Empirical vs. mechanistic models for primary infections of Plasmopara viticola*

Plasmopara viticola, the causal agent of grapevine downy mildew, requires fungicide treatments to avoid severe yield losses, so epidemiological models have been elaborated to better manage fungicide scheduling. Primary oosporic inoculum plays a key role in epidemic development, and some of these models have been elaborated following an empirical approach to define quantitative relationships between occurrences of primary infections and weather factors influencing them. Recently, a mechanistic dynamic model was elaborated which accounts for the biological effects of weather on the different stages of the primary infection chain, from the progressive breaking of dormancy in the overwintering oospore population to infection establishment. In this work, three widely used models (3–10 rule, EPI and DMCast) were compared with this model (named UCSC model) on the basis of their theoretical approaches and their accuracy was tested against real data collected over a 9‐year period in Sardinia (Italy). The two empirical models, 3–10 and EPI, seem to be too over‐simplified to simulate correctly a complex biological phenomenon such as the sexual stage of P. viticola they lack consideration of some key stages of the infection chain and simultaneously simulate various biological events, each of which are influenced by specific weather conditions. EPI, in particular, needs specific calibrations and ongoing processes of adaptation that make its use in disease warning difficult. DMCast overcomes these problems because of its mechanistic approach; nevertheless, the empirical method used to model oospore maturation requires validations and, as in the present case, modifications before practical use. The UCSC model, due to its fully mechanistic approach, does not present this problem and provides accurate dynamic simulations of the sexual stage of P. viticola, with a high degree of detail and requiring neither calibrations nor corrections.     

PLASMO: a simulation model for control of Plasmopara viticola on grapevine1

The problem of protecting grapevine against diseases is an old one, but in the last few years new techniques have been developed to reduce cost to the farmer and damage to the ecosystem. These are based on mathematical models describing the state of the plant-parasite environment system. A model for forecasting development of grapevine downy mildew (Plasmopara viticola) is presented. The input variables are temperature, rainfall and leaf wetness (determining infection by sporangia), and RH and temperature (for incubation period). The model also takes into account the limited survival of spores. The output is expressed as %, disease progress. Field validation tests, performed in 1990, 1991 and 1992 in several vineyards in Toscana (central Italy) showed a good correlation between observed and simulated infections. The model allowed the number of treatments to be reduced without any increase in downy mildew damage. It could in future be integrated with grapevine growth and development simulation models in an expert system to determine infected tissue area and thus the economic damage threshold.     

Glycosylation and oxidative dimerization of resveratrol are respectively associated to sensitivity and resistance of grapevine cultivars to downy mildew

Levels of resistance to Plasmopara viticola, from susceptible to highly resistant, of different grapevine cultivars were observed in vineyards and confirmed by the symptoms developed after inoculations. On the abaxial surface of infected leaves, P. viticola developed abundant sporangiophores on susceptible cultivars (Chasselas, Gamay, Gamaret and Pinot Noir), whereas on less susceptible cultivars (Seyval Blanc and Johanniter) the parasite produced few sporangiophores and some necrotic spots at the site of infection. On resistant cultivars (Bronner, Solaris, IRAC 2091), P. viticola induced a hypersensitive response and only necrotic spots were visible and the disease ceased to develop. Stilbenes were analyzed 4, 7, 24, 48 h post-infection (hpi) on small leaf samples cut from the site of infection. Large differences were observed between the cultivars at 24 and 48 hpi. Susceptible grapevines produced resveratrol and its glycoside, piceide. In contrast, resistant plants produced high concentration of ε- and δ-viniferin. Resveratrol and piceide have little or no toxicity activity against P. viticola, whereas δ-viniferin is highly toxic and can be considered an important marker for resistance of grapevine to downy mildew. The importance of oxidative dimerization of resveratrol in comparison to the extent of its glycosylation in defense reaction of grapevines against P. viticola is discussed.     

Warning system in Portugal – organization and technical basis of forecasting*

The forecasting and warning system in Portugal started in 1964 with a control programme applied against Plasmopara viticola, the most important pathogen of grapevine. Since then, studies and new techniques have been developed and applied to other important pests of vineyards and orchards, on the basis of epidemiological and climatic data that supports technical evaluation of the risk. In the last few years, mathematical models have been evaluated as an important part of the control strategy, integrated with biological data from field monitoring. At the moment, forecasting models are available for Plasmopara viticola, Venturia pirina, Venturia inaequalis and Cydia pomonella. The integration of forecasting models with field observations is used to formulate warnings that are sent out to farmers about the need to spray or not.     

葡萄叶提取物对葡萄霜霉病的防治作用及有效成分分析

为探究葡萄叶提取物对葡萄霜霉病的防治作用,测定了葡萄叶提取物及其有效物质对葡萄生单轴霉Plasmopara viticola孢子囊和游动孢子的抑制作用以及对葡萄霜霉病的防治效果,观察了其对葡萄生单轴霉在叶片中扩展、产生孢子囊能力的影响,并对葡萄叶提取物组成成分进行了分析。结果表明,葡萄叶提取物对葡萄生单轴霉孢子囊的形成和萌发均有明显的抑制作用。当葡萄叶提取物浓度为10 mg/mL时,其对孢子囊形成的抑制率为82.4%,葡萄生单轴霉孢子囊和游动孢子的萌发率仅为7.6%和1.1%,均显著低于无菌水处理。荧光显微观察发现,葡萄叶提取物可以抑制葡萄生单轴霉在叶片内的扩展。当葡萄叶提取物浓度为10 mg/mL和50 mg/mL时,对葡萄霜霉病的室内防治效果分别为80.1%和100.0%;且浓度为10 mg/mL的葡萄叶提取物对葡萄霜霉病的田间预防效果为67.8%,与烯酰吗啉处理无显著差异。液质联用技术检测显示,葡萄叶提取物中含有白藜芦醇和紫檀芪,其对葡萄生单轴霉均有显著的抑制作用,且紫檀芪的抑制效果强于白藜芦醇。     

Transboundary system for grapevine downy mildew risk evaluation on a territorial scale (P. Rada project)*

A wide and homogenous viticultural area traverses the border between Friuli Venezia Giulia (Italy) and the western part of Slovenia. Despite similar agricultural, climatic and geographical conditions on each side of the border, the management of agricultural activities and, in particular, the organization of phytosanitary campaigns is carried out separately and differently between the two countries incurring a waste of money, time and energy. The project P. Rada (EU Interreg IIIA Italy Slovenia 2000–06) was set up with the aim of establishing a common expert agrometeorological system to evaluate the development of Plasmopara viticola on a territorial scale. The required agrometeorological data are collected by a network of weather stations and spatially interpolated in a grid with a resolution of 500 × 500 m. The spatial dataset obtained is integrated with rainfall images processed by a polarimetric radar located in Fossalon di Grado (Gorizia, Italy). Once the territorial dataset has been completed, data are used to feed two agrometeorological models: SWEB, for the estimation of leaf wetness, and PLASMO for the simulation of grapevine downy mildew. The main output of the system is represented by meteorological (temperature, rainfall, leaf wetness) and epidemiological (total and current number of infections, days for the outbreak of the current infection) maps capable of supplying end users with detailed information useful for planning and management of grapevine protection activity.     

Site-specific field resistance of grapevine to <Emphasis Type="Italic">Plasmopara viticola</Emphasis> correlates to altered gene expression and was not modulated by the application of organic amendments

The influence of site on resistance of grapevine (cv. Chasselas) to Plasmopara viticola was evaluated. Grapevine leaves from three vineyards in the region of Lake Neuchatel (Switzerland) were tested for their susceptibility to P. viticola in the lab in five successive years (2004–2008), and the expression levels of four selected defence-related genes (Glucanase, Lipoxygenase 9, 9-cis epoxycarotenoid dioxygenase, Stilbene synthase) were studied in 1 year. In all 5 years of examination, differences between sites were substantial. In four out of 5 years, plants from site Hauvernier were much less susceptible to P. viticola than plants from site Auvernier. In another year, differences were less pronounced but still significant for one leaf age. Susceptibility of plants from a third site (Concise) varied from year to year. Differences in the genetic background were excluded by microsatellite analysis. Differences in susceptibility were mirrored in the constitutive expression pattern of four defence-related genes, with samples from the Hauterive site clearly separated from samples of the other two sites in redundancy analysis. Furthermore, it was evaluated whether site-specific resistance can be modulated by agronomic practices such as the application of organic amendments. In two commercial vineyards (cv. Pinot noir), soils had either not (control) or yearly (compost) been amended with a compost for the last 9 years. Leaves from plants grown in any of the two treatments did not differ in their susceptibility to P. viticola in both years of examination. Additionally, under controlled conditions, none of 19 different composts amended to the substrate of grapevine seedlings or cuttings affected their susceptibility to P. viticola, but 8 out of 19 composts reduced severity in the control bioassay Arabidopsis thaliana—Hyaloperonospora arabidopsidis, indicating that a modulation of site-specific susceptibility of grapevine plants by organic amendments is at the very least, difficult.     

Pre-symptomatic detection of <Emphasis Type="Italic">Plasmopara viticola</Emphasis> infection in grapevine leaves using chlorophyll fluorescence imaging

Plasmopara viticola is an economically important pathogen of grapevine. Early detection of P. viticola infection can lead to improved fungicide treatment. Our study aimed to determine whether chlorophyll fluorescence (Chl-F) imaging can be used to reveal early stages of P. viticola infection under conditions similar to those occurring in commercial vineyards. Maximum (F_V/F_M) and effective quantum yield of photosystem II (Φ_PSII) were identified as the most sensitive reporters of the infection. Heterogeneous distribution of F_V/F_M and Φ_PSII in artificially inoculated leaves was associated with the presence of the developing mycelium 3 days before the occurrence of visible symptoms and 5 days before the release of spores. Significant changes of F_V/F_M and Φ_PSII were spatially coincident with localised spots of inoculation across the leaf lamina. Reduction of F_V/F_M was restricted to the leaf area that later yielded sporulation, while the area with significantly lower Φ_PSII was larger and probably reflected the leaf parts in which photosynthesis was impaired. Our results indicate that Chl-F can be used for the early detection of P. viticola infection. Because P. viticola does not expand systemically in the host tissues and the effects of infection are localised, Chl-F imaging at high resolution is necessary to reveal the disease in the field.     

Rapid method for detecting resistance to a QoI fungicide in Plasmopara viticola populations

BACKGROUND: The increasing occurrence of Qo inhibitor (QoI)‐fungicide‐resistant Plasmopara viticola (Berk. & MA Curtis) Berl. & DeToni populations is becoming a serious problem in the control of grapevine downy mildew worldwide. RESULTS: The authors have developed a rapid method for detecting resistance to a QoI fungicide, azoxystrobin, in P. viticola populations using the nested PCR‐RFLP method. With this method, a glycine‐to‐alanine substitution was discovered at codon 143 in the cytochrome b gene of P. viticola populations found in Japan. CONCLUSION: It is proposed that the nested PCR‐RFLP method is a high‐speed, sensitive and reliable tool for detecting azoxystrobin‐resistant P. viticola populations. Copyright © 2009 Society of Chemical Industry     

Downy mildew resistance evaluation in 28 grapevine hybrids promising for breeding programs in Trentino region (Italy)

Downy mildew is a major grapevine disease caused by the biotrophic oomycete, Plasmopara viticola. Numerous disease resistance studies of diverse Vitis germplasm have been previously carried out to identify downy mildew resistance sources; however, ratings were mainly reported using leaf disc in vitro testing and foliage field assessment, or upon leaf and cluster field evaluations. In the current study, 28 grapevine hybrid cultivars were screened using leaf disc bioassay, for disease resistance characterization of both existing and wild-collected materials. 16 hybrids were identified as highly resistant or resistant, and will serve as relevant resistance donors in future pre-breeding and breeding programs. All grapevine hybrids were evaluated for foliar and cluster downy mildew resistance in an untreated field trial over three successive years. This study showed that the leaf disc bioassay provided some information on the resistance level of the genotypes under scrutiny, but it was a weak predictor of their resistance level under field conditions on leaves and even more on bunches. These findings are relevant to future applications in both traditional and marker-assisted breeding programs which promote sustainable viticulture.     

Method for rapid detection of the PvCesA3 gene allele conferring resistance to mandipropamid, a carboxylic acid amide fungicide, in Plasmopara viticola populations

BACKGROUND: The occurrence of carboxylic acid amide (CAA)‐fungicide‐resistant Plasmopara viticola populations is becoming a serious problem in the control of grapevine downy mildew worldwide. RESULTS: The authors have developed a method, which utilises PCR‐RFLP, for the rapid detection of resistance to the CAA fungicide mandipropamid in P. viticola populations. With this method, a glycine‐to‐serine substitution at codon 1105 of the cellulose synthase gene PvCesA3 of CAA‐fungicide‐resistant P. viticola was easily detected, although no resistant P. viticola was detected from 398 isolates in Japan. CONCLUSION: It is proposed that the PCR‐RFLP method is a reliable tool for the rapid detection of CAA‐fungicide‐resistant P. viticola isolates. Only 4 h was required from the sampling of symptoms to the phenotyping of fungicide resistance. Copyright © 2011 Society of Chemical Industry     

Monitoring of QoI fungicide resistance in Plasmopara viticola populations in Japan

BACKGROUND: The increasing occurrence of QoI fungicide resistance in Plasmopara viticola (Berk. & MA Curtis) Berl. & DeToni populations is becoming a serious problem in the control of grapevine downy mildew. In Japan, the existence of QoI‐fungicide‐resistant P. viticola was reported in 2009. RESULTS: The QoI fungicide resistance in P. viticola samples collected from vineyards in Japan in 2008 and 2009 was monitored. Resistant P. viticola were detected in the regions where QoI fungicides have been introduced in accordance with the pest management programme, whereas in Hokkaido vineyards, where QoI fungicides have not yet been introduced, QoI‐fungicide‐resistant P. viticola were not found. CONCLUSION: Japan comprises thousands of islands and is physically isolated from other countries by the sea. Monitoring the emergence, incidence and distribution of QoI fungicide resistance in P. viticola populations in Japan is necessary to improve pest management strategies for downy mildew disease in Japanese vineyards. Copyright © 2010 Society of Chemical Industry     

Endophytic Beauveria bassiana activates expression of defence genes in grapevine and prevents infections by grapevine downy mildew Plasmopara viticola

Fungal entomopathogens like Beauveria bassiana (Ascomycota: Hypocreales) are known as antagonists of insects with multiple functional and ecological roles, and have attracted increased attention as biocontrol agents in integrated pest management programmes. For some crop plants, it has been proven that endophytic B. bassiana, besides its entomopathogenic habit, can provide protection against plant pathogens or limit their damaging effects. However, for grapevine, limited knowledge is available on the influence of endophytic B. bassiana on fungal pathogens and about the mechanisms underlying putative protection effects. This study assessed the protective potential of endophytic B. bassiana against grapevine downy mildew Plasmopara viticola in greenhouse experiments. Three and seven days after a B. bassiana treatment, potted grapevine plants were inoculated with P. viticola and the evolving disease severity was assessed. Disease severity was significantly reduced in B. bassiana-treated plants compared to control plants, depending on the age of leaves. Furthermore, a microarray and an RT-qPCR analysis were performed to work out fundamental aspects of genes involved in the interaction between grapevine and B. bassiana. The results indicate an up-regulation of diverse defence-related genes in grapevine as a response to endophytic establishment of B. bassiana. Thus, endophytic establishment of an entomopathogenic fungus such as B. bassiana in grapevine plants would represent an alternative and sustainable plant protection strategy, with the potential for reducing pesticide applications in viticulture.     

Contribution of molecular studies to botanical epidemiology and disease modelling: grapevine downy mildew as a case-study

After being accidentally introduced from the USA at the end of the 19^th century, downy mildew caused by Plasmopara viticola (Berk. et Curt.) Berlese et De Toni became one of the most damaging diseases affecting Vitis vinifera in Europe. Downy mildew causes both direct and indirect losses and can lead to severe reduction of yield. Our understanding of the life cycle and epidemiology of P. viticola has been recently altered by molecular studies that revealed that the overwintering inoculum (i.e., the oospores) does more than initiate disease, as was previously thought. A mechanistic model was developed for predicting the entire chain of processes leading to primary infections, and this primary infection model was linked to other models of secondary infection cycles. The model for primary infections defines the length of the primary inoculum season and a seasonal oospore dose consisting of several cohorts of oospores that progressively mature. The model was evaluated by means of Bayesian analysis in both Italy and eastern Canada, and showed high sensitivity, specificity, and accuracy both for potted plants and vineyards. Fungicide applications are necessary to control downy mildew because preventive agronomic practices are not very effective, including host resistance. The use of warning systems based on weather-driven models leads to a reduction in the use and cost of chemicals and a reduction in their environmental impact.     

Characterization of resistance mechanisms activated by Trichoderma harzianum T39 and benzothiadiazole to downy mildew in different grapevine cultivars

Downy mildew, caused by Plasmopara viticola, is one of the most destructive diseases of grapevine and is controlled with intense application of chemical fungicides. Treatment with Trichoderma harzianum T39 (T39) or benzothiadiazole‐7‐carbothioic acid S‐methyl ester (BTH) has been previously shown to activate grapevine resistance to downy mildew and reduce disease symptoms in the Pinot noir cultivar. However, enhancement of plant resistance can be affected by several factors, including plant genotype. In order to further extend the use of resistance inducers against downy mildew, the physiological and molecular properties of T39‐ and BTH‐activated resistance in different cultivars of table and wine grapes were characterized under greenhouse conditions. T39 treatment reduced downy mildew symptoms, but the degree of efficacy differed significantly among grapevine cultivars. However, efficacy of BTH‐activated resistance was consistently high in the different cultivars. Expression profiles of defence‐related genes differed among cultivars in response to resistance inducers and to pathogen inoculation. T39 treatment enhanced the expression of defence‐related genes in the responsive cultivars, before and after P. viticola inoculation. A positive correlation between the efficacy of T39 and the expression level of defence‐related genes was found in Primitivo and Pinot noir plants, while different genes or more complex processes were probably activated in Sugraone and Negroamaro. The data reported here suggest that the use of a responsive cultivar is particularly important to maximize the efficacy of resistance inducers and new natural inducers should be explored for the less responsive cultivars.     

Activity of the New Fungicide Benthiavalicarb Against Plasmopara viticola and its Efficacy in Controlling Downy Mildew in Grapevines

Benthiavalicarb is a new fungicide active against Oomycetes fungal plant pathogens. The present study shows that benthiavalicarb is effective for controlling the Oomycete fungal pathogen Plasmopara viticola, which causes downy mildew in grapevines. The fungicide did not affect zoospore discharge from sporangia of P. viticola, but strongly inhibited zoospore encystment, cystospore germination in vitro and mycelial growth, together with sporangial production in vivo. Benthiavalicarb showed strong prophylactic and local activity in intact plants or detached leaves and low translaminar activity. The compound was not translocated from leaf to leaf in either a acropetal or basipetal direction. Benthiavalicarb applied at 1, 3 and 6 days post-inoculation protected grapevine plants against downy mildew and inhibited sporulation of the pathogen. Similar results were obtained on leaf disks if benthiavalicarb was applied up to 96 h post-inoculation. Benthiavalicarb diminished the sporulation of P. viticola when applied to established disease in the tissue. Benthiavalicarb remained active on leaves for a period up to 28 days. Two foliar applications of benthiavalicarb, 2 weeks apart, to field-grown grapevines inhibited downy mildew development and were as effective as the standard metalaxyl-Cu treatment in controlling the disease. A formulated mixture of benthiavalicarb + Folpet was similar or superior in performance to metalaxyl-Cu and the new strobilurin trifloxystrobin in controlling downy mildew. The effectiveness of benthiavalicarb makes it well suited for integration into a control programme against downy mildew disease in vineyards, and as a component to delay resistance buildup.     

An Aqueous Extract of the Dry Mycelium of Penicillium chrysogenum Induces Resistance in Several Crops under Controlled and Field Conditions

We have examined the effect of Pen, an aqueous extract of the dry mycelium of Penicillium chrysogenum, on plant–pathogen interactions. Pen controlled a broad range of pathogens on several crop plants under greenhouse and field conditions. Pen protected grapevine from downy and powdery mildew (caused by Plasmopara viticola and Uncinula necator), tomato from early blight (caused by Phytophthora infestans), onion from downy mildew (Peronospora destructor) and apple trees from apple scab (caused by Venturia inaequalis) to a similar extent as fungicides such as copper and sulphur or well-known inducers such as benzothiadiazole or β-aminobutyric acid. Pen had no major direct fungicidal effect and is thus supposed to protect plants by activating their defense mechanisms. The raw material for extraction of Pen was available in constant quality, a prerequisite for commercial application. Under certain conditions, Pen caused phytotoxic side effects. The symptoms mostly consisted of small necrotic spots or, more rarely, of larger necrotic areas. The development of the symptoms was dependent on several parameters, including concentration of Pen, the number of applications, the persistence on the plant tissue, the plant species and variety and environmental conditions. In grapevine, a partially purified fraction of Pen was much less toxic than the crude Pen extract, but protected the plants to a similar extent against P. viticola. Our data show that Pen has interesting and unique properties as a plant protection agent, but more research is needed to further reduce its phytotoxic side effects.     

„VitiMeteo Plasmopara“ – Prognosemodell zur Bekämpfung von Plasmopara viticola (Rebenperonospora) im Weinbau

Downy mildew, caused by Plasmopara viticola, is the most important disease of European grapevine (Vitis vinifera) in humid climates. On the basis of weather data the forcasting model “VitiMeteoPlasmopara” calculates the most important steps of the life cycle of downy mildew. A growth model for the simulation of leaf development is integrated in the pure forcasting model for downy mildew. “VitiMeteoPlasmopara” was elaborated by the Swiss Research Station ACW, Changins-Wädenswil and the State Institute of Viticulture and Enology Freiburg in Germany. Since 2003 the system is scientifically tested in Switzerland and Baden-Württemberg and applied in practice. Once or twice a day, the actual results of the forecasting system are visualised on the Internet. “Vitimeteo Plasmopara” is used to forecast downy mildew in Switzerland and in the south of Germany (Baden Württemberg), comprising approx. 42,000?ha of grapevine (15,000?ha CH, 27,000?ha D), and about 100 weather stations located in the area. The forecasting system is widely used by both advisory service and growers. It has proven a very useful tool to estimate the epidemiological situation and it allows the definition of spray intervals in relation with the epidemics. The open structure of the database allows the use and the integration of weather data into any other expert software moduls to forecast the development of other fungal diseases, pests or completely other models, for example irrigation models. “VitiMeteo Growth ”, a vine growth model, “VitiMeteo Insects”, a model for pests and “VitiMeteo DataGraph”, a software to analyze and present weather data, are already developed. The expert software moduls are summarized under designation “VitiMeteo Suite”.