Patent Application
20150133297
The present invention discloses new antimicrobial compositions to control plant diseases and to prevent microbial spoilage of crops.
It is estimated that about 25% of the world crop production is lost due to microbial spoilage, of which spoilage by fungi is by far the most important cause. Not only from an economical point of view, but also from a humane point of view it is of great importance to prevent spoilage of food products. After all, in many parts of the world people suffer from hunger.
Success in combating plant and crop diseases and in reducing the damage they cause to yields and quality depends greatly on the timely application of fungicides. The prolonged and frequent use of many fungicides such as e.g. benzamidazoles has contributed to reduce their effectiveness thanks to the development of phenomena of resistance.
Fungicides that inhibit ergosterol biosynthesis represent an important class of agricultural fungicides. They consist of various chemical groups, including the morpholines. The first of the morpholine fungicides, dodemorph, was introduced in 1965, followed four years later by tridemorph. Despite the development of new morpholines, these fungicides have not been immune to challenges in their development and maintenance. A large concern has been resistance development. Resistance to morpholine fungicides has been observed on several crops and diseases now (see Bagirova et al., 2001; Chabane, 1993; Dereviagina et al., 1999).
For many decades, the polyene macrolide antimycotic natamycin has been used to prevent fungal growth on food products such as cheeses and sausages. This natural preservative, which is produced by fermentation using Streptomyces natalensis, is widely used throughout the world as a food preservative and has a long history of safe use in the food industry. It is very effective against all known food spoilage fungi. Although natamycin has been applied for many years in e.g. the cheese industry, up to now development of resistant fungal species has never been observed.
Consequently, it can be concluded that there is a severe need for more effective antimicrobial compositions, e.g. antifungal compositions, for the treatment of fungal growth in and on plants and crops.
The present invention solves the problem by providing a new synergistic antimicrobial, e.g. antifungal, composition comprising a polyene antifungal compound and at least one antifungal compound from the family of morpholine fungicides. As used herein, the term “synergistic” means that the combined effect of the antifungal compounds when used in combination is greater than their additive effects when used individually.
In general, synergistic activity of two active ingredients can be tested in for example the analysis of variance model using the treatment interaction stratum (see Slinker, 1998). Relative efficacy can be calculated by means of the following formula: ((value of evolution status of untreated control−value of evolution status of composition)/(value of evolution status of untreated control))*100. An interaction coefficient can then be calculated by means of the following formula: ((relative efficacy of combination compound A+compound B)/(relative efficacy of compound A+relative efficacy of compound B))*100. An interaction coefficient larger than 100 indicates synergy between the compounds.
Alternatively, synergy can be calculated as follows: the antifungal activity (in %) of the individual active ingredients can be determined by calculating the reduction in mould growth observed on products treated with the active ingredients in comparison to the mould growth on products treated with a control composition. The expected antifungal activity (E in %) of the combined antifungal composition comprising both active ingredients can be calculated according to the Colby equation (Colby, 1967): E=X+Y−[(X·Y)/100], wherein X and Y are the observed antifungal activities (in %) of the individual active ingredients X and Y, respectively. If the observed antifungal activity (O in %) of the combination exceeds the expected antifungal activity (E in %) of the combination and the synergy factor O/E is thus >1.0, the combined application of the active ingredients leads to a synergistic antifungal effect.
The term “morpholine fungicide” as used herein includes morpholine fungicides, piperidine fungicides, cinnamic acid amide fungicides and spiroketal-amine fungicides.
In an embodiment of the invention, the at least one antifungal compound from the family of morpholine fungicides is selected from the group consisting of aldimorph, benzamorf, carbamorph, dimethomorph, dodemorph, fenpropidin, fenpropimorph, flumorph, piperalin, pyrimorph, spiroxamine and tridemorph.
In an embodiment the compositions may also contain two or more different antifungal compounds from the family of morpholine fungicides. It is to be understood that derivatives of antifungal compounds from the family of morpholine fungicides including, but not limited to, salts or solvates of antifungal compounds from the family of morpholine fungicides or modified forms of antifungal compounds from the family of morpholine fungicides may also be applied in the compositions of the invention. Examples of commercial products containing morpholine fungicides such as dimethomorph are products with the brand names Acrobat® and Forum®. Examples of commercial products containing morpholine fungicides such as fenpropidin are products with the brand names Instinct® and Patrol®. Examples of commercial products containing morpholine fungicides such as fenpropimorph are products with the brand names Corbel® and Volley®. Said commercial products can be incorporated in the present invention.
In an embodiment the polyene antifungal compound is selected from the group consisting of natamycin, nystatin, amphotericin B, trienin, etruscomycin, filipin, chainin, dermostatin, lymphosarcin, candicidin, aureofungin A, aureofungin B, hamycin A, hamycin B and lucensomycin. In a preferred embodiment the polyene antifungal compound is natamycin. In an embodiment the compositions may also contain two or more different polyene antifungal compounds. It is to be understood that derivatives of polyene antifungal compounds including, but not limited to, salts or solvates of polyene antifungal compounds or modified forms of polyene antifungal compounds may also be applied in the compositions of the invention. Examples of commercial products containing natamycin are the products with the brand name Delvocid®. Such products are produced by DSM Food Specialties (The Netherlands) and may be solids containing e.g. 50% (w/w) natamycin or liquids comprising between e.g. 2-50% (w/v) natamycin. Said commercial products can be incorporated in the compositions of the invention.
The composition of the present invention generally comprises from about 0.005 g/l to about 100 g/l and preferably from about 0.01 g/l to about 50 g/l of a polyene antifungal compound. Preferably, the amount is from 0.01 g/l to 3 g/l.
The composition of the present invention generally comprises from about 0.0001 g/l to about 2000 g/l and preferably from about 0.0005 g/l to about 1500 g/l of an antifungal compound from the family of morpholine fungicides. More preferably, the amount is from 0.001 g/l to 1000 g/l.
In an embodiment the composition of the present invention further comprises at least one additional compound selected from the group consisting of a sticking agent, a carrier, a colouring agent, a protective colloid, an adhesive, a herbicide, a fertilizer, a thickening agent, a sequestering agent, a thixotropic agent, a surfactant, a further antimicrobial compound, a detergent, a preservative, a spreading agent, a filler, a spray oil, a flow additive, a mineral substance, a solvent, a dispersant, an emulsifier, a wetting agent, a stabiliser, an antifoaming agent, a buffering agent, an UV-absorber and an antioxidant. A further antimicrobial antifungal compound may be an antifungal compound (e.g. imazalil, thiabendazole) or a compound to combat insects, nematodes, mites and/or bacteria. Of course, the compositions according to the invention may also comprise two or more of any of the above additional compounds. Any of the above mentioned additional compounds may also be combined with the polyene antifungal compound and/or the at least one antifungal compound from the family of morpholine fungicides in case the antifungal compounds are applied separately. In an embodiment the additional compounds are additives acceptable for the specific use, e.g. food, feed, medicine, cosmetics or agriculture. Additional compounds suitable for use in food, feed, medicine, cosmetics or agriculture are known to the person skilled in the art.
In a specific embodiment the further antimicrobial compound is a natural crop protection compound belonging to the group of phosphites, e.g. KH2PO3 or K2HPO3 or a mixture of both phosphite salts. Phosphite containing compounds as used herein means compounds comprising a phosphite group, i.e. PO3 (in the form of e.g. H2PO3−, HPO32− or PO33−) or any compound which allows the release of a phosphite ion including compounds such as phosphorous acid and phosphonic acid as well as derivatives thereof such as esters and/or alkali metal or alkaline earth metal salts thereof. In case the compositions of the present invention comprise a polyene antifungal compound (e.g. natamycin) and at least one phosphite containing compound, they preferably comprise 0.1 g or less lignosulphonate, more preferably 0.1 g or less polyphenol, per gram polyene antifungal compound. Preferably, they comprise 0.01 g or less lignosulphonate, more preferably 0.01 g or less polyphenol, per gram polyene antifungal compound. In particular, they are free of lignosulphonate and preferably free of polyphenol. Suitable examples of phosphite containing compounds are phosphorous acid and its (alkali metal or alkaline earth metal) salts such as potassium phosphites e.g. KH2PO3 and K2HPO3, sodium phosphites and ammonium phosphites, and (C1-C4) alkyl esters of phosphorous acid and their salts such as aluminum ethyl phosphite (fosetyl-Al), calcium ethyl phosphite, magnesium isopropyl phosphite, magnesium isobutyl phosphite, magnesium sec-butyl phosphite and aluminum N-butyl phosphite. Of course, mixtures of phosphite containing compounds are also encompassed. A mixture of e.g. KH2PO3 and K2HPO3 can easily be obtained by e.g. adding KOH or K2CO3 to a final pH of 5.0-6.0 to a KH2PO3 solution. As indicated above, precursor-type compounds which in the crop or plant are metabolized into phosphite compounds can also be included in the compositions of the present invention. Examples are phosphonates such as the fosetyl-aluminium complex. In e.g. a crop or plant the ethyl phosphonate part of this molecule is metabolized into a phosphite. An example of such a compound in the commercial ethyl hydrogen phosphonate product called Aliette® (Bayer, Germany). The ratio of phosphite to natamycin (in weight) in the compositions is in general between 2:1 to 500:1 (w/w), preferably between 3:1 to 300:1 (w/w) and more preferably between 5:1 to 200:1 (w/w). Compositions according to the invention may have a pH of from 1 to 10, preferably of from 2 to 9, more preferably of from 3 to 8 and most preferably of from 4 to 7. They may be solid, e.g. powder compositions, or may be liquid. The compositions of the present invention can be aqueous or non-aqueous ready-to-use compositions, but may also be aqueous or non-aqueous concentrated compositions/suspensions or stock compositions, suspensions and/or solutions which before use have to be diluted with a suitable diluent such as water or a buffer system. Alternatively, the compositions of the invention can also be used to prepare coating emulsions. The compositions of the present invention can also have the form of concentrated dry products such as e.g. powders, granulates and tablets. They can be used to prepare compositions for immersion or spraying of products such as agricultural products including plants, crops, vegetables and/or fruits. Of course, the above is also applicable when the polyene antifungal compound and the at least one antifungal compound from the family of morpholine fungicides are applied as separate compositions.
In a further aspect the invention relates to a kit comprising a polyene antifungal compound and at least one antifungal compound from the family of morpholine fungicides. The polyene antifungal compound and the at least one antifungal compound from the family of morpholine fungicides may be present in two separate packages, e.g. containers. The components of the kit may be either in dry form or liquid form in the package. If necessary, the kit may comprise instructions for dissolving the compounds. In addition, the kit may contain instructions for applying the compounds.
In a further aspect the invention pertains to a method for protecting a product against fungi by treating the product with a polyene antifungal compound and at least one antifungal compound from the family of morpholine fungicides. In addition, the product can be treated with other antifungal and/or antimicrobial compounds either prior to, concomitant with or after treatment of the products with the polyene antifungal compound and the at least one antifungal compound from the family of morpholine fungicides. The product may be treated by sequential application of the polyene antifungal compound and the at least one antifungal compound from the family of morpholine fungicides or vice versa. Alternatively, the product may be treated by simultaneous application of the polyene antifungal compound and the at least one antifungal compound from the family of morpholine fungicides. In case of simultaneous application, the compounds can be present in different compositions that are applied simultaneously or the compounds may be present in a single composition. In yet another embodiment the product may be treated by separate or alternate modes of applying the antifungal compounds. In an embodiment the invention is directed to a process for the treatment of products by applying the polyene antifungal compound and the at least one antifungal compound from the family of morpholine fungicides to the products. By applying the compounds fungal growth on or in the products can be prevented. In other words, the compounds protect the products from fungal growth and/or from fungal infection and/or from fungal spoilage. The compounds can also be used to treat products that have been infected with a fungus. By applying the compounds the disease development due to fungi on or in these products can be slowed down, stopped or the products may even be cured from the disease. In an embodiment of the invention the products are treated with a composition or kit according to the invention. In an embodiment the product is a food, feed, pharmaceutical, cosmetic or agricultural product. In a preferred embodiment the product is an agricultural product.
The polyene antifungal compound and the at least one antifungal compound from the family of morpholine fungicides, the compositions according to the invention and the kits according to the invention can be applied to the products by spraying. Other methods suitable for applying these compounds, compositions and kits in liquid form to the products are also a part of the present invention. These include, but are not limited to, dipping, watering, drenching, introduction into a dump tank, vaporizing, atomizing, fogging, fumigating, painting, brushing, dusting, foaming, spreading-on, packaging and coating (e.g. by means of wax or electrostatically). In addition, the antifungal compounds may also be injected into the soil. Spraying applications using automatic systems are known to reduce the labour costs and are cost-effective. Methods and equipment well-known to a person skilled in the art can be used for that purpose. The compositions according to the invention can be regularly sprayed, when the risk of infection is high. When the risk of infection is lower spray intervals may be longer. Depending on the type of application, the amount of polyene antifungal compound applied may vary from 5 ppm to 10,000 ppm, preferably from 10 ppm to 5,000 ppm and most preferably from 20 to 1,000 ppm. Depending on the type of application, the amount of the at least one antifungal compound from the family of morpholine fungicides applied may vary from 10 ppm to 5,000 ppm, preferably from 20 ppm to 3,000 ppm and most preferably from 50 to 1,000 ppm.
In a specific embodiment the agricultural product can be treated post-harvest. By using a polyene antifungal compound and the at least one antifungal compound from the family of morpholine fungicides the control of post-harvest and/or storage diseases is achieved for a long period of time to allow transport of the harvested agricultural product over long distances and under various storage conditions with different controlled atmosphere systems in respect of temperature and humidity. Post-harvest storage disorders are e.g. lenticel spots, scorch, senescent breakdown, bitter pit, scald, water core, browning, vascular breakdown, CO2 injury, CO2 or O2 deficiency, and softening. Fungal diseases may be caused for example by the following fungi: Blumeria spp., e.g. Blumeria graminis; Uncinula spp., e.g. Uncinula necator, Leveillula spp., e.g. Leveillula taurica; Podosphaera spp., e.g. Podosphaera leucotricha, Podosphaera fusca, Podosphaera aphanis; Microsphaera spp., e.g. Microsphaera syringae; Sawadaea spp., e.g. Sawadaea tulasnei; Mycosphaerella spp., e.g. Mycosphaerella musae, Mycosphaerella fragariae, Mycosphaerella citri; Mucor spp., e.g. Mucor piriformis; Monilinia spp., e.g. Monilinia fructigena, Monilinia laxa; Phomopsis spp., Phomopsis natalensis; Colletotrichum spp., e.g. Colletotrichum musae, Colletotrichum gloeosporioides, Colletotrichum coccodes; Verticillium spp., e.g. Verticillium theobromae; Nigrospora spp.; Botrytis spp., e.g. Botrytis cinerea; Diplodia spp., e.g. Diplodia citri; Pezicula spp.; Alternaria spp., e.g. Alternaria citri, Alternaria alternata; Septoria spp., e.g. Septoria depressa; Venturia spp., e.g. Venturia inaequalis, Venturia pyrina; Rhizopus spp., e.g. Rhizopus stolonifer, Rhizopus oryzae; Glomerella spp., e.g. Glomerella cingulata; Sclerotinia spp., e.g. Sclerotinia fruiticola; Ceratocystis spp., e.g. Ceratocystis paradoxa; Fusarium spp., e.g. Fusarium semitectum, Fusarium moniliforme, Fusarium solani, Fusarium oxysporum; Cladosporium spp., e.g. Cladosporium fulvum, Cladosporium cladosporioides, Cladosporium cucumerinum, Cladosporium musae; Penicillium spp., e.g. Penicillium funiculosum, Penicillium expansum, Penicillium digitatum, Penicillium italicum; Phytophthora spp., e.g. Phytophthora citrophthora, Phytophthora fragariae, Phytophthora cactorum, Phytophthora parasitica; Phacydiopycnis spp., e.g. Phacydiopycnis malirum; Gloeosporium spp., e.g. Gloeosporium album, Gloeosporium perennans, Gloeosporium fructigenum, Gloeosporium singulata; Geotrichum spp., e.g. Geotrichum candidum; Phlyctaena spp., e.g. Phlyctaena vagabunda; Cylindrocarpon spp., e.g. Cylindrocarpon mali; Stemphyllium spp., e.g. Stemphyllium vesicarium; Thielaviopsis spp., e.g. Thielaviopsis paradoxy; Aspergillus spp., e.g. Aspergillus niger, Aspergillus carbonarius; Nectria spp., e.g. Nectria galligena; Cercospora spp., e.g. Cercospora angreci, Cercospora apii, Cercospora atrofiliformis, Cercospora musae, Cercospora zeae-maydis.
Another aspect of the present invention relates to the use of a polyene antifungal compound and at least one antifungal compound from the family of morpholine fungicides to protect a product against fungi. As indicated above, the compounds may be used, e.g. applied, sequentially or simultaneously. In an embodiment the invention relates to a use, wherein a composition or kit according to the invention is applied to the product. In an embodiment the product is a food, feed, pharmaceutical, cosmetic or agricultural product. In a preferred embodiment the product is an agricultural product.
In a specific embodiment the polyene antifungal compound and at least one antifungal compound from the family of morpholine fungicides can be used in medicine, e.g. to treat and/or prevent fungal diseases. The polyene antifungal compound and at least one antifungal compound from the family of morpholine fungicides can for instance be used in the form of a pharmaceutical composition. The composition may further comprise pharmaceutically acceptable excipients. The antifungal compounds may be administered orally or parenterally. The type of composition is dependent on the route of administration.
A further aspect of the invention is directed to a product treated with a polyene antifungal compound and at least one antifungal compound from the family of morpholine fungicides. In an embodiment the product is treated with a composition or kit according to the invention. The invention is therefore directed to a product comprising a polyene antifungal compound and at least one antifungal compound from the family of morpholine fungicides. The treated products may comprise a polyene antifungal compound and at least one antifungal compound from the family of morpholine fungicides on their surface and/or inside the product. Alternatively, the treated products may comprise a coating comprising these compounds. In an embodiment the treated products comprise from 0.000001 to 200 mg/dm2, preferably 0.00001 to 100 mg/dm2, more preferably from 0.00005 to 10 mg/dm2 of the polyene antifungal compound on their surface. In a further embodiment they comprise from 0.000001 to 200 mg/dm2, preferably 0.00001 to 100 mg/dm2, more preferably from 0.00005 to 10 mg/dm2 of the at least one antifungal compound from the family of morpholine fungicides on their surface. In an embodiment the product is a food, feed, pharmaceutical, cosmetic or agricultural product. In a preferred embodiment the product is an agricultural product.
The term “food products” as used herein is to be understood in a very broad sense and includes, but is not limited to, cheese, cream cheese, shredded cheese, cottage cheese processed cheese, sour cream, dried fermented meat product including salamis and other sausages, wine, beer, yoghurt, juice and other beverages, salad dressing, cottage cheese dressing, dips, bakery products and bakery fillings, surface glazes and icing, spreads, pizza toppings, confectionery and confectionery fillings, olives, olive brine, olive oil, juices, tomato purees and paste, condiments, and fruit pulp and the like food products.
The term “feed products” as used herein is also to be understood in a very broad sense and includes, but is not limited to, pet food, broiler feed, etc.
The term “pharmaceutical product” as used herein is also to be understood in a very broad sense and includes products comprising an active molecule such as a drug, agent, or pharmaceutical compound and optionally a pharmaceutically acceptable excipient, i.e. any inert substance that is combined with the active molecule for preparing an agreeable or convenient dosage form.
The term “cosmetic product” as used herein is also to be understood in a very broad sense and includes products that are used for protecting or treating horny tissues such as skin and lips, hair and nails from drying by preventing transpiration of moisture thereof and further conditioning the tissues as well as giving good appearance to these tissues. Products contemplated by the term “cosmetic product” include, but are not limited to, moisturizers, personal cleansing products, occlusive drug delivery patches, nail polish, powders, wipes, hair conditioners, skin treatment emulsions, shaving creams and the like.
The term “agricultural products” as used herein is also to be understood in a very broad sense and includes, but is not limited to, cereals, e.g. wheat, barley, rye, oats, rice, sorghum and the like; beets, e.g. sugar beet and fodder beet; pome and stone fruit and berries, e.g. apples, pears, plums, apricots, peaches, almonds, cherries, strawberries, raspberries and blackberries; leguminous plants, e.g. beans, lentils, peas, soy beans; oleaginous plants, e.g. rape, mustard, poppy, olive, sunflower, coconut, castor-oil plant, cocoa, ground-nuts; cucurbitaceae, e.g. pumpkins, gherkins, melons, cucumbers, squashes, aubergines; fibrous plants, e.g. cotton, flax, hemp, jute; citrus fruit, e.g. oranges, lemons, grapefruits, mandarins, limes; tropical fruit, e.g. papayas, passion fruit, mangos, carambolas, pineapples, bananas, kiwis; vegetables, e.g. spinach, lettuce, asparagus, brassicaceae such as cabbages and turnips, carrots, onions, tomatoes, potatoes, seed-potatoes, hot and sweet peppers; laurel-like plants, e.g. avocado, cinnamon, camphor tree; or products such as maize, tobacco, nuts, coffee, sugarcane, tea, grapevines, hops, rubber plants, as well as ornamental plants, e.g. cut flowers, roses, tulips, lilies, narcissus, crocuses, hyacinths, dahlias, gerbera, carnations, fuchsias, chrysanthemums, and flower bulbs, shrubs, deciduous trees and evergreen trees such as conifers, plants and trees in greenhouses. It includes, but is not limited to, plants and their parts, fruits, seeds, cuttings, cultivars, grafts, bulbs, tubers, root-tubers, rootstocks, cut flowers and vegetables.
A method for preparing a composition as described herein is another aspect of the present invention. The method comprises adding a polyene antifungal compound to at least one antifungal compound from the family of morpholine fungicides. The compounds may for instance be added separately to an aqueous composition and mixed, followed, if necessary, by adjustment of the pH, viscosity, etc. If added separately, some or all of the separate compounds may be in powder form, but alternatively some or all may also be in liquid form. The compounds may for instance also be added to one another in powder form and mixed to obtain a powdered composition. The powdered composition may then be added to an aqueous composition.
Four organic, unripe (green) bananas are used per treatment. The peel of each banana is wounded thrice using a cork borer according to the method described by de Lapeyre de Bellaire and Dubois (1987). Subsequently, each wound is inoculated with 15 μl of a Fusarium proliferatum suspension containing 1×105 of spores/ml. After incubation for 4 hours at 20° C., each banana wound is treated with 100 μl of a freshly prepared aqueous antifungal composition comprising either natamycin (DSM Food Specialties, Delft, The Netherlands), aldimorph or both. In addition, the morpholine fungicides benzamorf, carbamorph, dimethomorph, dodemorph, fenpropidin, fenpropimorph, flumorph, piperalin, pyrimorph, spiroxamine and tridemorph alone or in combination with natamycin are tested. The antifungal compositions comprise 1.00% (w/w) methylhydroxyethylcellulose (MHEC), 0.40% (w/w) xanthan gum, 0.20% (w/w) anti-foaming agent, 0.30% (w/w) citric acid, 0.39% (w/w) lactic acid and 0.11% (w/w) potassium sorbate. The pH of the composition is 4.0. A composition without natamycin or a morpholine fungicide is used as control. The treated, unripe bananas are incubated in a closed box in the dark at 20° C. and a relative air humidity of 95%, which is obtained in the presence of a saturated Na2HPO4 aqueous solution. During the first 20 days of incubation, a ripe (yellow) banana is included in the closed box to elevate the ethylene gas level and thus induce ripening of the treated, unripe bananas.
During incubation, the degree of mould growth on the bananas is assessed in a twofold manner: (i) the number of moulded wounds per total of 12 wounds is counted; and (ii) the antifungal activity (in %) of the individual active ingredients is determined by calculating the reduction in mould growth observed on the banana wounds treated with the antifungal composition in comparison to the mould growth on the banana wounds treated with the control composition. The expected antifungal activity (E in %) of the combined antifungal composition comprising both active ingredients is calculated according to the Colby equation (Colby, 1967):
E=X+Y−[(X·Y)/100]
wherein X and Y are the observed antifungal activities (in %) of the individual active ingredients X and Y, respectively. If the observed antifungal activity (O in %) of the combination exceeds the expected antifungal activity (E in %) of the combination and the synergy factor O/E is thus >1.0, the combined application of the active ingredients leads to a synergistic antifungal effect.
The results clearly demonstrate that the antifungal composition comprising both natamycin and a morpholine fungicide protect bananas better against mould growth than natamycin or a morpholine fungicide alone.
Hence, the combination of natamycin and a morpholine fungicide has synergistic antifungal activity on bananas.
Treatment of Strawberries Twelve fresh, organic strawberries are used per treatment. Each strawberry is wounded with a 0.5 mm long cut and each wound is inoculated with 10 μl of a Botrytis cinerea suspension containing 1×105 of spores/ml. After a 2-hour incubation period at 20° C., each strawberry is dipped individually for 1 minute in a freshly prepared aqueous antifungal composition comprising either natamycin (DSM Food Specialties, Delft, The Netherlands), aldimorph or both. In addition, the morpholine fungicides benzamorf, carbamorph, dimethomorph, dodemorph, fenpropidin, fenpropimorph, flumorph, piperalin, pyrimorph, spiroxamine and tridemorph alone or in combination with natamycin are tested. The antifungal compositions also comprise 1.00% (w/w) methylhydroxyethylcellulose (MHEC), 0.40% (w/w) xanthan gum, 0.20% (w/w) anti-foaming agent, 0.30% (w/w) citric acid, 0.39% (w/w) lactic acid and 0.11% (w/w) potassium sorbate. The pH of the composition is 4.0. A composition without natamycin or a morpholine fungicide is used as control. The treated strawberries are incubated in a closed box in the dark at 20° C.
After incubation, the mould growth on the strawberries is assessed in a twofold manner: (i) the number of moulded strawberries per total of 12 strawberries is counted; and (ii) the antifungal activity (in %) of the individual and combined active ingredients is determined by calculating the reduction in mould growth observed on the strawberries treated with the antifungal composition in comparison to the mould growth on the strawberries treated with the control composition according to the Colby method described in Example 4 (Colby, 1967).
The results demonstrate that the antifungal composition comprising natamycin and a morpholine fungicide have a stronger antifungal activity on strawberries than natamycin or a morpholine fungicide alone.
Hence, the combined application of natamycin and a morpholine fungicide synergistically reduces mould growth on strawberries.
Ten fresh, organic mandarins are used per treatment. The peel of each mandarin is wounded once using a cork borer according to the method described by de Lapeyre de Bellaire and Dubois (1987). Subsequently, each wound is inoculated with 10 μl of a Penicillium italicum suspension containing 1×104 of spores/ml. After incubation for 2 hours at 20° C., the mandarins are dipped individually for 1 minute in a freshly prepared aqueous antifungal composition comprising either natamycin (DSM Food Specialties, Delft, The Netherlands), aldimorph or both. In addition, the morpholine fungicides benzamorf, carbamorph, dimethomorph, dodemorph, fenpropidin, fenpropimorph, flumorph, piperalin, pyrimorph, spiroxamine and tridemorph alone or in combination with natamycin are tested. In addition, the antifungal compositions comprise 3.1% (w/w) beeswax, 0.76% (w/w) glycerol, 0.66% (w/w) polyoxyethylene sorbitan monostearate (Tween 60), 0.03% (w/w) methylhydroxyethylcellulose (MHEC), 0.02% (w/w) xanthan gum, 0.02% (w/w) anti-foaming agent, 0.15% (w/w) citric acid and 0.01% (w/w) potassium sorbate. The pH of the composition is 4.0. A composition without natamycin or a morpholine fungicide is used as control.
The treated mandarins are incubated in a closed box in the dark at 20° C. and assessed on mould growth after 25, 28, 31 and 34 days of incubation. The antifungal activity (in %) of the individual and combined active ingredients is determined by calculating the reduction in mould growth observed on the mandarins treated with the antifungal composition in comparison to the mould growth on the mandarins treated with the control composition according to the Colby method (Colby, 1967) described in Example 1 and 2.
The results prove that the antifungal composition comprising natamycin and a morpholine fungicide is superior to the compositions comprising natamycin or a morpholine fungicide alone in preventing mould growth on mandarins.
Thus, the combined application of natamycin and a morpholine fungicide synergistically reduces mould growth on mandarins.
To demonstrate synergistic antifungal activity of the combination of natamycin with a morpholine fungicide against Botrytis cinerea, an in vitro assay is conducted using 96-well microtiter plates. The following compositions are tested:
Control (no active ingredient),
natamycin (DSM Food Specialties, Delft, The Netherlands),
a morpholine fungicide,
natamycin+a morpholine fungicide.
After filling each well of a microtiter plate with 92 μl of PCB medium, the active ingredient(s) are added from separate stock solutions prepared in PCB medium or methanol, which resulted in an intermediate volume of 100 μl per well. Subsequently, 100 μl of a Botrytis cinerea suspension prepared in PCB medium is used to inoculate each well with 2.5×103spores/ml. Each well thus contains a final volume of 200 μl and <1% of methanol, which does not affect growth of Botrytis cinerea (data not shown).
After incubation of the microtiter plates at 25° C., the in vitro antifungal activity (%) of the individual active ingredients is assessed by calculating the reduction in mould growth observed in the presence of the active ingredient in comparison to the mould growth observed in the absence of the active ingredient. The expected antifungal activity (E in %) of the active ingredient combination is calculated according to the Colby equation (Colby, 1967):
E=X+Y−[(X·Y)/100]
wherein X and Y are the observed antifungal activities (in %) of the individual active ingredients X and Y, respectively. If the observed antifungal activity (O in %) of the combination exceeds the expected antifungal activity (E in %) of the combination and the resulting synergy factor O/E is thus >1.0, the combined application of the active ingredients leads to a synergistic antifungal effect.
The results demonstrate that both the natamycin+morpholine fungicide combination have much stronger antifungal activity against Botrytis cinerea than natamycin and a morpholine fungicide individually.
Hence, the combined application of natamycin and a morpholine fungicide synergistically inhibits growth of Botrytis cinerea.
Twelve fresh, organic strawberries were used per treatment. Each strawberry was wounded with a 0.5 mm long cut and each wound was inoculated with 10 μl of a Botrytis cinerea suspension containing 1×105 of spores/ml. After a 3-hour incubation period at 20° C., each strawberry was dipped individually for 1 minute in a freshly prepared aqueous antifungal composition comprising either 500 ppm natamycin (DSM Food Specialties, Delft, The Netherlands), 600 ppm fenpropidin or both. Each antifungal composition also comprised 3.2% (w/w) beeswax, 0.8% (w/w) glycerol, 0.7% (w/w) polyoxyethylene sorbitan monostearate (Tween 60), 0.1% (w/w) polyoxyethylene sorbitan monooleate (Tween 80), 0.05% (w/w) methylhydroxyethyl-cellulose (MHEC), 0.03% (w/w) anti-foaming agent, 0.02% (w/w) xanthan gum, 0.02% (w/w) citric acid, 0.01% (w/w) lactic acid and 0.01% potassium sorbate. A composition without natamycin or fenpropidin was used as control. Each composition had a pH of 4. The treated strawberries were incubated in a closed box in the dark at 20° C. for 9 days.
During incubation, mould growth on the strawberries was assessed in a twofold manner: (i) the number of moulded strawberries per total of 12 strawberries was counted; and (ii) the antifungal activity (in %) of the individual and combined active ingredients was determined by calculating the reduction in mould growth observed on the strawberries treated with the antifungal composition in comparison to the mould growth on the strawberries treated with the control composition. The expected antifungal activity (E in %) of the combined antifungal composition comprising both active ingredients was calculated according to the Colby equation (Colby, 1967):
E=X+Y−[(X·Y)/100]
wherein X and Y are the observed antifungal activities (in %) of the individual active ingredients X and Y, respectively. If the observed antifungal activity (O in %) of the combination exceeds the expected antifungal activity (E in %) of the combination and the synergy factor O/E is thus >1.0, the combined application of the active ingredients leads to a synergistic antifungal effect.
The results in Table 1 (number of moulded strawberries per total of 12 strawberries) and Table 2 (antifungal activity) unequivocally demonstrate that the combined antifungal composition comprising 500 ppm natamycin and 600 ppm fenpropidin protected strawberries more effectively against mould growth than the compositions comprising natamycin or fenpropidin alone.
After 5 and 6 days of incubation, all 12 strawberries treated with either the control composition or fenpropidin alone showed mould growth, as did 11 and 12 strawberries treated with natamycin alone, respectively (see Table 1). However, when treated with the active ingredient combination of natamycin and fenpropidin, only 7 of the 12 strawberries were moulded on day 5 and only 10 of the 12 strawberries on day 6 (see Table 1).
Moreover, the observed antifungal activity exceeded the expected antifungal activity with approximately 5 to almost 40% between 4 and 9 days of incubation, which yielded synergy factors ranging from 1.4 to 7.0 (see Table 2).
Thus, the combined application of 500 ppm natamycin and 600 ppm fenpropidin leads to a strong synergistic reduction in mould growth on strawberries.
Eight fresh, organic oranges were used per treatment. Each orange was soaked in a 180 ppm hypochlorite solution for 10 minutes, then rinsed thoroughly with fresh tap water and dried. The peel of each disinfected orange was wounded once using a cork borer according to the method described by de Lapeyre de Bellaire and Dubois (1987). Subsequently, each wound was inoculated with 10 μl of a Penicillium italicum suspension containing 5×105 of spores/ml. After incubation for 3 hours at 20° C., each wound and the orange peel area of 1 cm around the wound was treated with in total 150 μl of a freshly prepared aqueous antifungal composition comprising either 500 ppm natamycin (DSM Food Specialties, Delft, The Netherlands), 500 ppm fenpropidin or both. Each antifungal composition also comprised 3.2% (w/w) beeswax, 0.8% (w/w) glycerol, 0.7% (w/w) polyoxyethylene sorbitan monostearate (Tween 60), 0.1% (w/w) polyoxyethylene sorbitan monooleate (Tween 80), 0.05% (w/w) methylhydroxyethyl-cellulose (MHEC), 0.03% (w/w) anti-foaming agent, 0.02% (w/w) xanthan gum, 0.02% (w/w) citric acid, 0.01% (w/w) lactic acid and 0.01% potassium sorbate. A composition without natamycin or fenpropidin was used as control. Each composition had a pH of 4. The treated oranges were incubated in a closed box in the dark at 20° C. and assessed on mould growth during a 28-day incubation period. The antifungal activity (in %) of the individual and combined active ingredients was determined by calculating the reduction in mould growth observed on the oranges treated with the antifungal composition in comparison to the mould growth on the oranges treated with the control composition according to the Colby method (Colby, 1967) as described in Example 5.
The results in Table 3 (antifungal activity) demonstrate that the active ingredient combination of 500 ppm natamycin and 500 ppm fenpropidin limited mould growth on oranges more effectively than natamycin or fenpropidin individually.
Between 22 and 28 days of incubation, the observed antifungal activity of the active ingredient combination of natamycin and fenpropidin exceeded antifungal activity with 5 to >20%, which yielded synergy factors >1.0 (see Table 3).
Thus, the combined application of 500 ppm natamycin and 500 ppm fenpropidin synergistically inhibits fungal growth on oranges.
The experiment was conducted as described in Example 6, except for the fact that each wounded and inoculated orange was treated with 150 μl of a freshly prepared aqueous antifungal composition comprising either 250 ppm natamycin (DSM Food Specialties, Delft, The Netherlands), 250 ppm fenpropidin or both. During the 26-day incubation period, the treated oranges were assessed on mould growth according to the two methods described in Example 5.
The results in Table 4 (number of moulded oranges per total of 8 oranges) and Table 5 (antifungal activity) reveal that the active ingredient combination of 250 ppm natamycin and 250 ppm fenpropidin was more successful in limiting mould growth on oranges than natamycin or fenpropidin alone.
After 18, 19 and 20 days of incubation, all 8 oranges treated with the control composition were moulded, as were 6 or 7 of the 8 oranges treated with either natamycin alone or fenpropidin alone. However, only 2 of the 8 oranges treated with the active ingredient combination of natamycin and fenpropidin were moulded on these three days (see Table 4).
Between days 21 through 26, all 8 oranges treated with the control composition displayed mould growth, in addition to 7 of the 8 oranges treated with either natamycin or fenpropidin alone. However, when both natamycin and fenpropidin were used for treatment, mould growth was observed for only 3 of the 8 treated oranges on days 21 and 22 and 5 of the 8 treated oranges on days 23 until 26 (see Table 4).
Moreover, the observed antifungal activity of the composition comprising both natamycin and fenpropidin was 10 to almost 40% higher than the expected antifungal activity between 18 and 26 days of incubation. Consequently, the synergy factor obtained on these days was always >1.0 and even increased from 1.1 on day 18 to 3.3 on day 26 (see Table 5).
Hence, a strong synergistic antifungal effect exists between 250 ppm natamycin and 250 ppm fenpropidin when applied in combination on oranges.
The experiment was conducted as described in Example 6, except for the fact that each wounded and inoculated orange was treated with 150 μl of a freshly prepared aqueous antifungal composition comprising either 500 ppm natamycin (DSM Food Specialties, Delft, The Netherlands), 400 ppm spiroxamine or both. During the 26-day incubation period, the treated oranges were assessed on mould growth according to the two methods described in Example 5.
The results in Table 6 (number of moulded oranges per total of 8 oranges) and Table 7 (antifungal activity) demonstrate the active ingredient combination of 500 ppm natamycin and 400 ppm spiroxamine had a higher antifungal activity on oranges than natamycin or spiroxamine individually.
After 14 through 16 days and 18 days of incubation, all 8 oranges treated with either the control composition or spiroxamine were moulded, as were 5 or 6 of the 8 oranges treated with natamycin alone. However, only 2 of the 8 oranges treated with the active ingredient combination of natamycin and spiroxamine were moulded between day 14 and 16 (see Table 6).
Between 19 and 23 days of incubation, all 8 oranges treated with either the control composition or spiroxamine alone showed mould growth, together with 6 of the 8 oranges treated with natamycin alone. Of the 8 oranges treated with both natamycin and spiroxamine, however, mould growth was observed for only 3 oranges on days 19 through 21, whereas 4 oranges were moulded on days 22 and 23 (see Table 6).
Moreover, the observed antifungal activity of the active ingredient combination of natamycin and spiroxamine exceeded the expected antifungal activity with >20 to >50% between 18 and 26 days of incubation. Consequently, the obtained synergy factors ranged from 1.5 to 4.6 (see Table 7).
Thus, the combined application of 500 ppm natamycin and 400 ppm spiroxamine leads to a surprisingly strong synergistic inhibition of mould growth on oranges.
The experiment was conducted as described in Example 6, except for the fact that each wounded and inoculated orange was treated with 150 μl of a freshly prepared aqueous antifungal composition comprising either 250 ppm natamycin (DSM Food Specialties, Delft, The Netherlands), 200 ppm spiroxamine or both. During the 26-day incubation period, the treated oranges were assessed on mould growth according to the two methods described in Example 5.
The results in Table 8 (number of moulded oranges per total of 8 oranges) and Table 9 (antifungal activity) clearly reveal that the active ingredient combination of 250 ppm natamycin and 200 ppm spiroxamine was more successful in limiting mould growth on oranges than natamycin or spiroxamine individually.
After 11 and 12 days of incubation, all 8 oranges treated with the control composition and 5 of the 8 oranges treated with natamycin alone showed mould growth, as did 7 and 8 oranges treated with spiroxamine alone, respectively. However, none of the 8 oranges treated with the active ingredient combination of natamycin and spiroxamine were moulded on days 11 and 12 (see Table 8).
On days 13 through 15, all 8 oranges treated with either the control composition or spiroxamine alone were moulded, as were 5 or 6 of the 8 oranges treated with natamycin alone, whereas only 1 of the 8 oranges treated with both natamycin and spiroxamine displayed mould growth (see Table 8).
After 16 and 18 days of incubation, all 8 oranges treated with either the control composition or spiroxamine alone showed mould growth, as did 6 of the 8 oranges treated with natamycin alone. Among the 8 oranges treated with both natamycin and spiroxamine, however, 5 were still mould free on day 16 and 4 on day 18 (see Table 8).
On days 19 through 26, mould growth was observed for all 8 oranges treated with either the control composition or spiroxamine alone and for 7 of the 8 oranges treated with natamycin alone. However, only 4 or 5 of the 8 oranges treated with both natamycin and spiroxamine were moulded (see Table 8).
Moreover, the observed antifungal activity of the composition comprising natamycin and spiroxamine was 5 to nearly 40% higher than the expected antifungal activity. Hence, the corresponding synergy factors all exceeded >1.0 and even increased from 1.1 on day 11 to 2.6 on days 25 and 26 (see Table 9).
In conclusion, the results of this example undoubtedly demonstrate the strong synergistic antifungal effect of 250 ppm natamycin and 200 ppm spiroxamine when applied in combination on oranges.
Ten fresh, organic sweet peppers were used per treatment. Each sweet pepper was soaked in a 180 ppm hypochlorite solution for 10 minutes, then rinsed thoroughly with fresh tap water and dried. The peel of each disinfected sweet pepper was wounded once using a cork borer according to the method described by de Lapeyre de Bellaire and Dubois (1987). Subsequently, each wound was inoculated with 10 μl of a Botrytis cinerea suspension containing 1×105 of spores/ml. After incubation for 3 hours at 20° C., each wound and the skin area of 0.5 cm around the wound was treated with in total 75 μl of a freshly prepared aqueous antifungal composition comprising either 200 ppm natamycin (DSM Food Specialties, Delft, The Netherlands), 150 ppm spiroxamine or both. Each antifungal composition also comprised 3.2% (w/w) beeswax, 0.8% (w/w) glycerol, 0.7% (w/w) polyoxyethylene sorbitan monostearate (Tween 60), 0.2% (w/w) polyoxyethylene sorbitan monooleate (Tween 80), 0.05% (w/w) methylhydroxyethyl-cellulose (MHEC), 0.03% (w/w) anti-foaming agent, 0.02% (w/w) xanthan gum, 0.02% (w/w) citric acid, 0.01% (w/w) lactic acid and 0.01% potassium sorbate. A composition without natamycin or spiroxamine was used as control. Each composition had a pH of 4.
The treated sweet peppers were incubated in a closed box in the dark at 20° C. and assessed on mould growth during incubation. The antifungal activity (in %) of the individual and combined active ingredients was determined by calculating the reduction in mould growth observed on the sweet peppers treated with the antifungal composition in comparison to the mould growth on the sweet peppers treated with the control composition according to the Colby method (Colby, 1967) as described in Example 5.
The results in Table 10 show that the combined antifungal composition comprising 200 ppm natamycin and 150 ppm spiroxamine protected sweet peppers more effectively against mould growth than the compositions comprising either natamycin or spiroxamine.
The observed antifungal activity exceeded the expected antifungal activity with approximately 5% on days 12 and 13, which yielded synergy factors >1.0 (see Table 10).
It can therefore be concluded that the combined application of 200 ppm natamycin and 150 ppm spiroxamine synergistically reduces mould growth on sweet peppers.
| Number | Date | Country | Kind |
|---|---|---|---|
| 12166260.5 | May 2012 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2013/058575 | 4/25/2013 | WO | 00 |
