WO2013000986A1

WO2013000986A1 - New antifungal compositions

Info

Publication number: WO2013000986A1
Application number: PCT/EP2012/062528
Authority: WO
Inventors: Jacobus Stark; De Angelique Rijk
Original assignee: Dsm Ip Assets B.V.
Priority date: 2011-06-30
Filing date: 2012-06-28
Publication date: 2013-01-03

Abstract

The present invention relates to new antifungal compositions and their use in the treatment of agricultural products.

Description

NEW ANTIFUNGAL COMPOSITIONS

Field of the invention

The present invention discloses new antimicrobial compositions to control plant diseases and to prevent microbial spoilage of crops.

Background of the invention

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.

Banana and plantains are ranked fourth after rice, wheat and maize in human consumption and constitute a major staple food crop for millions of people in developing countries. The banana plant is an herb belonging to the genus Musa and is grown in more than hundred countries worldwide. Most of the bananas are consumed by the local population, but also great volumes are exported to e.g. the USA, Europe and Japan.

Both bananas and plantain plants are sensitive to fungi such as fungi from the genus Fusarium and Mycosphaerella. Fungi from the genus Fusarium are known to cause Panama disease (also known as Fusarium wilt) of banana and plantain plants, while fungi from the genus Mycosphaerella cause the so-called Black and Yellow Sigatoka disease.

In WO 2005/074687 a new antifungal composition containing e.g. natamycin to prevent growth of fungi on banana plants in the field is described. The invention described in WO 2005/074687 offers a solution to protect banana plants in the field against pathogenic fungi. Besides fungal growth on banana and plantain plants in the field, spoilage of bananas and plantains by fungi after harvesting is an issue of major concern. This so called "crown rot" is caused by fungal infection after cutting the banana hand from the main stem. At this stage fungal spores and/or fungal mycelium fragments can easily enter the wound via the surface liquid. Sometimes fungi may also develop at the bottom of the banana, the place where the flower was present. Later during transport and/or ripening the spores can germinate and/or the fungal mycelium fragments can grow and the fungi grow further into the banana hand and will spoil the bananas. Crown rot can be caused by a number of fungal species. The most important species are Fusarium pallidoroseum, Fusarium proliferatum, Colletotrichum musae, Verticillium theobromae, Thielaviopsis paradoxa, Lasiodiplodia theobromae, Deightoniella torulosa and Fusarium roseum. Crown rot leads to considerable losses of bananas and plantains grown for local consumption and of exported bananas and plantains.

To prevent fungal damage, after harvesting at least all export bananas and plantains, but also many bananas and plantains for local consumption, receive a post- harvest treatment with an antifungal composition, e.g. by spraying the whole banana or by treating the crown with e.g. a brush. Examples of fungicides applied in today's practice are thiazoles, thiabendazole, benomyl, imidazoles such as imazalil or mixtures thereof.

EP 0 290 155 discloses an absorbent sheet material for application to plant wounds comprising a flexible laminate of a sheet material, a permeable layer, absorbent material and a fungicide e.g. thiabendazole.

US 4, 148,891 discloses antifungal preparations of polyene antibiotics dissolved in a mixture of a lower alkanol and a lower alkanoic acid for preventing or inhibiting fungal growth on agricultural products such as bananas.

Griffee and Pinegar (1974) examined several fungicides in the treatment of crown rot. Also pimaricin, now known as natamycin, was included in this study. From many other publications it is known that natamycin is a very effective antifungal compound. The authors indeed stated that natamycin showed a broad spectrum action. However, they also concluded that natamycin was not sufficiently active to give satisfactory control of fungi on bananas.

In spite of extensive treatment with the fungicides presently applied, spoilage problems still occur. It is known that fungi rather easily develop resistance to antifungal compounds, which is also the case for several fungi causing spoilage of bananas and plantains. When resistant strains develop, selection will take place and fungal problems will increase, leading to the use of even higher amounts of fungicides. Further, many of the fungicides applied on bananas and plantains cause environmental pollution and human health problems. Also workers safety is an important issue, as it is known that all farm workers come in close contact with high concentrations of these harmful fungicides.

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 was never observed.

Consequently, it can be concluded that there is a severe need for more effective, more environmental friendly, lower-toxicity and less harmful antimicrobial compositions, e.g. antifungal compositions, for the treatment of agricultural products such as for instance bananas and plantains.

Description of the invention

The present invention solves the problem by providing a new synergistic antimicrobial, e.g. antifungal, composition comprising a polyene antifungal compound and alum. 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.

"Alum" as used herein means a compound comprising the formula (A)(B)(D0₄)2-12H₂0, wherein A is a monovalent cation, B is a trivalent metal ion and D is selenium or sulphur. In an embodiment A is selected from the group consisting of K⁺, Rb⁺, Cs⁺, ΤΓ, NH₄ ⁺, CH₃NH₃ ⁺, HONH₃ ⁺, N₂H₅ ⁺ and Na⁺. In a preferred embodiment A is selected from the group consisting of K⁺, NH₄ ⁺ and Na⁺, with NH₄ ⁺ being preferred. In an embodiment B is selected from the group consisting of Al³⁺, Ga³⁺, ln³⁺, Ti³⁺, V³⁺, Cr³⁺, Mn³⁺, Fe³⁺, Co³⁺, lr³⁺, Rh³⁺, Mb³⁺ and Ru³⁺. In a preferred embodiment B is selected from the group consisting of Al³⁺ and Cr³⁺, with Al³⁺ being preferred. In an embodiment D is selected from the group consisting of Se and S. In a preferred embodiment D is S. The term "alum" as used herein also includes analogs of alum. Analogs of alum are either: a) a double sulphate or selenate having dodecahydrate hydration, i.e. a compound comprising the formula (A)2(D04)-(B)2(D0₄)₃-24H₂0, wherein A is a monovalent cation, B is a trivalent metal ion and D is selenium or sulphur. In an embodiment A is selected from the group consisting of K⁺, Rb⁺, Cs⁺, Tl⁺, NH₄ ⁺, CH₃NH₃ ⁺, HONH₃ ⁺, N₂H₅ ⁺ and Na⁺. In a preferred embodiment A is selected from the group consisting of K⁺, NH₄ ⁺ and Na⁺. In an embodiment B is selected from the group consisting of Al³⁺, Ga³⁺, ln³⁺, Ti³⁺, V³⁺, Cr³⁺, Mn³⁺, Fe³⁺, Co³⁺, lr³⁺, Rh³⁺, Mb³⁺ and Ru³⁺. In a preferred embodiment B is selected from the group consisting of Al³⁺ and Cr³⁺. In an embodiment D is selected from the group consisting of Se and S. In a preferred embodiment D is S, or

b) a double sulphate or selenate having another degree of hydration, i.e. a compound comprising the formula (A)-(B)(D0₄)₂-1 1 H₂0, wherein A is Na⁺ or K⁺, B is Al³⁺ and D is S (e.g. mendozite or kalinite); a compound comprising the formula (A)-(B)(D0₄)2-6H₂0, wherein A is CH₆N₃ ⁺ or (CH₃)₂NH₂ ⁺, B is Al³⁺ or Cr³⁺ and D is S {e.g. guanidinium alum or dimethylammonium alum); a compound comprising the formula (A)-(B)(D0₄)2-4H₂0, wherein A is K⁺, B is Fe³⁺ and D is S (e.g. goldichite); a compound comprising the formula (Α)-(Β)(0θ4)₂Ή₂Ο, wherein A is Τ , B is Pu³⁺ and D is S {e.g. thallium plutonium sulfate); a compound comprising the formula (A)-(B)(D0₄)₂, wherein A is K⁺, B is Fe³⁺ and D is S (e.g. yavapaiite), or

c) a compound comprising the formula (A)(D0₄)-(B)₂(D04)₃-22H₂0, wherein A is a divalent cation, B is a trivalent metal ion and D is selenium or sulphur. In an embodiment A is selected from the group consisting of Co²⁺, Ni²⁺, Mn²⁺, Mg²⁺, Fe²⁺ (e.g. wupatkiite, apjohnite, pickingerite, halotrichite). In an embodiment B is Al³⁺. In an embodiment D is S, or

d) a compound comprising the formula (A)₂(D0₄)-(B)(D0₄)-6H₂0, wherein A is a univalent cation, B is a divalent metal ion and D is S (e.g. a Tutton salt), or

e) a compound comprising the formula (A)₂(D0₄)-2(BD0₄), wherein A is a univalent cation, B is a divalent metal ion and D is S (e.g. langbeinites such as potassium magnesium sulphate).

In an embodiment the compositions may also contain two or more different alums.

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. 0.5-50% (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 0.001 % (w/v) to about 50% (w/v) alum and preferably from about 0.01 %(w/v) to about 15% (w/v) alum. 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 compound may be an antifungal compound (e.g. imazalil, thiabendazole) or a compound to combat insects, nematodes, mites and/or bacteria. In an embodiment the composition according to the invention comprises natamycin, alum, and thiabendazole, imazalil or both. 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 alum 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. KH₂P0₃ or K₂HP0₃ or a mixture of both phosphite salts. Phosphite containing compounds as used herein means compounds comprising a phosphite group, i.e. P0₃ (in the form of e.g. H₂P0₃ ^", HP0₃ ^2" or P0₃ ^3") 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. KH₂P0₃ and K₂HP0₃, sodium phosphites and ammonium phosphites, and (C1-C4) alkyl esters of phosphorous acid and their salts such as aluminum ethyl phosphite (fosetyl-AI), 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. KH₂P0₃ and K₂HP0₃ can easily be obtained by e.g. adding KOH or K₂C0₃ to a final pH of 5.0 - 6.0 to a KH₂P0₃ 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 5 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 alum are applied as separate compositions.

In a further aspect the invention relates to a kit comprising a polyene antifungal compound and alum. The polyene antifungal compound and alum 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, combining and/or applying the compounds. In an embodiment the kit also comprises an additional compound. Examples of additional compounds are given above. As an example, the kit may comprise a polyene antifungal compound such as natamycin, alum, and thiabendazole, imazalil or both. In a further aspect the invention pertains to a method for protecting a product against fungi by treating the agricultural product with a polyene antifungal compound and alum. In addition, the product can be treated with other antifungal and/or antimicrobial compounds either prior to or after treatment of the products with the polyene antifungal compound and alum. In an embodiment the product is further treated with thiabendazole, imazalil or both. The product may be treated by sequential application of the polyene antifungal compound and alum or vice versa. Alternatively, the product may be treated by simultaneous application of the polyene antifungal compound and alum. 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 compounds. In an embodiment the invention is directed to a process for the treatment of products by applying the polyene antifungal compound and alum 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 such as a banana, plantain or pineapple.

The compounds, the compositions according to the invention and the kits according to the invention can be applied to the products by spraying or fogging. 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, cascade treatment, watering, drenching, introduction into a dump tank, vaporizing, atomizing, fumigating, painting, brushing, dusting, foaming, spreading-on, packaging and coating (e.g. by means of wax or electrostatically).

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 alum applied may vary from 10 ppm to 50,000 ppm, preferably from 20 ppm to 40,000 ppm and most preferably from 50 to 25,000 ppm.

The composition is advantageously applied on crops after harvesting, preferably crops that show a wound after harvesting. In other words, the harvesting of the crops results in a wound and the wound is treated with a composition according to the invention. In general, the wound occurs when the crop, e.g. fruit such as banana, plantain or pineapple, is removed from the plant it is grown on. For instance, bananas and plantains cut from a banana plant have a wound that is a consequence of the cutting. Cutting can be done with a knife, a chopper, a hatchet or the like. It can be done by hand or automatically. So, in general the harvested crop, which is preferably fruit, is connected to its plant by a stalk which will be broken by mechanical force. Advantageously, they are liquids which can be applied by dipping, spraying or electrostatic spraying of e.g. bananas, plantains or pineapples. Alternatively, the wounds on the bananas, plantains or pineapples can also be treated using a paintbrush or e.g. a pad of cotton wool or cellulose pads impregnated with the antifungal compositions of the invention. The compositions of this invention can also be applied by using plaster-like carriers which are placed on the wound. Moreover, resin-like or wax-like compositions known in the art which cover the wound surface can be applied. In another aspect of this invention the antimicrobial compositions may be applied by means of a fruit coating. For all these treatments methods and equipment well-known to a person skilled in the art can be used. A main advantage of the brush method, plaster-like compositions and spraying resins on the wound surface is that the fungicide is directly applied on the wound tissue and not on the whole fruit. On the other hand, aqueous dipping, spraying or cascade treatment applications using automatic systems reduce the labour costs and are more cost-effective. In case the bananas are washed, the compositions of the invention can be applied by e.g. spraying if the bananas come out of the wash tanks. Wash tanks are used to remove dust and field heat from the bananas. In case the bananas are put into specific de-latexing tanks to remove the latex which is leaking from the wound originating from the cutting of the hands and consequently preventing latex drips on the bananas that may form ugly spots, the compositions of the invention can be applied by e.g. spraying if the bananas come out of the de-latexing tanks. Alternatively, the compositions of the present invention may be added to the washing or de-latexing solutions themselves. Thereafter, the hands may be cut into clusters of four to six fingers (bananas) which are washed again. Also during or after this additional washing step the compositions of the invention can be applied.

After treatment with the antimicrobial compositions of the invention the bananas may be packed in plastic, e.g. in the so-called mini wet-pack system commonly used for packaging of export bananas dip-treated in an antimicrobial composition. Alternatively, the bananas may be packed in boxes and shipped. By using a polyene antifungal compound and alum, 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.

Another aspect of the present invention relates to the use of a polyene antifungal compound and alum 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 such as for instance a banana or a plantain.

In a specific embodiment the polyene antifungal compound and alum can be used in medicine, e.g. to treat and/or prevent fungal diseases. The polyene antifungal compound and alum can for instance be used in the form of a pharmaceutical composition. The composition may further comprise pharmaceutically acceptable excipients. 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 alum. 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 alum. The treated products may comprise a polyene antifungal compound and alum 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 20,000 mg/dm², preferably 0.00001 to 15,000 mg/dm², more preferably from 0.00005 to 10,000 mg/dm² of alum or an analog thereof 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. In an embodiment the product further comprises at least an additional antimicrobial compound. In an embodiment the product further comprises thiabendazole, imazalil or both.

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. In a preferred embodiment, the agricultural product is a product that has a wound after harvesting such as a banana, a plantain or a pineapple.

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 alum. 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.

EXAMPLES

Example 1

Treatment of bananas with natamycin and alum

In this experiment freshly harvested bananas (variety Cavendish) were used. The bananas were obtained from a local grower in Costa Rica. Bananas with non-visible fungal disease symptoms were selected and surface-sterilized with a sodium hypochlorite solution and then rinsed with distilled water.

In each treatment, one exportation banana box (about 18 kg of weight) with an average number of clusters of 21 (the range was 19-22 clusters per treatment) was used. The bananas were inoculated with a mixture of spores of the phytopathogenic fungal species Colletotrichum musae (2 strains), Fusarium proliferatum (2 strains) and Fusarium pallidoroseum (2 strains). To obtain a representative cocktail of strains which cause crown rot in the field, the fungal strains were isolated from bananas in different times of the season (dry and rainy season). The fungal spore suspension was obtained using well-known methods and had a final concentration of approximately 10⁵ fungal spores per ml (equal for each of the six strains).

The crown of the bananas was inoculated with approximately 0.5 ml of the suspension of fungal spores. After incubation for 3 hours at room temperature, the banana crowns were sprayed using well-known methods with a composition comprising either 500 ppm of natamycin, or 2% (w/v) alum (NH₄AI(S0₄)₂-12 H₂0), or both. A composition without natamycin or alum was used as a control. All compositions contained 2.6% (w/v) potassium phosphite and 0.06% (w/v) xanthan gum and were prepared using well-known methods. The treated bananas were incubated for 26 days in closed boxes at 17°C.

After 26 days of incubation, the severity of crown rot disease of the treated bananas was assessed in a two-fold manner. Firstly, the crown rot incidence (in %) was determined from the number of bananas affected by the disease and used to calculate the reduction in the percentage of bananas affected by crown rot after treatment with the antifungal compositions compared to the control. Secondly, the average depth of the crown rot lesions (in cm) was determined by cutting the crown longitudinally and measuring the depth of the rot into the pedicles. The average lesion depths were used to calculate the reduction (in %) in lesion depth observed in the bananas treated with the antifungal compositions compared to those treated with the control. The expected reduction (E in %) in either the percentage of affected bananas or the lesion depth of the composition comprising both natamycin and alum 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 (see Table 1 ) clearly demonstrate that in a challenge test using high fungal contaminations, the compositions of the present invention can protect crops, e.g. fruits such as bananas, against fungal growth and further demonstrate that the compositions of the present invention show a synergistically enhanced antifungal activity compared to the antifungal activity of the active compounds when applied individually. Compared to the control, the use of alum alone caused a 1 % reduction in bananas with crow rot, whereas the use of natamycin alone resulted in a 5% reduction in bananas with crown rot. However, when using the combination of natamycin and alum, the observed reduction in the percentage of bananas affected by crown rot exceeded the expected reduction about 25%, which resulted in a synergy factor of 5.

In addition, the results clearly show that the disease severity of bananas treated with the combination comprising both natamycin and alum was much lower than that of the bananas treated wth natamycin or alum alone. Table 2 reveals that the lesions of the bananas treated with alum alone were even deeper than the lesions of the control bananas. In contrast, treatment with natamycin alone resulted in 15% less deep lesions. Moreover, treatment with the combination of natamycin and alum reduced the lesion depth with almost 60% compared to the control. Consequently, the observed reduction in lesion depth of the natamycin-alum combination was more than 40% higher than the expected reduction and a synergy factor of 4 was obtained.

Thus, the above-described results unequivocally demonstrate the strong synergistic antifungal effect of the combined application of natamycin and alum.

Table 1 : Percentage of bananas showing crown rot after 26 days of storage.

Table 2: Depth of the lesions caused by the crown rot after 26 days of storage.

Composition Depth of Reduction in depth of lesions Synergy Factor lesions (cm) compared to control (in %) (O/E)

Observed Expected

reduction (%) reduction (%) Control 2.10 0 - -

Natamycin 1.79 15 - -

Alum 2.37 0 - -

Natamycin + alum 0.91 57 15 4

REFERENCES

Colby SR (1967), Calculating synergistic and antagonistic responses of herbicide combination. Weeds 15: 20-22.

Griffee PJ and Pinegar JA (1974), Fungicides for control of the banana crown rot complex: in vivo and in vitro studies. Trop. Sci. 16: 107-120.

Slinker BK (1998), The Statistics of Synergism. Journal of Mol. and Cell. Cardiology 30:723-731 .

Claims

1. A composition comprising natamycin and alum.

2. A composition according to claim 1 , wherein alum comprises a compound comprising the formula (A)(B)(D0₄)₂-12H₂0, wherein A is a monovalent cation, B is a trivalent metal ion and D is selenium or sulphur.

3. A composition according to claim 1 or 2, wherein A is selected from the group consisting of K⁺, Rb⁺, Cs⁺, Tl⁺, NH₄ ⁺, CH₃NH₃ ⁺, HONH₃ ⁺, N₂H₅ ⁺ and Na⁺, B is selected from the group consisting of Al³⁺, Ga³⁺, ln³⁺, Ti³⁺, V³⁺, Cr³⁺, Mn³⁺, Fe³⁺, Co³⁺, lr³⁺, Rh³⁺, Mb³⁺ and Ru³⁺, and D is selected from the group consisting of Se and S.

4. A composition according to claim 1 , wherein alum is selected from the group consisting of:

a) a compound comprising the formula (A)₂(D0₄)-(B)₂(D0₄)₃-24H₂0, wherein A is a monovalent cation, B is a trivalent metal ion and D is selenium or sulphur, and wherein A is selected from the group consisting of K⁺, Rb⁺, Cs⁺, Τ , NH₄ ⁺, CH₃NH₃ ⁺, HONH₃ ⁺, N₂H₅ ⁺ and Na⁺, B is selected from the group consisting of Al³⁺, Ga³⁺, ln³⁺, Ti³⁺, V³⁺, Cr³⁺, Mn³⁺, Fe³⁺, Co³⁺, lr³⁺, Rh³⁺, Mb³⁺ and Ru³⁺ and D is selected from the group consisting of Se and S,

b) a compound comprising the formula (A)-(B)(D0₄)₂-1 1 H₂0, wherein A is Na⁺ or K⁺, B is Al³⁺ and D is S,

c) a compound comprising the formula (A)-(B)(D0₄)₂-6H₂0, wherein A is CH₆N₃ ⁺ or (CH₃)₂NH₂ ⁺, B is Al³⁺ or Cr³⁺ and D is S,

d) a compound comprising the formula (A)-(B)(D0₄)₂-4H₂0, wherein A is K⁺, B is Fe³⁺ and D is S,

e) a compound comprising the formula (A)-(B)(D0₄)₂-H₂0, wherein A is ΤΓ, B is Pu³⁺ and D is S,

f) a compound comprising the formula (A)-(B)(D0₄)₂, wherein A is K⁺, B is Fe³⁺ and D is S,

g) a compound comprising the formula (A)(D0₄)-(B)₂(D0₄)₃-22H₂0, wherein A is a divalent cation, B is a trivalent metal ion and D is selenium or sulphur, and wherein A is selected from the group consisting of Co²⁺, Ni²⁺, Mn²⁺, Mg²⁺, Fe²⁺, B is Al³⁺ and D is S,

h) a compound comprising the formula (A)2(D0₄)-(B)(D0 )-6H₂0, wherein A is a univalent cation, B is a divalent metal ion and D is S, and

i) a compound comprising the formula (A)₂(D0 )-2(BD0 ), wherein A is a univalent cation, B is a divalent metal ion and D is S.

5. A composition according to any one of the claims 1 to 4, wherein the composition 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.

6. A composition according to claim 5 comprising natamycin, alum and thiabendazole, imazalil or both.

7. A kit comprising natamycin and alum.

8. A method for protecting a product against fungi by treating the product with natamycin and alum.

9. A method according to claim 8, wherein the product is further treated with thiabendazole, imazalil or both.

10. A method according to claim 8 or 9, wherein the product is treated with a composition according to any one of the claims 1 to 6 or a kit according to claim 7.

1 1 . A method according to any one of the claims 8 to 10, wherein the product is an agricultural product.

12. A method according to claim 1 1 , wherein the product is a banana, plantain or pineapple.

13. A method according to claim 1 1 or 12, wherein the product is treated post-harvest.

14. Use of natamycin and alum to protect a product against fungi.

15. A product comprising natamycin and alum.

16. A product according to claim 15, wherein the product is an agricultural product.

17. A product according to claim 15 or 16, wherein the product further comprises thiabendazole, imazalil or both.