Natural products alone or with copper vs. grape downy mildew: efficacy, costs, Cu impact

In organic farming copper is one of the few fungicides that can be used and the only effective against downy mildew. Due to the problems of environmental impact associated with the use of this heavy metal, the European Union decided to restrict its use (Annex II Regulation EC n. 889/2008). In order to assess the possibility of replacing the copper or reduce the quantities used, we tested natural products and low rate copper formulations in field trials. The anti-downy mildew activity of these formulations has been studied in an organic vineyard. During the two years of activity we also estimated the economic cost of use of the products to assess their possible use in agricultural practice.     

Grapevine downy mildew control using reduced copper amounts in organic viticulture

Copper is an essential natural micronutrient. However, copper used as a plant protection product may have long-term consequences due to its accumulation in the soil. Limitations on copper use have therefore been defined in organic farming (Regulation EC 889/2008). In the light of new developments and evidence, the European Commission has planned to assess whether further restrictions are needed in the quantities of copper permitted. A two-year field trial was therefore set up with new copper formulations to evaluate the possibility of reducing the copper quantities applied with treatments and consequently to reduce copper soil residues. Plots were prepared, each containing 12 plants and repeated four times in randomized blocks. The test organism was Plasmopara viticola (Berk. and M.A. Curtis) Berl. and De Toni. Cupric formulations characterised by a low metallic content (Glutex CU 90 and Labicuper) were tested in comparison with a reference product (standard) and an untreated control. Evaluations of treatments were carried out periodically on 100 leaves and 100 bunches for each replicate. Data obtained were subjected to statistical analysis. Chemical analyses were performed to determine copper residues on leaves, grapes and soil. Samplings of leaves and grapes were carried out for each replicate. Soil samples were taken from 0-20 cm and 20-40 cm depth. Total copper was determined using spectrophotometry in atomic absorption by acetylene-air flame (FAAS at lambda = 324.8 microm). The results showed that the tested products were effective in controlling downy mildew with a lower copper dosage than with the cupric formulations used as a standard. Glutex CU 90 formulation led to an annual input of copper that was a little more than a third compared to the standard and Labicuper about a fifth or a sixth. At harvest, copper levels in grapes were much lower than RML (fixed at 50 mg/kg). With regard to the impact of cupric treatments on organic vineyard soil, no statistically significant differential increase in Cu residue was observed in soil between tested products versus untreated control. In conclusion, the environmental impact of copper in organic viticulture could be minimized through the new cupric formulations developed by agrochemical companies.     

Grapevine downy mildew control in organic farming

Cupric products at low dose and alternative compounds have been tested to control the downy mildew in an organic vineyard. It has found that copper compounds control downy mildew in a satisfactory way, reducing, at the same time, the dose of copper metal. The alternative products were not satisfactory to control Plasmopara viticola.     

Multicomponent reactions in fungicide research: the discovery of mandipropamid

Isocyanide-based multicomponent reactions of the Ugi- and Passerini-type have been valuable tools for the rapid exploration of the novel fungicidal compound classes of phenylglycinamides and mandelamides. Mandipropamid (6), which was discovered during this derivatisation, displays excellent activity against the economically important phytopathogens Phytophthora infestans (potato and tomato late blight) and Plasmopara viticola (grape downy mildew).     

Growth,Yield and Fruit Quality of Grapevines under Organic and Biodynamic Management

The main objective of this study was to determine growth, yield and fruit quality of grapevines under organic and biodynamic management in relation to integrated viticultural practices. Furthermore, the mechanisms for the observed changes in growth, yield and fruit quality were investigated by determining nutrient status, physiological performance of the plants and disease incidence on bunches in three consecutive growing seasons. A field trial (Vitis vinifera L. cv. Riesling) was set up at Hochschule Geisenheim University, Germany. The integrated treatment was managed according to the code of good practice. Organic and biodynamic plots were managed according to Regulation (EC) No 834/2007 and Regulation (EC) No 889/2008 and according to ECOVIN- and Demeter-Standards, respectively. The growth and yield of the grapevines differed strongly among the different management systems, whereas fruit quality was not affected by the management system. The organic and the biodynamic treatments showed significantly lower growth and yield in comparison to the integrated treatment. The physiological performance was significantly lower in the organic and the biodynamic systems, which may account for differences in growth and cluster weight and might therefore induce lower yields of the respective treatments. Soil management and fertilization strategy could be responsible factors for these changes. Yields of the organic and the biodynamic treatments partially decreased due to higher disease incidence of downy mildew. The organic and the biodynamic plant protection strategies that exclude the use of synthetic fungicides are likely to induce higher disease incidence and might partially account for differences in the nutrient status of vines under organic and biodynamic management. Use of the biodynamic preparations had little influence on vine growth and yield. Due to the investigation of important parameters that induce changes especially in growth and yield of grapevines under organic and biodynamic management the study can potentially provide guidance for defining more effective farming systems.     

Identification of effector genes from the phytopathogenic Oomycete Plasmopara viticola through the analysis of gene expression in germinated zoospores

Grapevine downy mildew caused by the Oomycete Plasmopara viticola is one of the most important diseases affecting Vitis spp. The current strategy of control relies on chemical fungicides. An alternative to the use of fungicides is using downy mildew resistant varieties, which is cost-effective and environmentally friendly. Knowledge about the genetic basis of the resistance to P. viticola has progressed in the recent years, but little data are available about P. viticola genetics, in particular concerning the nature of its avirulence genes. Identifying pathogen effectors as putative avirulence genes is a necessary step in order to understand the biology of the interaction. It is also important in order to select the most efficient combination of resistance genes in a strategy of pyramiding. On the basis of knowledge from other Oomycetes, P. viticola effectors can be identified by using a candidate gene strategy based on data mining of genomic resources. In this paper we describe the development of Expressed Sequence Tags (ESTs) from P. viticola by creating a cDNA library from in vitro germinated zoospores and the sequencing of 1543 clones. We present 563 putative nuclear P. viticola unigenes. Sequence analysis reveals 54 ESTs from putative secreted hydrolytic enzymes and effectors, showing the suitability of this material for the analysis of the P. viticola secretome and identification of effector genes. Next generation sequencing of cDNA from in vitro germinated zoospores should result in the identification of numerous candidate avirulence genes in the grapevine/downy mildew interaction.     

Microsatellite Markers for Characterization of Native and Introduced Populations of Plasmopara viticola, the Causal Agent of Grapevine Downy Mildew

We reported 31 microsatellite markers that have been developed from microsatellite-enriched and direct shotgun pyrosequencing libraries of Plasmopara viticola, the causal agent of grapevine downy mildew. These markers were optimized for population genetics applications and used to characterize 96 P. viticola isolates from three European and three North American populations.     

A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death

Sulfated laminarin (PS3) has been shown previously to be an elicitor of plant defense reactions in tobacco and Arabidopsis and to induce protection against tobacco mosaic virus. Here, we have demonstrated the efficiency of PS3 in protecting a susceptible grapevine cultivar (Vitis vinifera cv. Marselan) against downy mildew (Plasmopara viticola) under glasshouse conditions. This induced resistance was associated with potentiated H2O2 production at the infection sites, upregulation of defense-related genes, callose and phenol depositions, and hypersensitive response-like cell death. Interestingly, similar responses were observed following P. viticola inoculation in a tolerant grapevine hybrid cultivar (Solaris). A pharmacological approach led us to conclude that both callose synthesis and jasmonic acid pathway contribute to PS3-induced resistance.     

Control of Downy Mildew, Plasmopara viticola (de Bary) Berlese et de Toni, on Grapevine Leaves through Water Extracts from Composted Organic Wastes

Watery extracts of composted manure-straw-soil mixtures induced increased resistance of grapevine leaves against downy mildew, Plasmopara viticola, if applied by dipping or spraying. The extracts had no direct fungicidal or fungitoxic effects.     

Characterization of single-nucleotide-polymorphism markers for Plasmopara viticola, the causal agent of grapevine downy mildew

We report 34 new nuclear single-nucleotide-polymorphism (SNP) markers that have been developed from an expressed sequence tag library of Plasmopara viticola, the causal agent of grapevine downy mildew. This newly developed battery of markers will provide useful additional genetic tools for population genetic studies of this important agronomic species.     

Rationalization of pesticide treatments against powdery mildew of grape

The powdery mildew represents one of the diseases which affect the grape, it is diffused in all agricultural regions with variable intensity and epidemic course in operation of many microclimatic factors. The powdery mildew of grape is caused from Uncinala necator (Schw.) Burr. (nowadays named Erysiphe necator Schwein.); it is controlled with systemic therapy and contact chemicaL products. In some vineyards located in Latium (central Italy), different field trials have been carried out purposely to rationalize the treatments against E. necator. We have studied the powdery mildew infections through monitoring a set of environmental parameters, the evaluation of cultivar sensibility, the agricultural production method and the area characteristics. We have analysed the following environmental parameters monitoring every 15 minutes: precipitation, soil temperature, solar radiation, wind direction, wind speed, atmospheric relative humidity, atmospheric temperature, leaf wetness, soil humidity to cm 20 and soil humidity to cm 40. Besides, we have used Artificial Intelligence analysis techniques to try to forecast U. necator infections. Guideline EPPO/OEPP PP 1/4 (4) has been used. The trials were conducted in conventional and organic farms. In 2 conventional farms and in organic farm we have considered 1 untreated control thesis, in order to follow the course of infection, 1 standard farm reference thesis (standard), where the treatments were carried out according to the usual farm procedures and 1 thesis where the treatments were carried out according to examining the environmental data. In another conventional vineyard, we have considered only 1 untreated control thesis and 1 standard farm reference thesis (standard) to study disease trend. The achieved results have underlined the possibility (through the knowledge of data pedoclimatic and cultural) to position the treatments against the powdery mildew so that to reduce their number. The lower number of treatments that could follow as a result of environmental data to analyse could bring a series of evident economic and ecologic advantages for the farms.     

Characterization of Plasmopara-resistance in grapevine using in vitro plants

Although the exact mechanisms by which grapevine cells operate to reduce disease incidence caused by the downy mildew fungus Plasmopara viticola are not fully elucidated, our cytological results obtained from infected in vitro-plants confirm that enhanced disease resistance is associated with an expression of distinct reactions in a chronological order. An increased production of reactive oxygen species (superoxide radicals, 4-6 hours post infection, hpi) was followed by a hypersensitive response (6-8 hpi), an increased activity of peroxidase in cells flanking the infection area and in the vascular tissue (10-12 hpi) and an increased production, accumulation or conversion of phenolic compounds (12-15 hpi). These mechanisms seem also to be present in susceptible varieties as shown after an inoculation with non-host oomycetic pathogens on the basis of peroxidase activity, but they do not become activated after P. viticola infection. The investigation of the peroxidase activity in leaves at several time points after an infection with P. viticola indicated that there is a strong correlation between the POX activity in leaves of in vitro-plants and the resistance of grapevine plants to P. viticola in the field.     

Control of downy mildew (Pseudoperonospora cubensis) of greenhouse grown cucumbers with alternative biological agents

In organic cucumber production infection with downy mildew (Pseudoperonospora cubensis) is a major problem. Plant extracts from Glycyrrhiza glabra L. (licorice), a plant belonging to the family Fabaceae, and Salvia officinalis (sage) as well as cultures of the bacterium Aneurinibacillus migulanus were investigated for efficacy of disease control under commercial growing conditions. Contrary to bioassays, where sage extract and the microorganism showed highest activity, in the trials of 2008 G. glabra extract was more effective than sage extract or A. migulanus against P. cubensis. Parameters such as concentrations of the preparations or application intervals could have been the reason for this. In the following year's trial (2009) the concentration of these agents was therefore increased somewhat and plants were either treated in seven day application intervals or in ten day application intervals. In the semi-commercial trials of 2009 all alternative biological agents showed good efficacies up to around 80% against infection with downy mildew. The application interval seemed to have a marginal effect only. Again, the licorice extract tended to be the best agent.     

Expression analysis of defence-related genes in grapevine leaves after inoculation with a host and a non-host pathogen.

The expression of PR protein encoding genes and genes involved in the phenylpropanoid metabolism was analysed on grapevine leaves of susceptible and resistant cvs. in response to inoculation with the host-pathogen Plasmopara viticola and the non-host pathogen Pseudoperonospora cubensis, the downy mildew pathogen of cucumber. These experiments were conducted to elucidate whether or not grapevine plants susceptible to downy mildew exhibit an identical defence response after inoculation with the non-host pathogen. Expression analysis of defence-related genes revealed marked differences between the susceptible cultivar "Riesling" (Vitis vinifera) and the resistant cultivar "Gloire de Montpellier" (Vitis riparia). Whereas some genes seem to be expressed constitutively in "Gloire" or induced after an inoculation with both pathogens, expression of defence-related genes in Riesling was influenced mainly after inoculation with the non-host pathogen: PR-2, PR-3, PR-4, a PGIP gene, and especially genes encoding enzymes involved in anthocyanin biosynthesis (DFR, F3H, LDOX) were affected. Therefore, the occurrence of the respective products (flavans and other phenolics) in inoculated leaves was investigated with appropriate histological staining techniques. These stainings revealed a production of catechins and related phenolic compounds within the first 48 hai (hours after inoculation) with Ps. cubensis but not with P. viticola in Riesling, whereas in Gloire no further production was seen, which may be due to the high content of polyphenolics as observed in control leaves. In addition to the staining procedures, sporulation intensity was monitored on leaf discs. Pretreatments of leaf discs with Ps. cubensis led to a reduced browning reaction (as a result of a hypersensitive reaction) in Gloire and significantly reduced the intensity of sporulation in Riesling after a subsequent inoculation with P. viticola.     

Preliminary experiments on the use of oil-soluble copper compounds as fungicides

Tests were made of the solubility of a number of copper compounds in mineral and vegetable oils. Phytocidal tests on onion foliage showed that turpentine, pine oil and white spirit were damaging whereas cottonseed oil and white oil were innocuous. Copper salts in white oil, white spirit, turpentine or pine oil showed a higher fungicidal value than in cottonseed oil. In laboratory tests, a solution of copper 3:5-di- iso propyl salicylate was more fungicidal than Bordeaux mixture of the same copper concentration. In a small field trial, copper 3:5-di- ios propyl salicylate dissolved in white oil to give a concentration of 0.01% Cu was atomized three times at monthly intervals on to onion foliage without damage. The same material at 0.1% Cu in white oil was phytocidal. No control of onion downy mildew ( Peronospora schleideniana ) was obtained in this trial. The method of atomization was convenient and practicable for small-scale operation. It gave perfect wetting of the foliage and was very economical of material.     

INVESTIGATIONS ON FUNGICIDES

Twenty (aryloxythio)trichloromethanes were examined for in vitro fungicidal activity against six fungi. All compounds showed a direct fungistatic effect and some exhibited a marked fumigant action. Good protectant action against Alternaria solani on tomato and Botrytis fabae on broad bean was obtained with certain compounds, but none was better than the three standard protectants used for comparison. When supplied to plants through their roots, eight conferred significant systemic fungicidal protection against Alternaria solani in tomato, but there was no significant protection against Botrytis fabae in broad bean. In preliminary tests the 2:4:5-trichlorophenoxythio analogue gave promising results when examined as a fumigant fungicide for eradication of downy mildew in lettuce plants, for reducing lenticel rot in stored apples and for preventing blue-mould in oranges.     

Association of RGA-SSCP markers with resistance to downy mildew and anthracnose in grapevines

Downy mildew (Plasmopara viticola) and anthracnose (Sphaceloma ampelinum) are two major diseases that severely affect most grapevine (Vitis vinifera) cultivars grown commercially in Thailand. Progress of conventional breeding programs of grapevine for improved resistance to these diseases can be speeded up by selection of molecular markers associated with resistance traits. We evaluated the association between 13 resistance gene analog (RGA)-single-strand conformation polymorphism (SSCP) markers with resistance to downy mildew and anthracnose in 71 segregating progenies of seven cross combinations between susceptible cultivars and resistant lines. F(1) hybrids from each cross were assessed for resistance to downy mildew and anthracnose (isolates Nk4-1 and Rc2-1) under laboratory conditions. Association of resistance traits with RGA-SSCP markers was evaluated using simple linear regression analysis. Three RGA-SSCP markers were found to be significantly correlated with anthracnose resistance, whereas significant correlation with downy mildew resistance was observed for only one RGA-SSCP marker. These results demonstrate the usefulness of RGA-SSCP markers. Four candidate markers with significant associations to resistance to these two major diseases of grapevine were identified. However, these putative associations between markers and resistance need to be verified with larger segregating populations before they can be used for marker-assisted selection.     

Exploiting medicinal plants and phylloplane microflora for the management of grapevine downy mildew

Abstract

Grapevine downy mildew is the most devastating disease throughout the world causing huge monetary losses. Twenty medicinal plant extracts and six phylloplane microfloras were evaluated for their efficacy against sporangial germination of grapevine downy mildew pathogen Plasmopara viticola in vitro. The results revealed that the Neem Seed Kernel Extract (NSKE) at 5% significantly inhibited the sporangial germination (75.36%) of P. viticola. Among the phylloplane microflora Pseudomonas fluorescens was highly effective in reducing the sporangial germination (64.26%). Post inoculation spraying of NSKE (5%) and P. fluorescens (0.2%) effectively inhibited the disease development in the greenhouse. Three sprays with NSKE (5%) and phylloplane P. fluorescens (0.2%): first spray after initial appearance of disease and the second and third at 10 day intervals were found to be promising in reducing disease incidence in the field.     

Control of pearl millet downy mildew caused by Sclerospora graminicola with systemic fungicides in an artificially-contaminated plot

Among four fungicides, viz. metalaxyl (two formulations), fosetyl-Al, pro-pamocarb and cyomaxanil tested in vitro against sporangial germination inhibition of Sclerospora graminicola, cyomaxanil was found to be most inhibitory. In an artificially contaminated plot, when used as seed treatment or foliar spray for the control of downy mildew of pearl millet, only metalaxyl was effective. Metalaxyl 25 (Ridomil) and metalaxyl 35 (Apron) seed treatments protected the pearl millet plants from downy mildew up to 30 days. As a foliar spray, metalaxyl 25 used once at 20 days or twice after 20 and 38 days of plant growth gave less disease at harvest time. Seed treatment (metalaxyl 25 or 35) followed by one metalaxyl 25 spray was found to be effective in controlling the downy mildew. These treatments improved the growth of plants and yield significantly.