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.     

More years of field trials against Plasmopara viticola in organic viticolture

The organic viticulture assumes a decisive role in the national agricultural sector. More impelling problems in the management of organic vineyards are represented from the plants pathology defence and particularly of Plasmopara viticola containment. Copper represents one of the few usable fungicides in the organic farming and the only effective against downy mildew. With Regulation EC n. 473/2002, fixed maximum quantity usable of copper compounds, owing to the environment problems due to the copper accumulation in the soil. To reduce quantity of metal copper or replace it with natural products, are conducted field trials with copper compounds at a low rate or alternative to copper products. Besides, we are estimating possibility to reduce the operations against P. viticola optimizing fungicidal treatments. Field trials in the organic farms located near Rome, have been carried out. Guidelines EPPO/OEPP PP 1/31 (3) have been carried out. The results of the trials have showed that, using cupric products with low metallic content, to reduce copper quantities used, always allowed to respect the limits established by Regulation EC. The alternative products that were investigated have not guaranteed, instead, an adequate protection in high pressure of grapevine downy mildew. It was possible to reduce treatments against P. viticola through control of different environmental parameters. The trials confirm that the copper is indispensable for plant protection in organic farming as it is not possible to replace it with natural extracts substances. We can reduce, instead, the copper quantities used trough the use of new products with low quantity metal copper or through the evaluation of climatic and pedologic data that allow to rationalize the fungicidal treatments.     

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.     

The efficacy of spraying programmes and treatment methods against downy mildew in two grapevine cultivars in Kosovo

The present study aimed to assess the efficacy of applied programmes and treatment methods against downy mildew (Plasmopara viticola). Two vine cultivars (Frankovka and Game) were tested in an experimental field (in Rahovec, Kosovo) for a two-year period (2010–2011). The fungicides used were Quadris (azoxystrobin), Antracol EP-70 (propineb), Dithan M-45 (mancozeb), Ridomil (metalaxyl), Curzate (cymoxanil), Bordeaux mixture (calcium hydroxide and copper sulphate) and Mikal (fosetyl-aluminium) applied three treatment methods. The evaluation of the disease severity was performed using the McKinney index. In both tested cultivars and in both treatment years (2010 and 2011), all of the programmes tested significantly reduced the severity of grape downy mildew compared to the control. The highest efficacy against grape downy mildew was achieved with the combined use of Ridomil and Dithan (programme 3); this treatment was more than 80% effective in both cultivars and in both years.     

Evaluation of innovative products to reduce copper applications to control potato late blight in organic production systems

The main objective of this project, VETAB project, is to determine alternatives to massive copper utilization to control potato late blight (Phytophtora infestans (Mont.) de Bary) in organic systems. To reach such a target, we first performed a screening of candidate products and additives based on their efficiency in the laboratory, under controlled conditions. We evaluated a wide range of products: formulations with a low level of copper, antagonists suspensions, aminoacid extracts, plants extracts, potassium salts, sulphur formulation, organically stabilised peroxide and rhamnolipids. The product's suspensions were applied by vaporization on potato plants. Two different protocols of application were elaborated. To test the fungicide protection action, the product was applied four days before inoculation of the pathogen. To evaluate the defence stimulating effect, the product was applied several times during the plant growth before inoculation of the pathogen. The last vaporization was performed 4 days before inoculation. We also evaluated the resistance of the product to washing risk. Pathogen suspension was applied as droplets of 5 x 10(4) spo/ml on detached leaves. The leaves were then incubated (18 degrees C, RH > 90%, 6 days) in order to record symptoms development. The best results were obtained with formulations integrating reduced doses of copper and with potassium salts. In conclusion, a wide range of products and additives are proposed on the market but very few of those have a real efficiency. The performance of the most efficient products has to be confirmed in field trials.     

Synergism and phytotoxicity: The effects of tank-mix additives on the biological efficacy of Cu2+ against Venturia inaequalis and Podosphaera leucotricha

The wetting behaviour of the spray and the biological efficacy of Cu²⁺ active ingredients in agrochemical formulations may be enhanced by tank-mix additives. We investigated how three BREAK-THRU® additives (BT301: biodegradable, BT133 and BT420: bio-based and biodegradable) tank-mixed with commercial copper preparations influenced the spray distribution, leaf uptake and biological efficacy of copper additive mixtures against apple scab and apple powdery mildew under controlled conditions. We quantified the synergetic effects of these additives in foliar applications. In addition, we determined the phytotoxic potential and evaluated their impacts on photosynthetic activity, non-photochemical quenching and ROS activity. The additives BT301 and BT420 strongly reduced surface tension and contact angle of copper treatments. The fluorescence observations revealed that BT301 achieved better spreading of copper formulation with more complete coverage of the leaf surface than BT420 and BT133, whereas ‘coffee-ring’ spreading was observed with BT133. The additive BT301 showed an increase in relative fluorescence area, indicating higher ROS production as a signal of intra-cellular tissue activity. The photochemical efficiency of photosystem II (Fv/Fm) was not negatively influenced by copper or additive treatment. Thus, we observed no phytotoxic effects of copper-additive mixtures on apple leaves at treatment doses of 4 g Cu²⁺ L⁻¹. All copper treatments reduced apple scab infections significantly, by 53%–76%. Interestingly, addition of BT301 to copper preparations showed the strongest biological efficacy (83% reduction) against Venturia inaequalis, whereas addition of BT420 showed the strongest effect against Podosphaera leucotricha (89% infection reduction). The synergetic effects of additives on the biological efficacy without phytotoxic effects on plants may have potential for reducing copper loads in horticultural production systems.     

Micronized Copper Wood Preservatives: Efficacy of Ion,Nano, and Bulk Copper against the Brown Rot Fungus Rhodonia placenta

Recently introduced micronized copper (MC) formulations, consisting of a nanosized fraction of basic copper (Cu) carbonate (CuCO₃·Cu(OH)₂) nanoparticles (NPs), were introduced to the market for wood protection. Cu NPs may presumably be more effective against wood-destroying fungi than bulk or ionic Cu compounds. In particular, Cu- tolerant wood-destroying fungi may not recognize NPs, which may penetrate into fungal cell walls and membranes and exert their impact. The objective of this study was to assess if MC wood preservative formulations have a superior efficacy against Cu-tolerant wood-destroying fungi due to nano effects than conventional Cu biocides. After screening a range of wood-destroying fungi for their resistance to Cu, we investigated fungal growth of the Cu-tolerant fungus Rhodonia placenta in solid and liquid media and on wood treated with MC azole (MCA). In liquid cultures we evaluated the fungal response to ion, nano and bulk Cu distinguishing the ionic and particle effects by means of the Cu²⁺ chelator ammonium tetrathiomolybdate (TTM) and measuring fungal biomass, oxalic acid production and laccase activity of R. placenta. Our results do not support the presence of particular nano effects of MCA against R. placenta that would account for an increased antifungal efficacy, but provide evidence that attribute the main effectiveness of MCA to azoles.     

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.     

Assessing the activity of microbicides against bacterial spores: knowledge and pitfalls

Bacterial endospores (spores) have a higher intrinsic resistance to microbicides as compared to other microbial forms, most likely due to their impermeable outer layers and low water content. Though structural differences between the spores of various bacterial species may account for observed variations in their resistance to microbicides, flaws in methods for testing the sporicidal activity of microbicides often exaggerate the differences. This has major implications when considering the selection of one or more surrogates to assess microbicides against clinically relevant spore‐formers such as Clostridium difficile. The mounting significance of Cl. difficile as a pathogen is leading to a corresponding increase in the number of commercially available microbicidal formulations claiming activity against its spores without proper differentiation between the product's sporistatic and sporicidal actions. In this review we critically assess the situation and the implications of product claims on the field use of microbicidal products.     

On the activity loss of hydrolases in organic solvents: I. Rapid loss of activity of a variety of enzymes and formulations in a range of organic solvents

Dehydrated enzyme powders have been used extensively as suspensions in organic solvents to catalyze synthetic reactions. Prolonged enzyme activity is necessary to make such applications commercially successful. However, it has recently become evident that the stability and thus activity of many enzymes is compromised in organic solvents. Herein we explore the stability of various hydrolases (i.e., lipases from Mucor meihei and Candida rugosa, -chymotrypsin, subtilisin Carlsberg, and pig-liver esterase) and various formulations (lyophilized powder, cross-linked enzyme crystals, poly(ethylene glycol)-enzyme conjugates) in different organic solvents. The results show a roughly exponential activity decrease for all enzymes and formulations studied after exposure to organic solvents. Inactivation was observed independent of the enzyme, formulation details, and the solvent. In addition, no relationship was found between the magnitude of inactivation and the value of initial activity. Thus, quite active formulations lost their activity as quickly as less active formulations. The estimated half-times (t_1/2) for all enzymes and preparations ranged from 1.8 h for subtilisin C. co-lyophilized with methyl-β-cyclodextrin to 61.6 h for the most stable poly(ethylene glycol)--chymotrypsin preparation. The data here presented indicates that the inactivation is likely not related to changes in enzyme structure and dynamics.     

Compatibility of OMRI-certified surfactants with three entomopathogenic fungi

The Organic Materials Review Institute (OMRI) is a non-profit organisation providing an independent review of products intended for use in organic production systems to certify compliance with US national organic standards. Since all adjuvants to be used in organic agriculture production are required to meet these standards, OMRI's certified list of products is a convenient starting point when developing organic pest control formulations. In the current study, six OMRI-certified surfactants are tested for their compatibility with three common entomopathogens: Beauveria bassiana, Metarhizium brunneum and Isaria fumosorosea. The fungi were evaluated in two common propagule forms, solid-state produced conidia and liquid-media produced blastospores. The results show that most of the surfactants are compatible with the fungi at a high surfactant concentration (2% w/v). In general, the conidia showed a higher susceptibility (greater reduction in spore germination) to the surfactants than the blastospores under these conditions. In addition, the surface tension and foaming properties of the surfactants were determined.     

Effect of Organic Ligands on Accumulation of Copper in Hyperaccumulator and Nonaccumulator Commelina communis

Better understanding of copper uptake and accumulation regulation in plants is critical to the phytoremediation of copper contaminated soil. This study employed a 30-day pot experiment to assess the relationship between organic ligands and copper accumulation in plants. Hyperaccumulator and nonaccumulator varieties of Commelina communis were used, different organic ligands were applied, and the data of copper accumulation in shoots were collected. The six organic ligands included ethylenediaminetetraacetic acid and organic acids (formic acid, citric acid, malic acid, tartaric acid, and succinic acid). The results showed that organic ligands added to culture increased the copper accumulation both varieties. The results of the copper accumulation in shoots agreed with the study of the root uptake kinetics of copper influx. The addition of organic acids could increase copper accumulation in shoots because the copper influx in roots was increased. The results also indicated that the copper influx of hyperaccumulator roots was higher than that of nonaccumulator roots. This is one of the mechanisms by which a hyperaccumulator could amass large amounts of copper in its shoots. In this accumulation process, little effect on the leaf relative water content was in the hyperaccumulator and nonaccumulator of leaves and normal physiological condition of plants.     

Pearl millet downy mildew as affected by integrating seed dressing,sowing date and cultivar

The effects of seed dressing, sowing date and cultivar on incidence and severity of downy mildew of pearl millet induced by Sclerospora graminicola and yield were studied in a two-year field trial conducted at the Research farm of University of Maiduguri. The millet cultivars, Ex-Borno, SOSAT-C88, GB 8735 and Gwagwa were each dressed with metalaxyl at 0.75 and 1.50 g a.i./kg seed; and a batch of undressed seeds of each cultivar served as control. Both dressed and undressed seeds were used for dry-planting and wet-planting in early and late seasons. The results showed that seed dressing with the fungicides significantly (p ≤ 0.05) reduced the incidence and severity of downy mildew and increased grain yield. Dry-planting also significantly (p ≤ 0.01) increased grain yield irrespective of disease incidence. Delay in sowing led to a significant reduction in incidence and severity of downy mildew. Differences between the cultivars in relation to incidence and severity of downy mildew and grain yield were significant. SOSAT-C88 developed low or no downy mildew in both seasons. Sowing of dressed SOSAT-C88 as soon as rainfall established appeared most beneficial in the control of downy mildew. Dry- or wet-planting Ex-Borno dressed with any of the metalaxyl formulations proved to be effective for downy mildew management and for high yield.     

Optimisation and comparison of liquid and dry formulations of the biocontrol yeast <Emphasis Type="Italic">Pichia anomala</Emphasis> J121

The biocontrol yeast Pichia anomala J121 can effectively reduce mould growth on moist cereal grains during airtight storage. Practical use of microorganisms requires formulated products that meet a number of criteria. In this study we compared different formulations of P. anomala. The best way to formulate P. anomala was freeze-drying. The initial viability was as high as 80%, with trehalose previously added to the yeast. Freeze-dried products could be stored at temperatures as high as 30 °C for a year, with only a minor decrease in viability. Vacuum-drying also resulted in products with high storage potential, but the products were not as easily rehydrated as freeze-dried samples. Upon desiccating the cells using fluidised-bed drying or as liquid formulations, a storage temperature of 10 °C was required to maintain viability. Dependent on the type of formulation, harvesting of cells at different nutritional stresses affected the initial viabilities, e.g. the initial viability for fluidised-bed-dried cells was higher when the culture was fed with excess glucose, but for freeze-drying it was superior when cells were harvested after depletion of carbon. Using micro-silos we found that the biocontrol activity remained intact after drying, storage and rehydration for all formulations.     

Surfactin and fengycin contribute to the protection of a Bacillus subtilis strain against grape downy mildew by both direct effect and defence stimulation

Bacillus subtilis GLB191 (hereafter GLB191) is an efficient biological control agent against the biotrophic oomycete Plasmopara viticola, the causal agent of grapevine downy mildew. In this study, we show that GLB191 supernatant is also highly active against downy mildew and that the activity results from both direct effect against the pathogen and stimulation of the plant defences (induction of defence gene expression and callose production). High-performance thin-layer chromatography analysis revealed the presence of the cyclic lipopeptides fengycin and surfactin in the supernatant. Mutants affected in the production of fengycin and/or surfactin were thus obtained and allowed us to show that both surfactin and fengycin contribute to the double activity of GLB191 supernatant against downy mildew. Altogether, this study suggests that GLB191 supernatant could be used as a new biocontrol product against grapevine downy mildew.     

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.     

Comparison of theoretical and experimental data to evaluate substrate diffusional limitations for crown ether- and methyl-beta-cyclodextrin-activated serine protease subtilisin Carlsberg in tetrahydrofuran

Simple co-lyophilization of serine protease subtilisin Carlsberg with [12]-crown ether-4 (12-crown-4) or methyl-beta-cyclodextrin (MbetaCD) drastically increases its catalytic activity in organic solvents. We investigated whether the improved activity would cause substrate diffusional limitations. To experimentally assess the issue, the enzyme was inactivated with PMSF. Different amounts of active and inactive subtilisin were codissolved in 10 mM phosphate buffer (pH 7.8) followed by lyophilization with or without 12-crown-4 or MbetaCD. Initial rates for the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester and 1-propanol in anhydrous THF were plotted vs. the amount of active enzyme present in the formulations. For all three enzyme formulations a linear relationship was observed and the results clearly show that activation of subtilisin Carlsberg by crown ethers and MbetaCD did not cause diffusional limitations. This was somewhat surprising because theoretical models predicted such diffusional limitations for the activated formulations. However, investigation of the protein powder particles obtained after co-lyophilization with 12-crown-4 and MbetaCD revealed a drastically reduced particle size for these formulations when suspended in THF. The particle micronization afforded by the excipients prevented substrate diffusional limitations, a factor that should be taken into account when designing improved enzyme formulations for synthetic applications in organic solvents.     

pH dependence of copper geometry, reduction potential, and nitrite affinity in nitrite reductase

Many properties of copper-containing nitrite reductase are pH-dependent, such as gene expression, enzyme activity, and substrate affinity. Here we use x-ray diffraction to investigate the structural basis for the pH dependence of activity and nitrite affinity by examining the type 2 copper site and its immediate surroundings in nitrite reductase from Rhodobacter sphaeroides 2.4.3. At active pH the geometry of the substrate-free oxidized type 2 copper site shows a near perfect tetrahedral geometry as defined by the positions of its ligands. At higher pH values the most favorable copper site geometry is altered toward a more distorted tetrahedral geometry whereby the solvent ligand adopts a position opposite to that of the His-131 ligand. This pH-dependent variation in type 2 copper site geometry is discussed in light of recent computational results. When co-crystallized with substrate, nitrite is seen to bind in a bidentate fashion with its two oxygen atoms ligating the type 2 copper, overlapping with the positions occupied by the solvent ligand in the high and low pH structures. Fourier transformation infrared spectroscopy is used to assign the pH dependence of the binding of nitrite to the active site, and EPR spectroscopy is used to characterize the pH dependence of the reduction potential of the type 2 copper site. Taken together, these spectroscopic and structural observations help to explain the pH dependence of nitrite reductase, highlighting the subtle relationship between copper site geometry, nitrite affinity, and enzyme activity.     

Performance of copper naphthenate and its analogs as ground contact wood preservatives

For several years, an extensive research program investigating new preservative systems has been ongoing at Mississippi State University. This paper details our continuing experience with field testing of copper naphthenate and other copper carboxylate preservative systems. Results from field stake tests at an AWPA Zone 4 test site are presented. In general, copper carboxylates made with 'synthetic' acids yielded results equivalent to or only slightly lower than systems with straight naphthenic acids or nap acids amended with synthetic neodecanoic acid bottoms. Oil carrier formulations outperformed those in water or light organic solvents. All systems carried in oil would qualify as ground contact preservatives at the proper retention.