Memoria para optar al Título Profesional De Ingeniero Agrónomo / Esta investigación surge de la necesidad de encontrar nuevas alternativas para el control del oídio de la vid (Erysiphe necator Schw.) que puedan incorporarse en el concepto de manejo integrado de enfermedades en vides, utilizando productos de origen biológico, como el uso de bacterias antagónicas al hongo y extractos de plantas que tengan efecto inhibitorio de la enfermedad. El trabajo se realizó en un viñedo experimental de 5 años en los cultivares Thompson Seedless y Crimson Seedless ubicado en la Facultad de Ciencias Agronómicas de la Universidad de Chile.

In questo lavoro si intende indagare l'effetto degli anti-oidici più diffusi al fine di individuare nuovi approcci per la ricerca sui fungicidi. Le analisi statistiche mostrano che meptildinocap, zolfo, metrafenone e penconazolo, somministrati a dosi di etichetta, prevengono le infezioni di Erysiphe necator qualora applicati fino a 12 giorni prima dell'inoculazione. Meptildinocap e zolfo conservano una buona efficacia anche se applicati fino a 9 e 6 giorni dopo l'inoculazione. Meptildinocap neutralizza il massimo numero di conidi entro 6 giorni dal trattamento. La meta-analisi multivariata eseguita su prove di campo dimostra che tutti i fungicidi riducono la gravità delle infezioni su grappolo oltre l'80%, qualora applicati da 3 a 13 volte a dosi di etichette secondo il criterio fenologico. Nonostante cyflufenamid, quinoxyfen, fenarimol, fenarimol+zolfo, propiconazolo e kresoxim-metil+boscalid siano significativamente più efficaci dello zolfo, la loro applicazione ripetuta rappresenta una condizione favorevole all’insorgenza di resistenze. L’ approccio chemio-metrico all’ analisi dei dati ottenuti da microanalisi in microscopia SEM a raggi X mostra che meptildinocap induce modificazioni nei rapporti tra Ca e Al e P e S sulla parete dei Chasmoteci trattati. Tali modificazioni sono probabilmente dovute al dissolvimento della membrana plasmatica. Questi nuovi approcci possono rappresentare un'utile integrazione ai metodi già noti. / In this work the effect of the most widespread fungicides used for Erysiphe necator management is investigated in order to test new approaches to fungicide research. ANCOVA and Tukey’s post hoc tests showed that meptyldinocap, sulfur, metrafenone and penconazole administrated at label rates prevent E. necator infections when applied up to 12 days before inoculation. Meptyldinocap and sulfur preserve a good efficacy even when applied until 9 and 6 days after inoculation. Meptyldinocap significantly increases E. necator conidia mortality within 6 days exposure. The multi-treatment meta-analysis performed on field trials shows that all fungicides reduce disease severity over 80% on vine bunches when applied from 3 to 13 times at label rates with the phenological criterion. Although cyflufenamid, quinoxyfen, fenarimol, fenarimol+sulfur, propiconazole and kresoxim-methyl+boscalid are significantly more effective than sulfur their repeated application in vineyard represents an important E. necator resistance risk. The application of chemo-metric approach to data earned by SEM X-ray microanalysis shows that meptyldinocap leads to modifications in the relationships between Ca and Al and P and S in treated Chasmothecia wall. Such modifications are probably due to the plasma membrane disruption. The new approaches tested here may represent a useful integration of already known methods.

Orientador: Edson Luiz Furtado / Resumo: A seringueira (Hevea brasiliensis) é a terceira cultura florestal mais plantada no Brasil, e o estado de São Paulo é responsável por mais de 60% da produção de látex do país. É uma espécie nativa da região amazônica que possui hábito caducifólio, e durante o período de reenfolha é suscetível ao ataque de fitopatógenos foliares, que prejudicam e até limitam seu estabelecimento em determinadas regiões. Dentre os principais patógenos foliares da seringueira, destacam-se os fungos causadores de oídios, microrganismos biotróficos que infectam tecidos jovens, e podem causar desfolha, principalmente em condições de viveiro e jardim clonal. O agente causal do oídio da seringueira é descrito como Erysiphe quercicola, porém, devido a insuficientes descrições taxonômicas, morfológicas e moleculares desta doença, seu agente causal permanece em discussão, além da fase sexual deste patógeno carecer de qualquer registro neste hospedeiro. Portanto, o objetivo deste trabalho é caracterizar o oídio da seringueira, utilizando caracterizações morfológicas, análises moleculares e testes de patogenicidade. No período de julho a dezembro de 2019, foram coletadas amostras foliares de seringueira em diferentes estágios fenológicos infectadas com oídio na região de Botucatu, São Paulo. Para a caracterização morfológica, foram registradas e mensuradas características das hifas, conídios e conidióforos, que correspondem à fase assexuada do fungo. A caracterização molecular foi realizada através de análi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Rubber tree (Hevea brasiliensis) is the third most planted forest crop in Brazil, and the state of São Paulo is responsible for more than 60% of the country's latex production. It is a species native to the Amazon region that has a deciduous habit, and during the re-leafing period it is susceptible to the attack of leaf phytopathogens, which harm and even limit its establishment in certain regions. Among the main leaf pathogens of the rubber tree, there are the fungi that cause powdery mildew, biotrophic microorganisms that infect young tissues, and can cause defoliation, especially in nursery and clonal garden conditions. The causal agent of rubber mildew is described as Erysiphe quercicola, however, due to insufficient taxonomic, morphological and molecular descriptions of this disease, its causal agent remains under discussion, in addition to the sexual phase of this pathogen lacking any record in this host. Therefore, the objective of this work is to characterize powdery mildew mildew, using morphological characterizations, molecular analyzes and pathogenicity tests. From July to December 2019, leaf samples of rubber trees were collected at different phenological stages infected with powdery mildew in the Botucatu region, São Paulo. For morphological characterization, characteristics of hyphae, conidia and conidiophores, which correspond to the asexual phase of the fungus, were recorded and measured. Molecular characterization was performed through phylogenetic analyzes o... (Complete abstract click electronic access below) / Mestre

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.

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 / Powdery and downy mildew are two diseases of grapes that can cause large yield losses, and are usually controlled by regular fungicide applications. Development of fungicide resistance has been a growing challenge. Quinoxyfen is a protectant fungicide commonly used against powdery mildews. Unusual grape powdery mildew isolates that grew well on quinoxyfen-treated plants in the laboratory (designated as quinoxyfen lab resistance or QLR) were detected in a Virginia vineyard. In 2014, the first year of this study, 65% of powdery mildew isolates from parts of this vineyard that received no further quinoxyfen treatments had the QLR type of resistance, and this declined only slowly to 46% by the third year. Field trials were conducted in 2014, 2015, and 2016 to determine the efficacy of quinoxyfen in the presence of QLR. Powdery mildew control by quinoxyfen on both grape clusters and leaves was similar to, or only slightly less, than that provided by the standard anti-powdery mildew fungicides myclobutanil and boscalid in all three years. In order to gain a better understanding of the mode(s) of action and resistance mechanism(s) of quinoxyfen, gene expression of QLR and sensitive isolates, both in the presence and absence of quinoxyfen, was analyzed by nucleic acid sequencing. This study confirms previous research suggesting that quinoxyfen interferes with the important biological process signal transduction, and revealed additional gene targets of quinoxyfen. The phosphites are a group of fungicides commonly used to control grape downy mildew. Control failures after phosphite application have occasionally been suspected, and downy mildew isolates from vineyards with and without suspected control failures were tested in laboratory bioassays to determine if any level of resistance could be demonstrated. There was a limited range of sensitivity, and none of the isolates showed a notable loss of sensitivity. A field trial was conducted to determine the efficacy of one phosphite fungicide, Prophyt, applied at 14-day intervals under conditions favorable for disease development. Prophyt provided poor downy mildew control, suggesting that it has to be applied more frequently. Next-generation sequencing technologies were utilized to identify genetic markers for clade identification and generated a draft genome assembly of grape downy mildew, which improves the understanding of grape downy mildew genome. Grape downy mildew isolates collected from a vineyard in Virginia where mandipropamid provided poor control of downy mildew were bioassayed. The isolates tolerated mandipropamid rates well above the field rate, showing that they were indeed resistant. The mutation that confers mandipropamid resistance on other continents was found in the PvCesA3 gene of two resistant isolates.