WO2018102345A1

WO2018102345A1 - Use of the antifungal aureobasidin a in agriculture

Info

Publication number: WO2018102345A1
Application number: PCT/US2017/063599
Authority: WO
Inventors: Jon D. Polishook; Daniel P. RING; Sheo B. Singh; James Frederic Walter
Original assignee: Agrobiologics LLC
Current assignee: Agrobiologics LLC
Priority date: 2016-11-30
Filing date: 2017-11-29
Publication date: 2018-06-07
Anticipated expiration: 2019-05-30

Abstract

The present invention is concerned with the use of the natural product aureobasidin A (AbA) as an agricultural fungicide, compositions comprising aureobasidin A, and the use of such compositions to treat, control or prevent fungal infection in agricultural products, in particular, wherein the fungi are selected from the group consisting of Magnaporthe oryzae, Alternaria solani, Septoria triticum, Fusarium oxysporum, and Rhizoctonia solani.

Description

TITLE OF THE INVENTION

USE OF THE ANTIFUNGAL AUREOBASIDIN A IN AGRICULTURE

PRIORITY

This application claims the benefit of U.S. Provisional Application No.

62/427,855, filed November 30, 2016, the disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is concerned with the use of the natural product aureobasidin A (AbA) as an agricultural fungicide. The invention is also concerned with compositions comprising aureobasidin A (AbA), their preparation, and methods of using such compositions to control agricultural pests including, but not limited to, fungi selected from the group consisting of Magnaporthe oryzae, Alternaria solani, Septoria triticum, Fusarium oxysporum, and Rhizoctonia solani.

BACKGROUND OF THE INVENTION

In the agricultural industry there is a need to control plant pathogens that destroy crops and reduce yields. Farmers have traditionally employed a variety of methodologies to control such pests, one of which has been the use of compounds with antimicrobial activity, called fungicides, to protect the plants and prevent crop damage. There are many types of plant diseases, and foreign microorganisms can be introduced into new areas causing disease outbreaks. Current fungicides have traditionally been chemically synthesized compounds that have a limited spectrum of activity and often require repeated usage creating the potential for negative environmental impacts, which include contamination of foodstuffs, soil, surface and ground water and impact on native microbial and insect populations. In addition, pathogens often develop resistance to conventional fungicides, and as a result the agricultural industry continues to search for new agents with novel modes of action to combat disease resistance.

Aureobasidin A-R are antifungal cyclic depsipeptide antibiotics produced by Aureobasidium pullulans (Takesako et al, Journal of Antibiotics, 1991, 44, 919-924).

Aureobasidin A (AbA) is the most prominent, potent and studied member of this class of depsipeptides (Takesako et al, Journal of Antibiotics, 1991, 44, 919-924; Ikai et al, Journal of Antibiotics, 1991, 44, 925-933; and Takesako et al, Journal of Antibiotics, 1993, 46, 1414-1420). It was shown to have excellent activity against Candida, Aspergillus and Cryptococcus (U.S. Patent No. 5,698,670; Takesako et al, Journal of Antibiotics, 1993, 46, 1414-1420). AbA targets the inositol phophorylceramide (IPC) synthase, a key enzyme catalyzing sphingolipid synthesis in fungi (Cerantola et al., Molecular Microbiology 2009, 71, 1523-1537) but which is absent in mammals.

Liu et al. {International Journal of Food Microbiology 2007, 119, 223-229) have reported that AbA is active against five plant pathogenic fungi, including Penicillium digitatum, P. italicum, P. expansum, Botrytis cinerea and Monilinia fructicola. These pathogens cause disease in a variety of post-harvest fruit, and the authors suggested that AbA is a promising candidate to control post-harvest disease in fruit and vegetables. They noted that, of the five pathogens examined, P. digitatum was the most sensitive, whereas M. fructicola exhibited the least sensivtivity.

However, there remains an urgent need in the agricultural business for novel agents that can control plant disease caused by other fungi, such as of Magnaporthe oryzae, Alternaria solani, Septoria triticum, Fusarium oxysporum, and Rhizoctonia solani. Such agents should exhibit good efficacy, leave low residues after use, show lack of cross-resistance with existing fungicides, and display little or no toxicity toward non-target organisms.

Magnaporthe oryzae is a hemibiotrophic fungal pathogen that causes rice (Oryza sativa) blast, the most damaging disease on rice. Alternaria solani is a fungal pathogen that produces a disease in tomato and potato plants called early blight that is extremely destructive. Septoria tritici blotch (STB), caused by the ascomycete fungus Mycosphaerella graminicola (asexual stage: Septoria tritici), is one of the most important foliar diseases of wheat. Fusarium oxysporum is a pathogenic fungus common in soils around the world, and the cause of fusarium wilt, a deadly vascular wilting syndrome in plants. Fusarium oxysporum comprises over 120 known strains or "special forms" (formae speciales; f. sp.), each of which is specific to a unique plant host in which it causes disease. Collectively, these F. oxysporum strains infect and kill a large host range including many commercially harvested crops such as species in the Solenaceae family (tomatoes, peppers, potatoes, eggplant), watermelon, lettuce, legumes, beets, basil, strawberries, chrysanthemum, sugarcane, bananas, and multiple other species. Rhizoctonia solani causes a wide range of commercially significant plant diseases. It is one of the fungi responsible for Brown patch (a turfgrass disease), damping off (e.g., in soybean seedlings), as well as black scurf of potatoes, bare patch of cereals, root rot of sugar beets, belly rot of cucumber, sheath blight of rice, and many other pathogenic conditions.

Structure of aureobasidin A (AbA)

It has now been found that aureobasidin A is useful as an agricultural fungicide to treat, prevent, or control fungal infections in agricultural products, such as plants and seeds. Plants will include fruits and vegetables.

In one aspect of the present invention aureobasidin A has been found to be effective in treating, preventing, or controlling infections in agricultural products caused by the following fungi: Magnaporthe oryzae, Alternaria solani, Septoria triticum, Fusarium

oxysporum, and Rhizoctonia solani.

A second aspect of the present invention is concerned with agricultural compositions of aureobasidin A comprising an effective amount of the antifungal compound and an agriculturally acceptable carrier. Such compositions may additionally comprise one or more excipients selected from the group consisting of (a) one or more diluents, (b) one or more binders or binding agents, (c) one or more dispersing agents, (d) one or more emulsifying agents, (e) one or more surfactants or wetting agents, (f) one or more sticking agents, (g) one or more thickening agents, and (h) a pH adjuster. In one embodiment the surfactant is an anionic surfactant. In a second embodiment the surfactant is a non-ionic surfactant.

Aureobasidin A and agricultural compositions of aureobasidin A of the present invention may also be used in combination with one or more other agents useful to treat, prevent, or control agricultural pests in the field. In such combinations the compositions of the present invention and other active agents may be administered separately or in conjunction. In addition, the administration of one element may be prior to, concurrent to, or subsequent to the

administration of the other agent(s).

Examples of other active ingredients that may be administered in combination with compositions of the present invention, and either administered separately or in the same agricultural composition, include, but are not limited to:

(1) antifungal agents, such as Ilicicolin H, azoxystrobin and myclobutanil;

(2) plant growth regulators, such as dikegulac-sodium and trinexapac-ethyl; (3) herbicides, such as prodiamine and glyphosate;

(4) insecticides, such as bifenthrin and malathion; and

(5) acaricides, such as avermectin and kelthane.

The agricultural compositions of the present invention may contain about 0.1 to 95 percent by weight of aureobasidin A.

It is another aspect of the present invention to provide methods of treating, controlling, or preventing fungal infections on an agricultural product comprising applying to the agricultural product or plant the present agricultural compositions of aureobasidin A. The compositions of the present invention may be applied using a variety of methodologies, including soil drench, seed treatment, granular and foliar spray. With regard to the treatment of seeds, the compositions may alternatively be applied directly to the seed before planting of the seed or applied to the locus or soil in which the seeds are sown prior to the planting of the seed.

In such methods of the present invention the concentration of aureobasidin A to be applied to the agricultural product is about 0.001 to 1 percent by weight. In a class of this method the concentration of aureobasidin A to be applied to the agricultural product is about 0.001 to 0.01 percent by weight.

Another aspect of the present invention relates to the total amount of aureobasidin A to be applied per area of the field. In one class of this aspect about 2 to about 1000 grams of aureobasidin A are to be applied per acre. In a subclass of this class about 5 to 500 grams of aureobasidin A are to be applied per acre. In a second class of this aspect about 0.025 kilograms to about 5 kilograms of aureobasidin A are to be applied per hectare. In a subclass of this second class about 0.05 kilograms to about 1 kilogram of aureobasidin A are to be applied per hectare.

Another aspect of the present invention relates to the use of aureobasidin A and the compositions of the present invention to treat, control, or prevent fungal infections on an agricultural product.

It is another aspect of the present invention to provide for the use of aureobasidin A in the manufacture of a composition for use in treating, controlling, or preventing a fungal infection on an agricultural product.

The present invention also relates to a food, feed, or agricultural product treated with a composition of the present invention.

The present invention also relates to a process for the treatment of an agricultural product which comprises applying a composition of the present invention to such agricultural product or plant.

The present invention also relates to the use of a fermentation broth derived from strains of Aureobasidium pullulans as a live cell or cell suspension to treat, control, or prevent a fungal infection on an agricultural product. Such fermentation broths contain the antifungal agent aureobasidin A, which need not be isolated, purified, and re-formulated for use as an agricultural fungicide.

These and other aspects will become readily apparent from the detailed description of the invention which follows.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is concerned with the use of the naturally occurring antifungal aureobasisin A (AbA) to control the growth of agricultural pests including, but not limited to, Magnaporthe oryzae, Alternaria solani, Septoria triticum, Fusarium Oxysporum, and Rhizoctonia solani.

Aureobasidin A can be used to control a variety of plant diseases and can be applied in a variety of methodologies, including soil drench, seed treatment, granular and foliar spray. It can be readily formulated into a variety of compositions, including, but not limited to, emulsifiable concentrates, inverse emulsions, microemulsions, dispersible granules, wettable powders, dusts, and granules.

Aureobasidin A offers several advantages over existing synthetic fungicides. Since it is derived from natural sources, it already occurs in the environment and biological processes to degrade the compound are already present in the environment. Unlike many conventional fungicides, aureobasidin A does not contain any heavy metals or halogens and therefore is totally biologically degradable. Moreover, aureobasidin A has a different mechanism of action from existing commercial fungicides and therefore can be used to reduce fungicide resistance found in certain plant diseases.

The term "agricultural product" as used herein is 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, and soybeans; oleaginous plants, e.g. rape, mustard, poppy, olive, sunflower, coconut, castor-oil plant, cocoa, and ground-nuts; cucurbitaceae, e.g. pumpkins, gherkins, melons, cucumbers, squashes, and aubergines; fibrous plants, e.g. cotton, flax, hemp, and jute; citrus fruit, e.g. oranges, lemons, grapefruits, mandarins, and limes; tropical fruit, e.g. papayas, passion fruit, mangos, carambolas, pineapples, bananas, and kiwis;

vegetables, e,g. spinach, lettuce, asparagus; brassicaceae, such as cabbage 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, Gerbia, 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.

The following Examples are provided to illustrate the invention and are not intended to be construed as limiting the scope of the invention in any manner.

EXAMPLE 1

Suspensions of mycelial fragments or conidia of a fungal species, prepared either freshly from liquid cultures of the fungus or from cryogenic storage, were directly mixed into nutrient broth. Dilutions of AbA (from 0.03 to 180 ppm) were diluted with 0.025% Tween 20, and 10 μΐ. of this solution was pipetted into a microtitre plate (96-well format). The nutrient broth containing the fungal spores/mycelia fragments was then added to give a final

concentration of AbA. The test plates were incubated in the dark at 24°C and 96% relative humidity. The inhibition of fungal growth was determined photometrically after 2-7 days, depending on the pathosystem, and percent antifungal activity relative to the untreated check was calculated. The results are shown in Table 1 below. AbA demonstrated activity against

Magnaporthe oryzae, Alter naria solani, Septoria triticum, Fusarium Oxysporum, and

Rhizoctonia solani.

TABLE 1

Percent Control of Pathogens by AbA Treatment

EXAMPLE 2 For the production of aureobasidin A, Aureobasidium pullulans is grown and cultivated as described in Takesako et al., "Aureobasidins, New antifungal antibiotics taxonomy, fermentation, isolation and properties" Journal of Antibiotics 1991, 44, 919-924. For example, Aureobasidium pullulans is inoculated in 100 mL of liquid medium composed of yeast nitrogen base (Difco) 0.67% and glucose (2%) in a 500 mL Erlenmeyer flask. The flask is shaken at 25°C for two days to give a seed culture. One-liter seed culture is transferred into 200-liter fermenter containing 100 liters of medium consisting of glucose 2%, ammonium sulfate 0.5%, potassium hydrogen phosphate

sodium chloride 0.01%,

microgram/mL, and

microgram/mL. Twenty liters of a medium containing glucose 10%, ammonium sulfate 2.5%, polypepton 5%, potassium hydrogen phosphate

sodium chloride

microgram/mL, and

microgram/mL is supplemented to the culture. The fermentation is further continued at 25°C for 78 hours with 120-liters/minute aeration and 100 rpm agitation. The fermentation broth from producer strains can be processed in several different ways depending on the utility. In one method, the mycelia are separated and extracted by ethanol, methanol, acetone or ethyl acetate. The extract is concentrated and aureobasidin A is purified by standard chromatographic methods as described by Takesako et al, Journal of Antibiotics, 1991, 44, 919-924. In a second method, the fermentation broth is lyophilized to remove water to afford a dry powder. The cells in the fermentation broth are broken if necessary by addition of organic solvent, such as methanol, ethanol, acetone, and the like, and then evaporated to dryness to afford a powder. The fermentation broth obtained directly or after breaking the cells with solvent is sprayed onto the plant by foliar treatment, mixed with seeds, or used as a soil inoculant. In addition, broth powders generated using either of the two methods is mixed with appropriate agent(s) and sprayed by foliar treatment, mixed with seeds, or used as a soil inoculant.

EXAMPLES OF FORMULATIONS OF AUREOBASIDIN A FOR AGRICULTURAL USE

Specific embodiments of a formulation of aureobasidin A for use in agriculture are provided below. The final amounts (wt. %) of concentrate components are set forth in Table 2 below.

While the invention has been described and illustrated in reference to specific embodiments thereof, those skilled in the art will appreciate that various changes, modifications, and substitutions can be made therein without departing from the spirit and scope of the invention. It is intended therefore that the invention be limited only by the scope of the claims which follow and that such claims be interpreted as broadly as is reasonable.

Claims

WHAT IS CLAIMED IS:

1. An agricultural composition comprising an agriculturally effective amount of aureobasidin A and an agriculturally acceptable carrier.

2. The composition according to Claim 1 additionally comprising one or more excipients selected from the group consisting of (a) a diluent, (b) a dispersing agent, (c) an emulsifying agent, (d) a wetting agent, (e) a surfactant, and (f) a sticking agent.

3. The composition according to Claim 1 formulated in the form of an emulsifiable concentrate, an emulsion, or a microemulsion.

4. The composition according to Claim 1 or 2 further comprising an agriculturally effective amount of one or more agents selected from the group consisting of an antifungal agent, a plant growth regulator, a herbicide, an insecticide, and an acaricide.

5. A method for treating, controlling, or preventing a fungal infection on an agricultural product wherein said fungal infection is selected from the group consisting of Magnaporthe oryzae infection, Alternaria solani infection, Septoria triticum infection, Fusarium oxysporum infection, and Rhizoctonia solani infection comprising the application to said agricultural product the composition according to Claims 1 to 4.

6. Use of the composition according to Claim 1 to treat, control, or prevent a fungal infection on an agricultural product.

7. Use of aureobasidin A in the manufacture of a composition for use in treating, controlling, or preventing a fungal infection on an agricultural product.

8. A food, feed, or agricultural product treated with a composition according to Claim 1 or 2.

9. The method according to Claim 5 wherein said agricultural product is a plant or seed.

10. The method according to Claim 5 wherein the application is directly to the agricultural product.

11. The method according to Claim 11 wherein the application is by foliar spray.

12. A process for the treatment of an agricultural product which comprises the application of a composition according to Claim 1 or 2 to said agricultural product.

13. Use of a fermentation broth derived from strains of Aureobasidium pullulans as a live cell or cell suspension to treat, control, or prevent a fungal infection on an agricultural product.

14. The use of Claim 13 wherein said fungal infection is selected from the group consisting of Magnaporthe oryzae infection, Alternaria solani infection, Septoria triticum infection, Fusarium oxysporum infection, and Rhizoctonia solani infection.