Resumo: A ervilha (Pisum sativum L.) é um dos alimentos mais produzidos no mundo. Uma das principais doenças que ocorrem na cultura é o oídio, causada por Erysiphe pisi DC. O controle da doença tem sido realizado por meio de pulverizações com fungicidas, no entanto, o uso de cultivares resistentes deve ser pensado como uma alternativa viável e de efetivo controle no manejo da doença. O trabalho objetivou elucidar a herança da resistência ao oídio na cultivar de ervilha MK-10 e caracterizar alguns aspectos histológicos da infecção de E. pisi nessa cultivar. Para o estudo da herança, as gerações F1, F2, retrocuzamentos e geração F3 de MK-10 com duas populações suscetíveis foram avaliadas em ambiente protegido e a epidemia foi aberta. Nas avaliações histológicas, os seguintes parâmetros foram avaliados: % de germinação dos conídios, % de conídios germinados que formaram apressório, % de conídios germinados com apressório que estabeleceram colônia (% colonização) e número de haustórios por colônia. Para comparar as razões de segregação obtidas no estudo da herança da resistência, adotou-se o teste do Qui-quadrado (X²) e para os dados das análises histológicas, utilizou-se o teste Tukey a 5% de probabilidade. Com base nas segregações obtidas no estudo da herança e nas análises histológicas realizadas, concluiu-se que a resistência de MK-10 ao oídio é devida a um par de alelos recessivos, provavelmente o er1er1 e que a resistência é expressa na fase de pré-penetração, completada por uma morte celular localizada pós-penetração. / Abstract: The pea (Pisum sativum L.) is the most produced foods in the world. One of the main diseases that occur in the culture is the powdery mildew, caused for Erysiphe pisi DC. The control of the disease has been carried through by means of sprayings with fungicides, however, the use to cultivars resistants must be thought as a viable alternative and of effective control about the handling of the disease. The work objectified to elucidate the inheritance of the resistance to powdery mildew in cultivar of pea MK-10 and to characterize some histological aspects of the infection of E. pisi in this cultivar. For the study of the inheritance, the F1, F2, generations, backcrosses and F3 generations of MK-10 with two susceptible populations had been evaluated in protected environment, the epidemic were open. In the histological evaluations, the following parameters had been evaluated: percentage of germination, percentage of germinated sporelings forming aprressorium, percentage of germinated sporelings with appressorium that established a colony (% colonization) and number of haustoria for colony. To compare the gotten reasons of segregation in the study of the inheritance of the resistance, the test of Qui-square (X²) was adopted and for the data of the histological analyses, the Tukey test was used 5% of probability. On the basis of the segregations gotten in the study of the inheritance and the carried through histological analyses, were concluded that the resistance of MK-10 to the powdery mildew must to a pair of recessive alleles probably er1er1 and that the resistance is express in the pre-penetration phase, completed for a cellular death located post-penetration. / Orientador: Marcelo Agenor Pavan / Coorientador: Norberto da Silva / Banca: Tiyoko Nair Hojo Rebouças / Banca: Edson Luiz Furtado / Mestre

Resumo: O controle químico tem sido utilizado freqüentemente com baixa eficiência para o fungo Didymella bryoniae, causador da doença crestamento gomoso ou cancro da haste em meloeiro. Porém, alternativas de controle como uso da enxertia e manipulação da nutrição devem ser estudadas. Avaliou-se porta-enxertos e concentrações de potássio na resistência de meloeiro rendilhado a Didymella bryoniae em ambiente protegido. No primeiro experimento, quatro porta-enxertos (melão „Dinero‟, melancia „Ojakkyo‟, abóbora „Strong Tosa‟ e cabaça „Calabash TI-191‟) foram inoculados com discos de micélio (7 mm de diâmetro) de quatro isolados de Didymella bryoniae (Dbr 34, Dbr 36, Dbr 37 e Dbr 39) em caule previamente ferido. Baseado no tamanho da lesão, o porta-enxerto „Dinero‟ foi susceptível, „Strong Tosa‟ moderadamente resistente, „Ojakkyo‟ e „Calabash TI-191‟ resistentes e o isolado Dbr 37 se mostrou mais agressivo dentre os isolados. No segundo experimento, o híbrido de meloeiro „Bônus II‟ foi enxertado nos porta-enxertos „Dinero‟ e „Strong Tosa‟, inoculadas com o isolado Dbr 37 do fungo e submetidas a diferentes concentrações de potássio (0; 62,5; 125; 187,5; 250 mg L-1), tanto plantas enxertadas como pé-franco. As plantas do híbrido „Bônus II‟ enxertadas foram resistentes ao fungo e as pé-franco susceptíveis. As concentrações de potássio não influenciaram no tamanho da lesão e na sobrevivência das plantas, porém influenciaram na condutância estomática e transpiração das plantas. / Abstract: Chemical control of the fungus Didymella bryoniae, causal agent of gummy stem blight of melon, is frequently inefficient. Alternative methods as grafting on resistant rootstocks and nutrition handling must be studied. Different rootstocks and potassium concentrations were tested to control the disease on net melon grown under plastic house conditions. In a first experiment four rootstocks (melon „Dinero‟, watermelon „Ojakkyo‟, squash „Strong Tosa‟ and calabash „TI-191‟) were tested for resistance inoculated by disks mycelium (7 mm diameter) with four isolates ( Dbr 34, Dbr 36, Dbr 37 and Dbr 39) of D. bryoniae in stem previously injured. Based on lesion size „Dinero‟ proved to be susceptible to the fungus, „Strong Tosa‟ moderately resistant and „Ojakkyo‟ and „TI-191‟ resistant and the isolated Dbr 37 proved more aggressive among isolates. In a second experiment hybrid net melon „Bonus II‟ was grafted on rootstocks „Dinero‟ and „Strong Tosa‟, inoculated with isolate Dbr 37 of the fungus and submitted to different potassium concentration (0; 62,5; 125; 187,5; 250 mg L-1), inoculated both grafted plants as non grafted. Grafted plants of „Bonus II‟ were resistant to the fungus and the non grafted ones susceptible. Potassium concentrations did not influenced size of the lesion on the stem and also in the plants survival, however influence on stomatal conductance and transpiration plants. / Orientador: Rumy Goto / Coorientador: Edson Luiz Furtado / Coorientador: Dirceu Maximino Fernandes / Banca: César Júnor Bueno / Banca: Haydée Siqueira Santos / Mestre

De nombreux et divers micro-organismes vivent dans les tissus interne et externe desfeuilles des plantes, la phyllosphère. Ils influencent de nombreux traits, les interactions biotiques,le flux d’énergie, la tolérance au stress de leur hôte et en fin de compte la valeur sélectivede leurs hôtes. Il a été montré que plusieurs traits quantitatifs de plantes structurentla communauté microbienne de la phyllosphère. Ainsi des Loci de ces traits quantitatifs(Quantitative Trait Loci QTL) liés à la structure de cette communauté étaient attendus.L’objectif principal de ce travail était de rechercher des régions génomiques chez le chêne(Quercus robur L.), dont l’effet se prolonge jusqu’au niveau de la communauté, influençantainsi le microbiote de la phyllosphère. Tout d’abord, nous avons étudié la composition etle réseau d’interactions du microbiote de la phyllosphère partant un intérêt particulier àErysiphe alphitoides, un agent pathogène majeur pour les chênes. Nous avons montré quel’infection par E. alphitoides est accompagnée par des changements dans la composition dela communauté fongique foliaire, mais pas dans le composition de la communauté bactérienne.Nous avons souligné certains partenaires d’interaction d’E. alphitoides et nous avonsmontré que le réseau d’interactions microbiennes, contrairement aux résultats précédents,été dominé par des interactions positives. Ensuite nous avons effectué une analyse QTLde descripteurs de la communauté microbienne dans une population de pleins frères. Nousavons trouvé 8 QTL correspondant à des traits de la communauté microbienne: compositionfongique et bactérienne, diversité fongique, pourcentage de Erysiphe alphitoides. Troisd’entre eux sont colocalisés avec un QTL de sensibilité à E. alphitoides, suggèrent un fortdéterminisme génétique de la résistance à l’oïdium chez Q. robur. Enfin, nous présentonsles résultats préliminaires d’une étude d’association génétique et discutons nos résultatsavec une perspective évolutive. / Numerous and various microorganisms inhabit inner and outer tissues of plant leaves, thephyllosphere. They influence many plant traits, biotic interactions, energy flux, host stresstolerance and ultimately the fitness of their hosts. Many plant quantitative traits wereshown to structure the phyllosphere microbial community. Hence quantitative trait loci(QTLs) linked to the structure of this community were expected. The main objective ofthis work was to search for genomic regions in oak (Quercus robur L.), whose effect extendsto the community level, influencing the phyllosphere microbiota. First, we studied thecomposition and the interaction network of oak phyllosphere microbiota with specific focuson Erysiphe alphitoides, a major biotrophic pathogen of oak. We showed that infection byE. alphitoides is accompanied by changes in the foliar fungal community composition butnot in the bacterial community composition. We highlighted likely interaction partners ofE. alphitoides and we showed that the complex microbial interaction network, in contrastto previous findings, was dominated by positive interactions. Next we performed QTLanalysis of microbial community descriptors in a full-sib mapping population of oak. Wefound 8 QTLs for microbial community traits: fungal and bacterial composition, fungaldiversity, and percentage of Erysiphe alphitoides reads. Three of these QTLs colocalizedwith a QTL for powdery mildew sensibility, suggesting for strong genetic determinism ofpowdery mildew resistance in Q. robur. Finally we present preliminary results of a geneticassociation study and discuss our findings within an evolutionary perspective.

MSCAGR / Department of Plant Production

This study was undertaken to determine the sensitivity of grape downy mildew (DM, Plasmopara viticola) and powdery mildew (PM, Erysiphe necator) to commonly used single-site fungicides in Virginia and nearby states. DM and PM isolates were collected from 2005 to 2007. In grape leaf disc bioassays, 92% of the DM isolates were QoI (azoxystrobin)-resistant, but none were resistant to mefenoxam. Eighty-two percent of the PM isolates were QoI-resistant, but none were resistant to boscalid and quinoxyfen. The frequency of the G143A point mutation, which confers high levels of QoI resistance, was quantified in DM and PM isolates by real-time PCR. Most of the QoI-resistant DM and PM isolates contained >95% of the 143A allele. QoI-sensitive DM isolates contained less than 1% of 143A. One out of 145 and 14 out of 154 QoI-resistant DM and PM isolates (able to grow on azoxystrobin concentration ï ³ 1 µg/ml), respectively, contained less than 1% 143A. Most PM isolates exhibited reduced sensitivity to five DMI fungicides when compared to a sensitive subgroup (n=9) and compared to published reports for unexposed populations; the resistance factor (median EC50 of the entire isolate collection divided by median EC50 of sensitive subgroup) was highest for tebuconazole (360) and myclobutanil (350), followed by triflumizole (79), triadimefon (61), and fenarimol (53). Sensitivities to all five DMI fungicides, but also azoxystrobin, were moderately to strongly correlated (pairwise r-values ranging from 0.60 to 0.88). / Master of Science in Life Sciences

Development of fungicide resistance in fungal and oomycete pathogens is a serious problem in grape production. Quinoxyfen is a fungicide widely used against grape powdery mildew (Erysiphe necator). In 2013, E. necator isolates with reduced quinoxyfen sensitivity (designated as quinoxyfen lab resistance or QLR) were detected in Virginia. Field trials were conducted in 2014, 2015, and 2016 at the affected vineyard to determine to what extent quinoxyfen might still contribute to disease control. Powdery mildew control by quinoxyfen was good, similar to, or only slightly less, than that provided by myclobutanil and boscalid in all three years. The frequency of QLR in vines not treated with quinoxyfen declined only slowly over the three years, from 65% to 46%. Information about the mode of action of quinoxyfen is limited; previous research suggests that quinoxyfen interferes with the signal transduction process. We profiled the transcriptomes of QLR and sensitive isolates in response to quinoxyfen treatment, providing support for this hypothesis. Additional transcriptional targets of quinoxyfen were revealed to be involved in the positive regulation of the MAPK signaling cascade, pathogenesis, and sporulation activity. Grape downy mildew (Plasmopara viticola), another important grape pathogen, is commonly controlled by phosphite fungicides. A field trial and laboratory bioassays were conducted to determine whether P. viticola isolates from vineyards with suspected control failures showed reduced sensitivity against phosphite fungicides. Prophyt applied at 14-day intervals under high disease pressure provided poor downy mildew control in the field. Next-generation sequencing technologies were utilized to identify 391,930 single nucleotide polymorphisms (SNPs) and generated a draft P. viticola genome assembly at ~130 megabase (Mb). Finally, field isolates of P. viticola collected from a Virginia vineyard with suspected mandipropamid control failure were bioassayed. The EC50 values of the isolates were >240 μg.ml-1 for mandipropamid, well above the field rate. The PvCesA3 gene of two resistant isolates was sequenced revealing that these isolates had a GGC-to-AGC substitution at codon 1105, the same mutation that has been found associated with CAA resistance elsewhere. / PHD

Afin d'améliorer nos pratiques agricoles dans le contexte d'une agriculture durable, plusieurs agents de lutte biologique (ALB) ont été développés, testés et sont maintenant utilisés dans le monde pour combattre les pertes de rendements causées par les maladies. Blumeria graminis f. sp. hordei ( Bgh) est l'agent pathogène responsable du blanc de l'orge et peut réduire les rendements de cette culture jusqu'à 40%. Un champignon épiphyte, Pseudozyma flocculosa, a été découvert et identifié en 1987 en association étroite avec le blanc du trèfle. Les chercheurs ont alors remarqué que ce champignon exhibait une forte activité antagoniste contre le blanc en détruisant les structures de l'agent pathogène. Suite à d'autres travaux, il est apparu que ce comportement antagoniste était dirigé contre tous les membres des Erysiphales et semblait lié à la synthèse d'un glycolipide antifongique soit la flocculosine. Toutefois, on n'est toujours pas parvenus à associer l'efficacité de l'ALB avec la production de ce glycolipide. Ces observations suggèrent que d'autres facteurs seraient impliqués lorsque les deux protagonistes, l'ALB et le blanc, sont en contact. L'objectif principal de ce projet était donc de chercher d'autres mécanismes moléculaires pouvant expliquer l'interaction P. flocculosa-blanc et orge, en faisant une analyse transcriptomique complète des trois protagonistes en même temps. L'interaction tripartite a été échantillonnée à différents temps suivant l'inoculation de P. flocculosa sur des feuilles d'orge présentant déjà une intensité de blanc d'environ 50%. Les échantillons de feuilles prélevés ont ensuite été utilisés pour l'extraction de l'ARN qui ont été ensuite transformés en ADNc pour la préparation des librairies. Cinq répliquats ont été effectués pour chaque temps et le tout a été séquencé à l'aide de séquençage par synthèse Illumina HiSeq. Les séquences obtenues (reads) ont ensuite été analysées à l'aide du logiciel CLC Genomics Workbench. Brièvement, les séquences obtenues ont été cartographiées sur les trois génomes de référence. Suite à la cartographie, les analyses d'expression ont été conduites et les gènes exprimés de façon différentielle ont été recherchés. Cette étape a été conduite en portant une attention particulière aux gènes codant pour un groupe de protéines appelées CSEP pour “candidate secreted effector proteins” qui seraient possiblement impliquées dans l'interaction tripartite. Parmi les protéines exprimées de façon différentielle en présence du blanc ou en absence de ce dernier, nous avons pu constater que certaines CSEP étaient fortement exprimées en présence du blanc. Ces résultats sont prometteurs et nous offrent une piste certaine pour l'élucidation des mécanismes impliqués dans cette interaction tripartite.

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 description available.