but the growth-enhancing properties of bacterial based BCAs make economic sense and would make them attractive to growers. Treatment A (Streptomyces spp.) had the most number of fruit for the entire growing period and the best overall yield (kg yield) again. Two of the BCA / silicon treatments have marginally better PM control compared to that of the control (E) treatment, although not statistically significant. Treatment E (control) has the highest average fruit mass in this instance but does not have the highest yield (kg yield) when compared to treatments A and B, possibly due to the growth-enhancing properties of most of these BCAs. Therefore, most of these BCA treatments give fairly inconsistent results that vary possibly according to season, humidity and temperature, making it difficult to predict their efficacy. Using combinations or weekly alternations of these BCAs with extremes of climatic adaptation will probably be the most reliable method of obtaining consistent results. Bacterial BCAs are shown to have lower humidity requirements and produce the most consistent results in terms of fruit number, yield and fruit mass and a combination of bacterial and fungal based BCAs would possibly be the best as this would control PM and yet still have the growth enhancing properties from the bacterial based BCAs. From the research, it can be said that some BCAs in trials 1 and 2 produce results similar to that of the control in terms of percentage leaf area covered by PM and some are shown to have improved yields. Results produced from certain BCA treatments are thus equal to the control; yet provide an environmentally friendly alternative to synthetic fungicides. Silicon is listed as a beneficial element rather than an essential element; however, literature claims it to be highly effective in treating PM in cucurbits. Results from trials 1 and 2 show that control of PM is possible in most cases, when a holistic approach is used. This approach includes a cucumber variety with a high PM tolerance, optimum nutrition, cultural practices and silicon in combination with the BCAs. A complete change of management practices is necessary to implement such a BCA program. / Agriculture, Animal Health and Human Ecology / M. Tech. (Nature Conservation)

No presente trabalho, a metodologia NBS-profiling foi utilizada para o desenvolvimento de marcadores RGA (Resistance Genes Analogs) em duas populações de Phaseolus vulgaris, sendo uma derivada do cruzamento entre Bat 93 e Jalo EEP558 (BJ) e a outra derivada do cruzamento entre Carioca e Flor de Mayo (CFM). Uma vez identificados, foram mapeados 32 marcadores RGA na população BJ e 40 na população CFM. Nove dos marcadores alocados no mapa de ligação da população BJ se localizaram em proximidade a clusters de resistência, já identificados por outros pesquisadores. Foi realizado o sequenciamento de 32 dos RGA detectados, sendo 16 da cada população. Cinco sequências oriundas da população BJ e três sequências da população CFM apresentaram similaridade com proteínas de resistência identificadas em P. vulgaris, Glycine max e Medicago truncaluta. O mapa de ligação aqui gerado para a população CFM foi utilizado para o posicionamento de QTL (Quantitative Trait Loci) de resistência à mancha-angular (Pseudocercospora griseola (Sacc.) Crous & Braun) e ao oídio (Erysiphe polygoni DC.). Um total de 12 QTL foi mapeado, cinco associados à resposta à mancha angular e sete à resposta ao oídio. / In the presenty study, the NBS-profiling method was used for the development of RGA (Resistance Gene Analogs) markers in two populations of Phaseolus vulgaris, one derived from a cross between \'Bat 93\' and \'Jalo EEP558\' (BJ) and the other derived from a cross between \'Carioca\' and \'Flor de Mayo (CFM). After their identification, 32 RGA markers were mapped on the BJ population and 40 on the CFM population. Nine of the markers assigned to the linkage map of the BJ population were located in the proximity to clusters of resistance already identified by other researchers. We carried out the sequencing of 32 out of the RGA detected, being 16 from each population. Five sequences derived from the BJ population and three sequences from the CFM population showed similarity to resistance proteins identified in P. vulgaris, Glycine max and Medicago truncaluta. The linkage map here generated for the CFM population was used for the positioning of QTL (Quantitative Trait Loci) for resistance to angular leaf spot (Pseudocercospora griseola (Sacc.) Crous & Braun) and powdery mildew (Erysiphe polygoni DC.). Twelve QTL were mapped, five associated to the response to the angular leaf spot and seven to the powdery mildew.

A videira é uma das plantas mais antigas cultivadas pela humanidade, sendo que no Brasil a uva é a terceira fruta com maior volume de produção, atrás apenas do cultivo das bananas e das laranjas. Apesar da produção rentável, principalmente aos pequenos produtores, o parreiral é susceptível a várias doenças cujo manejo compromete até 59% dos gastos do produtor. No estado de São Paulo, dentre as doenças, três têm destaque: a antracnose (causada pelo Sphaceloma ampelinum), o míldio da videira (causado pelo Plasmopara viticola) e a ferrugem (causada pelo Phakopsora euvitis). Os produtores utilizam controle químico de forma intensa e preventiva, chegando a 100 aplicações de fungicidas em um ciclo de até 120 dias. Os principais fungicidas utilizados são os inibidores da quinona oxidase (QoI), que agem impedindo o transporte de elétrons do citocromo b ao citocromo c1 na cadeia respiratória da mitocôndria. Porém, existem relatos de resistência ao fungicida aplicado no campo em diversos países. As substituições G143A, G137R e F129L na sequência da proteína citocromo b impedem que o fungicida se ligue ao seu sítio alvo. As mutações que levam às substituições estão localizadas em uma das regiões chamada hotspot do gene citocromo b (cytB). Visto que, pela carência de estudos, a resistência genética a esses fungicidas nunca foi relatada no Brasil, o objetivo principal desse trabalho foi sequenciar e caracterizar a região hotspot em isolados de míldio, ferrugem e antracnose provenientes de parreirais do estado de São Paulo. Foram selecionados 35 isolados de 11 locais diferentes; desses, 11 isolados de míldio foram considerados geneticamente resistentes, pois apresentam a mutação para o resíduo alanina na posição 143, e 4 isolados foram considerados geneticamente sensíveis. Os dois isolados de ferrugem selecionados também foram considerados geneticamente sensíveis. Pela estratégia de Genome Walking foi possível sequenciar 65% do gene cytB de um dos isolados brasileiros de P. viticola; foram encontrados poucos polimorfismos e nenhum íntron na sequência analisada. Os resultados obtidos com esse estudo podem servir de suporte para a tomada de decisões de manejo mais adequadas para a realidade da viticultura brasileira; além disso, são importantes para futuros estudos sobre a evolução do patógeno com a pressão seletiva exercida pelos fungicidas. / Grapevine is one of the most ancient cultivated plants and its fruit, grape, is notably important in Brazil, since it is the third most produced, only behind banana and citrus. Although it is rentable especially to smallholders, the vineyard is often attacked by several pathogens and the damages induced by them can compromise up to 59% of the producers\' expenses in order to keep the diseases under control. In Sao Paulo state there are three important diseases that attack vineyards: anthracnose (caused by Sphaceloma ampelinum), downy mildew (caused by Plasmopara viticola) and rust (caused by Phakopsora euvitis). Pest management practices used by the producers relies on intensive and preventive use of fungicides, in which the culture is sprayed 100 times per vineyard\'s growth cycle (that last approximately 120 days). One of the most used fungicides are the quinone oxidase inhibitors (QoI), that act by blocking the electron transport chain at the mitochondria binding at the Qo site of the cytochrome b (cytB) complex. However, there are several reports of the presence of resistant strains in different countries. Resistance is caused by the aminoacids substitutions F129L, G137R and G143A in the cytochrome b protein sequence, that prevent the fungicide molecule binding to its target site. The mutations in the cytB gene that lead to these substitutions are harbored in a region called hotspot for fungicide resistance. Since this type of study was never reported in Brazil, the main purpose of this work was to sequence and characterize the hotspot region of different isolates from anthracnose, downy mildew and rust. Thirty five isolates from eleven different locations were choosen for the study. Eleven of them harbored the mutation that lead to the substitution G143A; these were then considered genetically resistant to the QoI fungicides. On the contrary, four downy mildew and the two rust isolates were considered sensitive to the QoI fungicides, since none of the aminoacids substitutions were observed. Also, by using a technique named Genome Walking it was possible to sequence 65% of cytB gene from a Brazilian downy mildew isolate. In this sequence were found few polymorphisms and none intron. These study findings are unique for Brazilian isolates and might be useful to provide reliable support for the pest management decisions regarding the reality that is found at the vineyards in Brazil. Furthermore, the results presented here are important to the comprehension of pathogen\'s evolution when suffering from a selective pressure caused by the intensive use of fungicides.

No presente trabalho, a metodologia NBS-profiling foi utilizada para o desenvolvimento de marcadores RGA (Resistance Genes Analogs) em duas populações de Phaseolus vulgaris, sendo uma derivada do cruzamento entre Bat 93 e Jalo EEP558 (BJ) e a outra derivada do cruzamento entre Carioca e Flor de Mayo (CFM). Uma vez identificados, foram mapeados 32 marcadores RGA na população BJ e 40 na população CFM. Nove dos marcadores alocados no mapa de ligação da população BJ se localizaram em proximidade a clusters de resistência, já identificados por outros pesquisadores. Foi realizado o sequenciamento de 32 dos RGA detectados, sendo 16 da cada população. Cinco sequências oriundas da população BJ e três sequências da população CFM apresentaram similaridade com proteínas de resistência identificadas em P. vulgaris, Glycine max e Medicago truncaluta. O mapa de ligação aqui gerado para a população CFM foi utilizado para o posicionamento de QTL (Quantitative Trait Loci) de resistência à mancha-angular (Pseudocercospora griseola (Sacc.) Crous & Braun) e ao oídio (Erysiphe polygoni DC.). Um total de 12 QTL foi mapeado, cinco associados à resposta à mancha angular e sete à resposta ao oídio. / In the presenty study, the NBS-profiling method was used for the development of RGA (Resistance Gene Analogs) markers in two populations of Phaseolus vulgaris, one derived from a cross between \'Bat 93\' and \'Jalo EEP558\' (BJ) and the other derived from a cross between \'Carioca\' and \'Flor de Mayo (CFM). After their identification, 32 RGA markers were mapped on the BJ population and 40 on the CFM population. Nine of the markers assigned to the linkage map of the BJ population were located in the proximity to clusters of resistance already identified by other researchers. We carried out the sequencing of 32 out of the RGA detected, being 16 from each population. Five sequences derived from the BJ population and three sequences from the CFM population showed similarity to resistance proteins identified in P. vulgaris, Glycine max and Medicago truncaluta. The linkage map here generated for the CFM population was used for the positioning of QTL (Quantitative Trait Loci) for resistance to angular leaf spot (Pseudocercospora griseola (Sacc.) Crous & Braun) and powdery mildew (Erysiphe polygoni DC.). Twelve QTL were mapped, five associated to the response to the angular leaf spot and seven to the powdery mildew.

Plant immune system is entirely based on the immunities of the individual cells in which systemic signals originate from the infection sites. Powdery mildew disease is one of the agents causing these infection sites, resulting in significant yield losses, if disease develops. Understanding the molecular basis of plant-pathogen interactions is the new trend for fighting against plant pathogens, since classical methods used in selection of resistant plants are becoming less and less efficient nowadays. Thus, finding out the genes which are responsible in plant&rsquo / s resistance is becoming very important. In this thesis, effect of &lsquo / General Control Nondepressible-2&rsquo / (GCN2) homolog protein in barley defense mechanism was aimed to be studied. The GCN2 of yeast was v previously identified in our laboratory as an interacting protein when the yeast cDNA library was screened with a putative yellow rust R gene (Yr10) fragment. There are reports available in the literature for the function of GCN2 protein, which makes it a good candidate for a role in disease resistance. Thus, the barley homologue of GCN2 might have a role in the R protein mediated early disease response of which may be proceeding via Programmed Cell Death (PCD). In order to observe such function of HvGCN2 in barley, silencing of its expression via Virus Induced Gene Silencing (VIGS) was investigated. Therefore, the GCN2 homologue was found to function as dampening the severity of the disease. The silencing with triple technical replicates was observed in 5 of the 6 samples, at an average of 43.2% by qRT-PCR analysis. The pathogen growth levels at different time points were analyzed under light microscope on the silenced and the control samples by measuring the primary and secondary hyphae lengths. The total of 24 seedlings and 292 individual spores were analyzed, and then the level of disease formation was quantitated with 603 primary hyphae and 106 secondary hyphae measurements. Up to 25% hyphae growth rate differences between the control and silenced groups were observed with a probability value less than 0.05 on t-test.

Powdery mildew (PM) is an important foliar disease of many crops, occurring under both greenhouse and field conditions. The application of biological control and soluble silicon (Si) against PM has received increasing acceptance as a result of increased environmental and public concern over the use of fungicides for disease management, and because many key fungicides are no longer effective because of resistance problems. However, success with these control options depends on the development of effective antagonists and understanding how best to use Si in agriculture. Potential antagonists of PM were isolated from naturally infected leaves of different plants. A total of 2000 isolates were tested in a preliminary screening on detached leaves of zucchini. The best 30 isolates showing consistent results were further tested under greenhouse conditions for their efficacy against PM of zucchini. In a greenhouse trial, 23 isolates provided disease control to levels of 30 to 77%. Application of 29 isolates resulted in significant reductions in values of area under disease progress curve (AUDPC). The best five isolates were identified as Clonostachys rosea (Link) Schroers, Samuels, Seifert & Gams (syn. Gliocladium roseum) (Isolate EH), Trichothecium roseum (Pers.) Link (syn. Cephalothecium roseum) (Isolate H20) and Serratia marcescens (Bizio) (Isolates B15, Y15 and Y41). Three adjuvants (Break-ThruR (BK), PartnerR (PR) and Tween-80R (T-80)) were compared for their ability to improve efficacy of spray application of silicon (Si) and biocontrol agents (BCAs) against PM. Both BK and PR improved the efficacy of Si significantly (P < 0.05). Microscopic studies showed that BK affected PM fungi directly and enhanced the deposition of BCAs on the pathogen. Break-ThruR was only toxic to the pathogen mycelia when used at > 0.25 m. .-1, but phytotoxic to zucchini plants when used at > 0.45m. .-1. However, it did not affect the c.f.u. of bacterial BCAs. Use of BK at 0.2-0.4 m. .-1 can be recommended to assist spray application of Si (at 750 mg .-1) or BCAs for improved control of PM. The effect of concentration, frequency of application and runoff of Si sprays applied to the foliage was evaluated for control of PM of zucchini. Silicon (250-1000 mg .-1) + BK (0.25 m. .-1), was sprayed onto zucchini plants at frequencies of 1-3 wk-1. Spraying Si reduced the severity of PM significantly (P < 0.05). Regardless of the concentration of Si, the best results were obtained when the frequency of the treatment was increased, and when spray drift or spray runoff were allowed to reach the rhizosphere of the plants. When Si was applied onto leaves, direct contact between the spray and the pathogen resulted in mycelial death. Part of the spray (i.e., drift and runoff) was absorbed by plant roots, and subsequently played an important role in the health of the plants. If affordable, soluble Si should be included in nutrient solutions of hydroponics or supplied with overhead irrigation schemes when PM susceptible crops are grown. Under greenhouse conditions, application of BCAs, with or without Si, reduced the severity and development of PM significantly (P < 0.001). Application of Si significantly reduced the severity and AUDPC values of PM (P < 0.05 for both parameters). Silicon alone reduced the final disease level and AUDPC values of PM by 23-32%, and improved the efficacy of most BCAs. In the course of the investigation, antagonistic fungi consistently provided superior performances to bacterial isolates, providing disease control levels of up to 90%. Higher overall disease levels reduced the efficacy of Si against PM, but did not affect the efficacy of BCAs. Under field conditions, Si alone reduced disease by 32-70%, Isolate B15 reduced disease by 30-53% and Isolate B15 + Si reduced disease by 33-65%. Other BCAs applied alone or together with Si reduced the disease level by 9-68%. Most BCAs reduced AUDPC values of PM significantly. For most antagonists, better efficacy was obtained when Si was drenched into the rhizosphere of the plant. However, efficacy of some of the BCAs and Si were affected by environmental conditions in the field. Repeated trials and better understanding of how to use Si and the BCAs, in terms of their concentration and application frequency, and their interactions with the plant and the environment, are needed before they can be used for the commercial control of PM. Elemental analysis was conducted to determine the impact of differing application levels of silicon (Si) in a form of potassium silicate (KSi) in solution in terms of Si accumulation and selected elements in different tissues of zucchini and zinnia and growth of these plants, and to study the effect of PM on the levels of selected elements in these two plant species. Plants were grown in re-circulating nutrient solutions supplied with Si at different concentrations and elemental composition in different parts were analysed using EDX and ICP-OES. Increased levels of Si in the solution increased the levels of Si in leaves and roots of both plants without affecting its distribution to other plant parts. In zucchini, the roots accumulated the highest levels of Si, substantially more than in the shoots. In contrast with zinnia, accumulation of Si was highest in the leaves. Accumulation of potassium (K) in shoots of both plants increased with increased levels of KSi in the nutrient solution. However, K levels in flower of zinnia, fruits of zucchini and roots of both plants remained unaffected. Increased level of Si reduced accumulation of calcium (Ca) in both plants. Adding Si into the nutrient solution at 50 mg .-1 resulted in increased growth of zucchini and increased uptake of P, Ca, and Mg by both plant species. However, application of higher levels of Si did not result in any further biomass increase in zucchini. Levels of Si in the nutrient solution had no effects on elemental composition and characteristics of the fruits of zucchini. In both plant species, the presence of PM on the leaves of plants resulted in these leaves accumulating higher levels of Si and Ca, but less P, than leaves of uninfected plants exposed to the same levels of soluble Si. The highest concentrations of Si were observed in leaf areas infected with PM, and around the bases of trichomes. For optimum disease control and maximum accumulation of different elements in these two plants, hydroponic applications of Si at 50-150 mg .-1 is recommended. Five selected biocontrol agents and potassium silicate, used as source of soluble Si, were tested under hydroponic conditions at various concentrations against PM of zinnia (Glovinomyces cichoracearum (DC) Gelyuta, V.P.). Application of BCAs resulted in reductions in final disease level and AUDPC values of PM by 38-68% and 30-65%, respectively. Both severity and AUDPC values of PM were reduced by 87-95% when plants were supplied with Si (50-200 mg .-1). It is proposed that the provision of a continuous supply of Si and the ability of this plant species to accumulate high levels of Si in its leaves were the major reasons for the good response of zinnia to Si treatments against PM. Silicon played a protective role before infection and suppressed development of PM after infection. The combination of the best selected BCAs and Si can be used as an effective control option against PM of zinnia when grown in hydroponic system. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

A videira é uma das plantas mais antigas cultivadas pela humanidade, sendo que no Brasil a uva é a terceira fruta com maior volume de produção, atrás apenas do cultivo das bananas e das laranjas. Apesar da produção rentável, principalmente aos pequenos produtores, o parreiral é susceptível a várias doenças cujo manejo compromete até 59% dos gastos do produtor. No estado de São Paulo, dentre as doenças, três têm destaque: a antracnose (causada pelo Sphaceloma ampelinum), o míldio da videira (causado pelo Plasmopara viticola) e a ferrugem (causada pelo Phakopsora euvitis). Os produtores utilizam controle químico de forma intensa e preventiva, chegando a 100 aplicações de fungicidas em um ciclo de até 120 dias. Os principais fungicidas utilizados são os inibidores da quinona oxidase (QoI), que agem impedindo o transporte de elétrons do citocromo b ao citocromo c1 na cadeia respiratória da mitocôndria. Porém, existem relatos de resistência ao fungicida aplicado no campo em diversos países. As substituições G143A, G137R e F129L na sequência da proteína citocromo b impedem que o fungicida se ligue ao seu sítio alvo. As mutações que levam às substituições estão localizadas em uma das regiões chamada hotspot do gene citocromo b (cytB). Visto que, pela carência de estudos, a resistência genética a esses fungicidas nunca foi relatada no Brasil, o objetivo principal desse trabalho foi sequenciar e caracterizar a região hotspot em isolados de míldio, ferrugem e antracnose provenientes de parreirais do estado de São Paulo. Foram selecionados 35 isolados de 11 locais diferentes; desses, 11 isolados de míldio foram considerados geneticamente resistentes, pois apresentam a mutação para o resíduo alanina na posição 143, e 4 isolados foram considerados geneticamente sensíveis. Os dois isolados de ferrugem selecionados também foram considerados geneticamente sensíveis. Pela estratégia de Genome Walking foi possível sequenciar 65% do gene cytB de um dos isolados brasileiros de P. viticola; foram encontrados poucos polimorfismos e nenhum íntron na sequência analisada. Os resultados obtidos com esse estudo podem servir de suporte para a tomada de decisões de manejo mais adequadas para a realidade da viticultura brasileira; além disso, são importantes para futuros estudos sobre a evolução do patógeno com a pressão seletiva exercida pelos fungicidas. / Grapevine is one of the most ancient cultivated plants and its fruit, grape, is notably important in Brazil, since it is the third most produced, only behind banana and citrus. Although it is rentable especially to smallholders, the vineyard is often attacked by several pathogens and the damages induced by them can compromise up to 59% of the producers\' expenses in order to keep the diseases under control. In Sao Paulo state there are three important diseases that attack vineyards: anthracnose (caused by Sphaceloma ampelinum), downy mildew (caused by Plasmopara viticola) and rust (caused by Phakopsora euvitis). Pest management practices used by the producers relies on intensive and preventive use of fungicides, in which the culture is sprayed 100 times per vineyard\'s growth cycle (that last approximately 120 days). One of the most used fungicides are the quinone oxidase inhibitors (QoI), that act by blocking the electron transport chain at the mitochondria binding at the Qo site of the cytochrome b (cytB) complex. However, there are several reports of the presence of resistant strains in different countries. Resistance is caused by the aminoacids substitutions F129L, G137R and G143A in the cytochrome b protein sequence, that prevent the fungicide molecule binding to its target site. The mutations in the cytB gene that lead to these substitutions are harbored in a region called hotspot for fungicide resistance. Since this type of study was never reported in Brazil, the main purpose of this work was to sequence and characterize the hotspot region of different isolates from anthracnose, downy mildew and rust. Thirty five isolates from eleven different locations were choosen for the study. Eleven of them harbored the mutation that lead to the substitution G143A; these were then considered genetically resistant to the QoI fungicides. On the contrary, four downy mildew and the two rust isolates were considered sensitive to the QoI fungicides, since none of the aminoacids substitutions were observed. Also, by using a technique named Genome Walking it was possible to sequence 65% of cytB gene from a Brazilian downy mildew isolate. In this sequence were found few polymorphisms and none intron. These study findings are unique for Brazilian isolates and might be useful to provide reliable support for the pest management decisions regarding the reality that is found at the vineyards in Brazil. Furthermore, the results presented here are important to the comprehension of pathogen\'s evolution when suffering from a selective pressure caused by the intensive use of fungicides.

La vigne est soumise à la pression de nombreux bioagresseurs dont des parasites obligatoires tels que l’oïdium et le mildiou. La lutte contre les maladies causées par les pathogènes biotrophes nécessite une utilisation souvent intensive de fongicides. Le vignoble consomme à lui seul 16% des fongicides commercialisés chaque année en France. Pour réduire leur impact environnemental qui conduit à l’acquisition de la résistance aux pesticides des pathogène et la présence de résidus dans les vins et dans l’atmosphère, des efforts doivent être entrepris pour développer des stratégies de protection innovante de remplacement ou complémentaire permettant de réduire les intrants pesticides.Les stimulateurs des défenses des plantes permettent de limiter le développement des bioagresseurs en conditions contrôlées. Toutefois, leurs efficacités in natura sont variables et souvent décevantes. Suite au grand nombre de produits potentiellement stimulateurs des défenses des plantes, et à l’intérêt que leur portent les viticulteurs, il est nécessaire de disposer de connaissances et d’outils qui permettent d’évaluer leus efficacités et mieux connaitre leurs potentiels de protection du vignoble. Pour ce faire, une méthode d’évaluation de l’efficacité de produits potentialisateurs ou éliciteurs a été développée au niveau biologique, moléculaire (expression de gènes impliqués dans les défenses) et biochimique (analyses qualitatives et quantitatives des polyphénols), nommée "BioMolChem". Cette méthode a permis d’évaluer l’efficacité de deux phosphonates et d’un analogue de l’acide salicylique, sur différents génotypes et phénotypes de mildiou de la vigne et d’oïdium. Cette approche méthodologique "BioMolChem" a permis d’établir des corrélations entre l’expression de gènes de défense, la présence de certains stilbènes et une efficacité des défenses de Vitis vinifera cv. Cabernet-Sauvignon vis-à-vis de l’oïdium et du mildiou. Les modifications des patrons d’expression des 19 gènes suivis dans les feuilles de vigne et les profils HPLC de polyphénols révèlent des mécanismes de défense multigéniques et complexes. Ainsi, les réactions de défense de la plante sont-elles modulées, en fonction de l’éliciteur considéré, mais aussi en fonction de la diversité phénotypique et génétique des agents pathogènes contre lesquels elle se défend. Ces défenses se caractérisent par une sur-expression d’un ensemble de gènes de défense et une accumulation de composés phénoliques spécifiques.Les marqueurs (gènes et molécules) ainsi identifiés, la méthode "BioMolChem" a été appliquée in natura et a conforté, pour partie, les résultats obtenus au laboratoire. Dans des conditions de fortes pressions parasitaires, il est donc possible de protéger les feuilles et les grappes, à l’aide de SDP et des essais d’association ou d’alternance avec des fongicides conventionnels montrent l’intérêt potentiel de l’emploi des SDP au vignoble. Chemin faisant, dans le cadre d’une viticulture innovante et durable, les SDP et la méthode "BioMolChem" ont été appliqués sur des génotypes hybrides (Vitis vinifera x Muscadinia rotundifolia). Nous révélons que selon le niveau de résistance intrinsèque des génotypes (plus ou moins résistants à l’oïdium et au mildiou), il est possible d’augmenter le niveau de la résistance exprimée par élicitation. Ainsi, les SDP pourraient-ils s’avérer des alliés d’intérêt pour l’utilisation de variétés partiellement résistantes et limiter potentiellement le contournement des QTL de résistance. L’ensemble de ce travail, à but appliqué, a conduit à l’obtention de résultats qui nous permettent de mieux comprendre comment la vigne réagit aux SDP dans son environnement agronomique. Leur exploitation et leur finalisation devraient nous permettre d’exploiter et de mettre en place une utilisation des éliciteurs mieux adaptée, à des stratégies alternatives ou complémentaires de la gestion des bioagresseurs de la vigne. / Powdery (Erysiphe necator) and downy mildew (Plasmopara viticola) are very important grapevine diseases (Vitis vinifera). These two biotrophic pathogens, which are native to the United States, infect green vine tissues and cause significant economic loss as well as environmental damage through the repetitive applications of fungicides. To reduce their environmental impact efforts should be made to develop strategies to protect innovative alternative or complementary to reduce pesticide inputs.In this study, the efficacy and the role of Benzothiadiazole (BTH), a salicylic acid analogue, and two phosphonate derivatives strengthen plant defence mechanisms against various isolates of downy and powdery mildews (Plasmopara viticola and Erysiphe necator). These compounds showed differences in their efficacy depending on the variability of mildews and highly dependent on plant genetics, environmental conditions and selection pressure. The plant defense stimulation could be an alternative or additional method to traditional pest management in the grapevine.Tools “BioMolChem” were developed to better assess the defence status of the plant defences in vitro and in natura. Transcript kinetics of selected defence-related genes and polyphenol contents profiles, during Vitis vinifera-biotrophic pathogen interaction, were characterized, and the impact of pathogen diversity was investigated in the absence or presence of elicitation. In vineyard, under strong pathogen pressures, it is thus possible to protect leaves and clusters, with SDP and assays of association or alternation with conventional fungicides show the potential interest of the use of these SDP in the vineyard.The grapevine defense mechanisms are complex, depending on the elicitor, leading to the coordinated accumulation of pathogenesis-related proteins (PR), the production of phytoalexins, and the reinforcement of plant cell walls.On the way, within the framework of an innovative and sustainable viticulture, the SDP was applied to hybrid genotypes (V. vinifera x M. rotundifolia). We reveal that according to the level of intrinsic resistance of the genotypes (more or less resistant to powdery and to downy mildew), it is possible to increase the level of the expressed resistance. The SDP could become allies of interest in the use of partially resistant grapevine varieties.The present findings provide insights into the potential use of transcripts and stilbenes as markers of the defense status of grapevine leaves with or without elicitation or infection, which should allow us to exploit and develop a better use of elicitors in alternative or complementary strategies in grapevine pest management.

Les variétés traditionnelles de vigne nécessitent de très nombreux traitements phytosanitaires pour lutter contre les maladies cryptogamiques qui touchent leurs parties herbacées, comme le mildiou et l’oïdium, causés par Plasmopara viticola et Erysiphe necator respectivement. Ces traitements, coûteux et préjudiciables pour l'environnement, pourraient être réduits par l’emploi de variétés résistantes. La vigne cultivée européenne (Vitis vinifera, 2n=38) est très sensible au mildiou et à l'oïdium. En conséquence, la résistance doit être introduite à partir d’autres Vitaceae ayant un niveau de résistance plus élevé à ces maladies. Plusieurs origines de résistance ont déjà été observées et inventoriées, en particulier chez l’espèce d’origine américaine Muscadinia rotundifolia (2n=40). Ces facteurs sont d'un intérêt majeur pour la sélection de variétés résistantes. Cependant, lors du processus d'introgression, des difficultés à obtenir des pépins viables en F1 ainsi que des anomalies phénotypiques dans les descendances en rétrocroisement ont été constatées. Afin d’optimiser la gestion des résistances provenant de cette espèce dans les programmes d’amélioration variétale, il est nécessaire de comprendre l’organisation génétique et génomique de M. rotundifolia, et de compléter la connaissance des facteurs de résistance issus de cette espèce. Dans ce contexte, les objectifs de la thèse sont : (i) de réaliser une analyse comparative des génomes de V. vinifera et M. rotundifolia et (ii) d’identifier de nouveaux facteurs de résistance chez M. rotundifolia utilisables à terme en sélection. Pour cela, une carte génétique de M. rotundifolia a été développée à partir d’une population de 200 individus issue de l’autofécondation de M. rotundifolia cv. Regale. Parallèlement, la même population a été testée pour son niveau de résistance au mildiou et à l’oïdium. Une carte génétique couvrant 950 cM a été réalisée. Elle comprend 191 marqueurs microsatellites répartis sur les 20 chromosomes de M. rotundifolia, et permet de conclure à un niveau de macrosynténie très élevé avec V. vinifera. Le groupe de liaison 20 de M. rotundifolia correspondrait à la partie inférieure du groupe de liaison 7 de V. vinifera. Par ailleurs, un QTL de résistance au mildiou a été détecté sur le groupe de liaison 18 de M. rotundifolia, au niveau d’une région riche en gènes de type NBS-LRR, et un nouveau QTL de résistance majeur à la l’oïdium a été mis en évidence sur le groupe de liaison 14 de M. rotundifolia. Ce QTL, nommé Ren5 pour ‘Resistance to Erysiphe necator 5’, montre une action précoce dans l’arrêt de la croissance du mycélium du pathogène, dès l’établissement des premiers stades de biotrophie du champignon. De plus, le QTL Ren5 a été confronté à deux souches supplémentaires d’E. necator, appartenant aux deux groupes d’oïdium retrouvés dans vignobles européens, contre lesquelles il reste efficace. Les données de cartographie génétique générées pour M. rotundifolia dans ce travail, ainsi que la mise en évidence de Ren5 et de son mode d’action, permettront d’améliorer la gestion des facteurs de résistance issus de cette espèce pour la sélection de variétés résistantes au mildiou et à l’oïdium. / Grapevine requires numerous harmful plant-health treatments, in particular to control downy and powdery mildews, caused by Plasmopara viticola and Erysiphe necator, respectively. One way to reduce the use of fungicides in viticulture is to create resistant grapevine cultivars. European cultivated grapevine (Vitis vinifera, 2n=38) is susceptible to mildews, whereas related species such as Muscadinia rotundifolia (2n=40) exhibit high levels of resistance. In breeding programs, resistance factors from these related species are introduced into the susceptible elite varieties. Nevertheless, difficulties in obtaining viable seeds in the siblings of Vitis x Muscadinia crosses are observed. In order to optimize the management of resistance factors from M. rotundifolia in breeding programs, it is necessary to understand the genomic organisation of this species, and to complete the knowledge of these factors. Thus, the main objectives of this work are : (i) making a comparative analysis of V. vinifera and M. rotundifolia genomes and (ii) identifying new resistance factors from M. rotundifolia. For this purpose a framework genetic map of M. rotundifolia cv. Regale has been created, using a 200 individual S1 population. This population has also been screened for its resistance to downy and powdery mildew. A 950 cM genetic map has been generated, including 191 SSR markers distributed across the 20 chromosomes of M. rotundifolia. A high level of macrosynteny has been observed between the Muscadinia map and the genetic maps available for V. vinifera. Linkage group (LG) 20 of M. rotundifolia matches with the lower part of V. vinifera LG7. Furthermore, a QTL for resistance to downy mildew has been identified on M. rotundifolia LG18, and a major QTL for resistance to powdery mildew has been mapped on LG14. The latest, called Ren5 for ‘Resistance to Erysiphe necator 5’, acts during an early stage to stop E. necator’s mycelium growth, since the first stages of biotrophy have been established for the fungus. Moreover, Ren5 has been confronted to two additional powdery mildew strains, belonging to the two European groups of E. necator, and it remained efficient. Gathering knowledge about the genetic organization of M. rotundifolia, and the mechanism and spectrum of action of newly identified resistance factors such as Ren5, will be useful to optimize the management of M. rotundifolia resistance traits in breeding programs aiming to create new resistant varieties to downy and powdery mildews