Grapevine powdery mildew is caused by Erysiphe necator, an Ascomycete fungus and an obligate biotroph restricted to growth on its grapevine host. Biotrophic pathogens form a stable association with host cells without directly causing cell death, and take up nutrients from, in the case of powdery mildew ( PM ), host epidermal cells ( Rumbolz et al., 2000 ). As the fungus grows, its increasing biomass becomes a strong nutrient sink capable of altering assimilate flow and storage in the host. To identify host genes that may mediate nutrient delivery to powdery mildew infected tissues and therefore may contribute to disease susceptibility, a candidate gene approach using degenerate and RT - PCR, and a nontargeted approach using microarray analysis was instigated. Once identified, " susceptibility genes " could be targeted for manipulation to provide alternative resistance strategies based on reduced susceptibility in the future. In addition to genes encoding pathogenesis and stress related proteins, microarray analysis revealed that transcript levels of a putative metal transporter and a cell wall structural protein were elevated in infected berry skin, while aquaporin water channels and genes associated with photosynthesis were generally repressed. Degenerate PCR was used to isolated new cell wall invertase, monosaccharide and amino acid transporter genes and initial RT - PCR revealed that expression of genes involved in sugar mobilisation were the most significantly modulated by powdery mildew infection. Previously unreported hexose transporters ( HTs ), ( VvHT3, VvHT4 and VvHT5 ) and a cwINV ( VvcwINV ) had been isolated from cDNA prepared from powdery mildew infected grapevine leaves. Full length clones of grapevine HTs and cwINV were obtained by RACE PCR. Heterologous expression of the three new HTs in yeast confirmed that VvHT4 and VvHT5 mediated glucose uptake, while VvHT3 did not function in the yeast system. However, transient expression of a translational fusion of the VvHT3 protein with green florescence protein in onion epidermal cells indicated that it is targeted to the plasma membrane of plant cells. Quantitative RT - PCR analysis of these new genes, together with previously reported grapevine HTs and cytoplasmic and vacuolar invertases, indicated that expression of VvcwINV and VvHT5, were significantly up - regulated by PM infection, while a vacuolar invertase was strongly down - regulated by PM infection. Invertase activity assays were in agreement with these findings, showing elevated sucrolytic activity in insoluble fractions and reduced sucrolytic activity in soluble fractions. These results suggest that apoplasmic phloem unloading of sucrose in the infected leaf is elevated and that VvHT5 is induced to recover the additional hexoses from the apoplasm. Basic localisation studies indicated that VvHT5 and VvcwINV are not induced specifically in powdery mildew infected leaf regions, but are induced in a more diffuse distribution within infected leaves. To determine if induction of VvHT5 and VvcwINV is specific to PM infection or if other stimuli may also mediate these responses, leaves were inoculated with downy mildew or stressed by wounding. Transcript levels of VvHT5 and VvcwINV were elevated by wounding and downy mildew infection, suggesting that the induction of these genes may be part of a general stress response. To explore the signalling pathways that may underlie these responses, leaves were treated with the plant growth regulators ethylene, jasmonate and abscisic acid. Exogenous application of ethylene and methyl jasmonate only marginally affected the expression of the genes studied, however foliar application of abscisic acid ( ABA ) induced gene expression changes similar to those observed in response to powdery mildew infection and wounding. Promoter sequences of VvHT3, VvHT4, VvHT5 and VvcwINV were isolated and analysed for the presence of regulatory elements. Compared with the promoters of VvHTs that were not induced by pathogen infection or wounding, the VvHT5 and VvcwINV promoters contained numerous motifs associated with induction by ABA including ABRE, Myc and Myb binding elements. The path of sugar loading into the mesocarp of grape berries during ripening is still poorly understood and few molecular components associated with this process have been described. Quantitative RT - PCR was used to monitor the expression of five HTs and VvcwINV during Cabernet sauvignon and Shiraz berry development and ripening. Of the three new HTs reported here, the expression of VvHT3 is most consistent with a potential role in sugar loading, while VvHT5 is induced late in this process. VvcwINV transcript levels were high pre - ripening and also during the later stages of ripening, therefore based on this expression pattern, a role for this enzyme during ripening is not clearly evident. These results are discussed in terms of an apoplasmic step in phloem unloading in ripening grape berries. This study has provided new insights into the molecular and biochemical processes associated with the formation of carbohydrate sink metabolism in response to stress stimuli, and sugar delivery to grape berries during ripening. ABA - dependant pathways may mediate the stress - associated induction of VvcwINV and VvHT5, presumably to recruit additional carbohydrates to the affected organ to energise repair and defence responses. At this stage it is unknown if this response is beneficial to pathogen nutrition, however potentially, modification of genes associated with carbohydrate sink metabolism could provide an alternative way to engineer resistance to this pathogen. / Thesis (Ph.D.)--School of Agriculture, Food and Wine, 2006.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 76-86).

La peronospora della vite è causata dall’Oomicete eterotallico Plasmopara viticola ed è ampiamente diffusa in tutte le aree caratterizzate da primavere miti e piovose. Gli organi svernanti di questo patogeno sono le oospore, che rappresentano l’unica fonte di inoculo per le infezioni primarie. Recenti studi molecolari hanno evidenziato l’importanza delle infezioni primarie e suggerito una revisione dell’epidemiologia di P. viticola. Questa tesi è costituita dagli studi epidemiologici realizzati per indagare il processo di maturazione e germinazione delle oospore mirati all’elaborazione di un modello dinamico per la simulazione delle infezioni primarie di P. viticola. Il modello è stato successivamente validato in diverse aree viticole italiane e, inoltre, utilizzato come base per un warning system in tempo reale per la gestione dei trattamenti in vigneto. / The heterothallic Oomycete Plasmopara viticola represents the causal agent of downy mildew of grapevine (Vitis spp.). The unique source of inoculum is represented by the overwintering sexual spores, originated by the fusion between anteridium and oogonium, so called oospores. Despite their critical impact on the epidemiology of this disease, knowledge about oospores presents some inconsistencies that are engaged in the present dissertation. Initially, the effect of water moistening the grape leaf litter holding overwintering P. viticola oospores was investigated. A close relationship was found between vapour pressure deficit (VPD in hPa) and aW (water activity) of the leaf litter, so that when VPD is lower than 2.13 hPa there is sufficient water for oospores to develop. Results showed that moisture of the leaf litter due to the water flow from the atmosphere makes the oospore development possible also during non rainy periods. Then, the effects of environmental conditions on the variability in germination dynamics of Plasmopara viticola oospores were studied over five years. The germination course was determined indirectly as the relative infection incidence (RII) occurring on grape leaf discs kept in contact with oospores sampled from a vineyard between March and July. The time elapsed between the 1st of January and the infection occurrence was expressed as physiological time, using sums of hourly rates from a temperature-dependent function only in hours when VPD was not a limiting factor (hydro-thermal time, HT). The Gompertz equation calculated over hydro-thermal time produced a consistent modelling of the general relationships between the germination dynamics of a population of P. viticola oospores and weather conditions. It represents the relative density of the seasonal oospores that should have produced sporangia when they have experienced favourable conditions for germination. Finally, a dynamic model for Plasmopara viticola primary infections on grapevine was elaborated according to a mechanistic approach. Development of the sexual stage of the pathogen was split into different state variables, in which changes from one state to another were regulated by rates depending on environmental conditions. The conceptual model was based on the definition of a primary inoculum season, a seasonal oospore (inoculum) dose, and its division into many coeval cohorts. Each cohort progresses along the primary infection cycle (production and survival of sporangia, release, survival and dispersal of zoospores, infection, appearance of disease symptoms) simultaneously, with a time step of one hour. The model was evaluated by comparing model predictions with disease onset in: i) 100 vineyards of Northern, Southern and Insular Italy (1995 to 2007); ii) 42 groups of potted grapevine plants exposed to inoculum (2006 to 2008). Most of the wrong positive predictions occurred in early season, when the host was in the earlier growth stages, or when the oospore germination was triggered by isolated weak rain events. Considering that neither calibration nor empirical adjustment of model parameters were necessary to obtain accurate simulation, it was concluded that this model produces a reasonable approximation of the primary infection processes underlying oospore development. A warning system based on such a model and on short-term weather forecasts was developed and its use was evaluated in experimental vineyards over a 3-year period in North Italy. An unsprayed control was compared with a “warning” treatment (fungicides were applied when the warning system predicted an infection), a “threshold” treatment (fungicides were applied as in the warning treatment, but only for the oospore cohorts higher than a fixed threshold), and the grower’s schedule. Average efficacy in decreasing disease incidence on leaves compared to the unsprayed control was > 90% for all treatments. On the average, 6.8 sprays were applied following the grower’s schedule; use of the warning system reduced applications by about one half (warning treatment) or two third (threshold treatment). The grower’s schedule was the most expensive control strategy, with average of 337 €/ha; the average saving was 174 and 224 €/ha for the warning and the threshold treatments, respectively. The value of this dissertation consists in two relevant and connected aspects. From one side the studies performed on the oospore maturation and germination allowed to better understand and clarify a key point of the downy mildew epidemics still wrapped by a lack of information. From the other side the model elaborated during this thesis represents a practical and efficient tool that leads to the reduction both of growers’ costs and chemical input in the environment.

The effect of temperature and duration of leaf wetness (DLW) on sporulation of Bremia lactucae was determined for lettuce cv. Ithaca. A single spore isolate of B. lactucae was produced and was identified using lettuce differential lines each of which had known gene for resistance. Potted lettuce plants were inoculated with the isolate and incubated at 15 C, at 100% relative humidity for 24 h and then at a relative humidity lower than 70%. Seven days later, the plants were submitted to five different temperatures (5, 10, 15, 20 and 25 C) and six durations of leaf wetness (4, 6, 8, 10, 12 and 14 h). The number of spores produced was determined at the end of each wet period. After 4 h of incubation no spores were observed at any of the temperatures. Highest number of spores was found at 10 and 15 C for more than 10 h of DLW. The rate of sporulation rapidly increased between 8 and 10 h for all the temperatures, including 25 C, where the amount of spores produced was very low. The observed number of spores was transformed into proportion of maximum sporulation (PMS) by providing each data by the maximum number of spores observed for each experiment. The Richards model was used to describe sporulation as a function of leaf wetness duration and the rate and maximum value expressed as a function of temperature. This approach resulted in a three-dimensional equation that explained 87% of the variation in the PMS. Spore viability was also estimated for each temperature and DLW. It was zero after 6 h of incubation and reached almost the maximum after 10 h for all the temperatures. The sporulation model was validated under field conditions and it predicted high, medium, low, or no sporulation in 8 out of 11 times.

La protection des vignes contre les maladies cryptogamiques telles que le mildiou est assurée majoritairement par des fongicides de synthèse dont certains posent des problèmes environnementaux et sanitaires. L’induction de résistance de la vigne par des éliciteurs des réactions de défenses pourrait permettre de réduire leur utilisation. Toutefois, l’efficacité de cette stratégie est avérée en conditions de serres, mais reste plus variable au vignoble. L’efficacité des éliciteurs est conditionnée par le niveau de réponse défensif de la plante, et plus globalement, par son état physiologique. Dans ce contexte et dans le cadre de ce travail intégré au projet FUI IRIS+, l’objectif était de vérifier si des biostimulants, via leurs effets sur la physiologie de la plante, étaient susceptibles d’augmenter son niveau de réponse aux éliciteurs. Ce projet impliquait en amont le criblage d’éliciteurs et de biostimulants efficaces sur vigne. L’activation des défenses par des éliciteurs a un coût métabolique et énergétique que la plante doit assumer. Dans un premier temps, nous avons utilisé le modèle « suspensions cellulaires » afin de comparer l’impact de deux éliciteurs oligosaccharidiques sur le métabolisme primaire de la vigne. Des analyses enzymatiques et métaboliques ont permis de montrer que l’oligogalacturonide, qui a eu la plus forte capacité à activer le métabolisme secondaire associé à la défense, comparé à la laminarine, est également celui qui a eu l’impact le plus marqué sur le métabolisme primaire ; notamment sur certains glucides et acides aminés. Ainsi, l’élicitation mobilise des ressources, impacte le métabolisme primaire de la vigne et son efficacité pourrait être conditionnée par l’état physiologique global de la plante. Dans un second temps, un criblage a été réalisé afin d’identifier de nouveaux éliciteurs potentiels parmi sept fournis par le partenaire industriel. Des tests de protection contre Plasmopara viticola (agent du mildiou) réalisés sur boutures herbacées en conditions de serres, ont permis de retenir un extrait de plante codé SDN3. Les autres ont été écartés car déjà utilisés en laboratoire (l’un d’entre eux), insuffisants en terme d’efficacité, instables ou phytotoxiques. Des études in vitro et in planta ont révélé que l’activité biologique de SDN3 est liée à deux modes d’action : par activation des défenses et par effet direct contre l’agent pathogène. Dans un troisième temps, un criblage de cinq biostimulants potentiellement efficaces sur vigne était à réaliser mais aucune méthodologie de test n’était disponible au laboratoire. Ainsi, quatre dispositifs de complexités différentes ont dû être mis au point, permettant le suivi phénotypique des parties aériennes et/ou racinaires (et dans certains cas physiologique) : systèmes « godets », « rhizotron plan », « tubes » et un autre système appelé « X » (non décrit car lié à une protection industrielle). Seuls deux modèles ont montré un intérêt. En effet, le modèle « tube » a permis de montrer une augmentation du poids frais moyen par racine primaire en réponse à un apport par la voie racinaire de l’un des produits testés, codé BS3. Le modèle « X » a également mis en évidence une accélération de l’ouverture du bourgeon, et une augmentation du nombre de racines primaires en réponse à BS3 appliqué par voie racinaire. La mise au point d’applications d’éliciteurs et d’infection par P. viticola est en cours de finalisation afin de vérifier l’hypothèse de départ à l’aide de la combinaison de BS3 avec SDN3. / The protection of vineyards against cryptogamic diseases such as downy mildew is mainly ensured by synthetic fungicides, which cause serious environmental and health problems. The induction of resistance by elicitors could allow to reduce their use. However, even if their efficacy is demonstrated in greenhouse conditions, it remains quite variable in field conditions. Indeed, the efficacy of an elicitor depends on the plant’s ability to respond, or more generally, on the latter’s physiological status. In this context, part of the FUI project IRIS+, the aim of this present work is to evaluate whether biostimulants, through their effect on the plant’s physiology, would be able to increase their responsiveness to elicitors. The activation of defenses implies a metabolic and energetic cost that the plant must get in charge. First of all, we used a cell suspension model in order to compare two oligosaccharidic elicitors on the primary metabolism of grapevine. Enzymatic and metabolic analyses showed that the oligogalacturonide, which had a stronger impact on secondary metabolism related to defense, compared to laminarin, also showed a more notable impact on primary metabolism, particularly on some sugars and amino acids. The elicitation of grapevine defenses effectively turned out to require resources. Secondly, a screening was carried out to select the most effective elicitor among seven products that were provided by the company. Protection assays against Plasmopara viticola on herbaceous cuttings allowed us to identity SDN3 as the most interesting candidate. In vitro and in planta studies revealed that the mode of action of SDN3 relied on both the activation of defenses and a direct effect against the pathogen. Lastly, as no protocol, nor methodology were available in our laboratory to screen biostimulants, four systems were developed, in order to monitor phenotypic traits of aerial and/or root system: the “pot” model, the “rhizotron” model, the “tubes”, and another termed “X” (no description allowed because of an industrial protection). Only the “X” model showed potential interests, since it allowed to display biostimulating effects such as the acceleration of the bud opening, and the increase of the mean number of primary roots, in response to BS3, applied to the roots. The development of the protocol to apply the elicitor and to infect with P. viticola spores are currently in progress, in order to assess the initial hypothesis by using the combination of BS3 and SDN3.

Atualmente, a videira Niagara Rosada é a variedade de uva de mesa mais cultivada no Estado de São Paulo, Brasil. Contudo, sua produtividade e custo de produção têm sido afetados pela ocorrência de doenças fúngicas, principalmente o míldio, causado pelo fungo Plasmopara viticola. Para o controle dessa doença, têm sido feitas pulverizações com fungicidas, as quais muitas vezes em excesso. Dentre as variáveis meteorológicas que influenciam a ocorrência de doenças fúngicas nas plantas, a duração do período de molhamento (DPM) é uma das mais importantes. A presença de molhamento sobre a superfície das plantas provê a água requerida pelos patógenos para o processo de germinação e infecção do tecido foliar. Visando a subsidiar sistemas agrometeorológicos de alerta fitossanitário, cuja finalidade é possibilitar a racionalização do uso de fungicidas, os objetivos do presente estudo foram: determinar a posição da videira com a DPM mais longa e sua relação com a DPM medida na posição padrão, a 30 cm de altura sobre o gramado; avaliar a estimativa da DPM sobre o gramado por diferentes modelos a partir de dados meteorológicos obtidos em uma estação meteorológica padrão e verificar as relações entre a DPM estimada para o gramado e a DPM medida no vinhedo; e correlacionar a epidemiologia do míldio (P. viticola) durante o ciclo da videira Niagara Rosada, sem controle químico, com as variáveis DPM medida no vinhedo (posição da videira com a DPM mais longa), DPM estimada na posição padrão sobre o gramado pelo melhor modelo, assim como com outras variáveis meteorológicas. A DPM foi medida em posição padrão sobre o gramado e em quatro diferentes posições da videira: topo da planta com a face superior do sensor voltada para sudoeste e nordeste (Topo-SW e Topo-NE) e altura dos cachos de uva com a face superior do sensor voltada para sudoeste e nordeste (Dossel-SW e Dossel-NE). A epidemiologia do míldio foi avaliada sem controle químico, utilizando-se escala de notas de 0 a 4, com nove níveis de severidade, para as folhas e cachos da videira. No estudo da variabilidade espacial da DPM, não houve diferença significativa entre a parte mais alta (1,6 m) e a parte mais baixa (1,0 m) da videira, assim como entre as faces sudoeste e nordeste das plantas. Ao se analisar a relação entre a DPM sobre o gramado e a DPM nas diferentes posições da videira obtida por meio de regressão linear simples, observou-se uma boa correlação, com R2 = 0,88. Na avaliação dos modelos de estimativa da DPM, o modelo CART foi o que teve o melhor desempenho sobre o gramado, sendo que esta estimativa também apresentou uma boa correlação com a DPM medida no interior do vinhedo. Isso permitiu concluir que é possível estimar a DPM no vinhedo de Niagara Rosada a partir de medidas ou estimativas da DPM na estação meteorológica padrão. Na modelagem do desenvolvimento do míldio, a DPM esteve presente nas melhores correlações com a severidade do míldio na videira Niagara Rosada, mostrando a grande importância desta variável para a ocorrência de doenças fúngicas nos vinhedos. / Nowadays, the \'Niagara Rosada\' grapevine is the most cultivated table grape variety in the State of São Paulo, Brazil. However, yield and production cost of this grapevine have been affected by fungal diseases, mainly downy mildew, caused by Plasmopara viticola fungus. For controlling this disease, producers have been applied an excessive number of sprays with fungicides. Among the meteorological variables that influence the occurrence of fungal plant diseases, leaf wetness duration (LWD) is one of the most important. The wetness presence on plant surface provides the water required by the phatogens to germinate and to infect leaf tissues. Aiming to subsidize the plant disease warning systems, which has as purpose to rationalize the use of fungicides in the vineyards, the objectives of the present study were: to determine the canopy position of the Niagara Rosada table grape with longer LWD and its correlation with measured standard LWD over turfgrass; to estimate LWD over turfgrass considering different models with data from a standard weather station, and to evaluate the correlation between estimated LWD over turfgrass and LWD measured in the vineyard; and to correlate downy mildew occurrence in the vineyard, without chemical control, with measured LWD at the vineyard (canopy position with longer LWD), with estimated LWD in standard condition over turfgrass for the best model, and with other meteorological variables. LWD was measured in standard condition over turfgrass and in four different canopy positions of the vineyard: at the top of the plants, with sensors facing southwest and northeast (Top-SW and Top-NE), and at the grape bunches height, with sensors facing southwest and northeast (Bottom-SW and Bottom-NE). The downy mildew epidemiology during the grapevine cycle was evaluated without chemical control, using scores ranging from 0 to 4, with nine severity levels, for leaves and bunches of grapevine. When the spatial variability of LWD was studied, no significant difference was observed between the top (1.6 m) and the bottom (1.0 m) of the canopy and also between the southwest and northeast face of the plants. The analysis of the relationship between standard LWD over turfgrass and crop LWD in different positions of the grape canopy showed a define correlation (R2 = 0.88). Among the LWD estimative methods, CART was the one with the best performance to estimate LWD over turfgrass. The results from this model also presented a good correlation with measured LWD inside the vineyard, showing that LWD can be estimated for this crop with data from a nearby standard weather station. For the downy mildew modeling, LWD also present the best correlations with disease severity in the \'Niagara Rosada\' vineyard, showing the great importance of this variable for fungal diseases occurrence in this crop.

Cheatgrass (Bromus tectorum) is an invasive annual grass common in several semiarid plant communities in the western U.S. B. tectorum presence increases fire frequency and size, reducing species diversity, and leading to annual species-dominated systems with inconsistent livestock forage potential and degraded wildlife habitat value. Most efforts to manage B. tectorum-dominated rangelands have focused on controlling the plant itself rather than addressing the causes of vegetation change. An alternative approach, ecologically-based invasive plant management (EBIPM), identifies treatments that can alter factors associated with the causes of succession, leading to a more desirable vegetation state. This study utilized the EBIPM framework to design a large-scale demonstration project, which implemented a series of manipulation treatments (mowing, prescribed fire, imazapic herbicide, and seeding with perennial species) to suppress B. tectorum and promote desirable species. The treatments were implemented at two semiarid shrubland sites in northwestern Utah. Treatments were evaluated by measuring resident vegetation cover, density, aboveground biomass, and litter and soil seed banks. Herbicide was most effective in reducing B. tectorum cover, density, and biomass, while fire was effective in reducing seed density in the litter seed bank. Treatment interactions were rarely significant; however, by combining fire and herbicide, increased B. tectorum control was achieved. Seedlings of seeded perennial grasses emerged in all treatments; however, establishment by the end of the first growing season was greatest in treatments involving fire. The results of this study indicate that using a decision-making framework to select a series of treatments that alter the causes of succession can improve the management of B. tectroum-dominated rangelands.

Quinoa (Chenopodium quinoa Willd.) is a pseudocereal native to the Andean region of South America and a staple crop for subsistence farmers in the altiplano of Bolivia and Peru. Downy mildew is the most significant disease of quinoa caused by the pathogen Peronospora farinosa f.sp. chenopodii Byford. This disease greatly impacts quinoa crops with yield losses up to 99%. As fungicides are expensive for farmers, the development of resistant cultivars appears to be the most efficient means for controlling downy mildew. The quinoa germplasm bank contains high amounts of genetic diversity, some of which exhibit mildew resistance. Methods for evaluating mildew severity are important for finding resistant genotypes that are useful in breeding programs. The main objectives of this study were to evaluate and investigate downy mildew resistance in quinoa through several different methods. A simple inoculation method was developed for downy mildew disease assessment by placing a damp piece of cheesecloth on a leaf, pipetting a known spore solution onto the cloth, and subjecting the plants to specific humidity cycles in a growth chamber. After inoculation of five quinoa-breeding lines in a growth chamber, accession 0654 was found to be the most resistant, while genotypes NL6 and Sayana showed moderate resistance. Each of these genotypes displayed some potential for resistance breeding programs. Investigation of the growth and development of P. farinosa through resistant and susceptible quinoa genotypes revealed fewer sporangiophores, hyphal strands, and haustoria among leaf tissues of accession 0654 than in the susceptible Chucapaca cultivar. Peronospora farinosa growth was detected in leaf, petiole, and stem tissues with polymerase chain reaction (PCR) using ITSP primers designed from the internal transcribed spacer (ITS) region of the pathogen. Scanning electron microscopy (SEM) also revealed that P. farinosa penetrated stomata via appressoria, secreted extracellular matrices during sporangia germination, grew intercellularly in leaf and petiole tissues, and exited leaf tissue through stomata. Future research requiring knowledge of resistant quinoa genotypes, P. farinosa growth and development, or inoculation methods for large numbers of small quinoa plants would benefit from this report.

No description available.

No description available.