WO2014085576A1 - Synergistic combinations of fungicides and physical membrane disrupting agents and methods of use - Google Patents

Abstract

The present invention relates to compositions comprising a synergistic fungicidal combination of a polyene fungicide and physical membrane disrupting agents other than a lipopeptide. The present invention relates to compositions comprising a synergistic fungicidal combination of a lipopeptide and physical membrane disrupting agents other than a polyene fungicide. The invention also includes methods for using such compositions in controlling fungal pathogens.

Description

SYNERGISTIC COMBINATIONS OF FUNGICIDES AND PHYSICAL

MEMBRANE DISRUPTING AGENTS AND METHODS OF USE

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This patent application claims priority to U.S. Provisional Patent Application No. 61/730,959, filed November 28, 2012, and U.S. Provisional Patent Application No.

61/731,059, filed November 29, 2012, the disclosures of both of which are hereby incorporated by reference.

FIELD OF INVENTION

[0002] The present invention relates to synergistic combinations of polyene fungicides with physical membrane disrupting agents other than a lipopeptide, to improve the fungicidal activity of both components. The present invention also relates to synergistic combinations of a lipopeptide with physical membrane disrupting agents other than polyene fungicides, to improve the fungicidal activity of both components.

BACKGROUND OF INVENTION

[0003] Fungicides have myriad uses, including for crop protection; as food, feed, and cosmetics preservatives; and as pharmaceuticals for both human and veterinary applications. Crop yield reduction, food-borne diseases and fungal infections of both humans and animals are a problem in both developed and developing countries. Therefore, improvements to the efficacy of existing fungicides, especially those that are environmentally friendly and are not susceptible to development of fungal resistance are highly desirable.

[0004] Polyene fungicides are antifungal antibiotics that have been used in all of the aforementioned fields. They may be obtained through fermentation of Streptomyces species, including but not limited to Streptomyces natalensis, which is commonly found in soil. Activity of polyene fungicides derives, in part, from their ability to damage cell membranes by forming complexes with ergosterol. Numerous studies have confirmed that the potential for development of fungi resistant to natamycin is very low. Further, polyene fungicides have negligible toxicity, as they do not affect the cholesterol present in mammalian cells.

[0005] Non-ribosomal peptides, including cyclic amphiphilic lipopeptides include surfactins, iturins and fengycins; are well-recognized for their antimicrobial properties and have been used in the field of crop protection. Because of their mode of action, they also have potential uses in biopharmaceutical and other biotechnology applications. Lipopeptides may be obtained through fermentation of various soil bacteria, including Bacillus subtilis and Bacillus amyloliquefaciens. Lipopeptides, similarly to polyene fungicides, kill fungi by disrupting cell membranes. The potential for the development of fungal resistance to these compounds is expected to be very low since they act directly upon membrane lipids and not on a single site protein target. Further, lipopeptides are environmentally friendly and of low risk to workers and consumers; in fact, crops treated with lipopeptide-containing Bacillus strains may be harvested on the day of treatment. There exists a continuing need in the art for new fungicide

compositions.

SUMMARY OF INVENTION

[0006] The present invention relates to synergistic combinations of polyene fungicides with physical membrane disrupting agents other than a lipopeptide, to improve the fungicidal activity of both components. The present invention also relates to synergistic combinations of a lipopeptide with physical membrane disrupting agents other than polyene fungicides, to improve the fungicidal activity of both components.

[0007] In one embodiment, the polyene fungicide is one or more of natamycin, nystatin, amphotericin B, aureofungin, filipin and lucenosomycin and/or derivatives of each of these polyene fungicides. In another embodiment, the composition includes more than one polyene fungicide.

[0008] In one embodiment, the lipopeptide component of the synergistic fungicidal combination may be part of a fermentation product produced by a lipopeptide -producing microorganism, may be a crude extract of such fungicidal fermentation product, or may be purified or semi -purified from such fermentation product. In other embodiments the lipopeptides are synthetic or semi- synthetic {e.g. , a parent lipopeptide is obtained from a microorganism and is derivatized). In some embodiments, the lipopeptide-producing microorganism is a Bacillus species bacteria. In other embodiments, the lipopeptide-producing microorganism is a

Streptomycete . In still other embodiments, the lipopeptide-producing microorganism is a Paenibacillus species bacteria.

[0009] In a particular embodiment, the lipopeptide-producing bacteria is Bacillus subtilis, Bacillus amyloliquefaciens or any other Bacillus species that produces one or more lipopeptides. In yet another embodiment, the lipopeptides produced by such Bacillus species are from one or more of the following families: surfactin-type compounds, iturin-type compounds and fengycin-type compounds. Bacillus species that produce lipopeptides include, but are not limited to amyloliquefaciens, cereus, thuringiensis, coagulans, pumilus, licheniformis; others will be known to those of skill in the art. In a particular embodiment, the lipopeptide-producing bacteria is Bacillus subtilis QST713. In some embodiments, the Bacillus subtilis may be Bacillus subtilis QST713 and its variants; Bacillus amyloliquefaciens strain D747, Bacillus subtilis MBI600, Bacillus subtilis Y1336, Bacillus amyloliquefaciens strain FZB42, or Bacillus subtilis var. amyloliquefaciens FZB24. In one embodiment, the composition is comprised of at least one physical membrane disrupting agent and a lipopeptide-containing fermentation product. In another embodiment, the lipopeptide-containing fermentation product is from a Bacillus species bacteria, such as those described herein.

[0010] In some embodiments the lipopeptide component of the compositions of the present invention is comprised of one more of the following compounds: surfactin-type compounds, fengycin-type compounds, iturin-type compounds and fusaricidins. Iturin-type compounds suitable for the present invention include one or more of the following compounds: bacillomycin D, bacillyomycin F, bacillomycin L, bacillomycin LC (also known as

bacillopeptin), mycosubtilin, iturin A, iturin AL, and iturin C. Fengycin-type compounds suitable for the present invention are fengycin A, fengyci B, plipastatin A, plipastatin B, and agrastatins as described in U.S. Patent No. 6,291,426. Surfactin-type compounds suitable for the present invention are esperin, lichenysin, pumilacidin and surfactin. In a particular embodiment, the lipopeptide component includes one or more of iturin-type compounds, such as iturin As, mycosubtilin and/or bacillomycin, fengycin-type compounds and surfactin. In yet another embodiment the lipopeptide component includes at least two of the following compounds:

iturins, fengycin-type compounds and surf ac tins.

[0011] In some embodiments, at least one physical membrane disrupting agent is an antimicrobial peptide. In a particular embodiment, the physical membrane disrupting agent is a cyclotide, melittin, gramicidin S, Gramicidin D, or a combination thereof. In another embodiment, the physical membrane disrupting agent is a lipopeptide such as a fusaricidin. In another embodiment, the physical membrane disrupting agent is not an azole. In another embodiment, the physical membrane disrupting agent is not a lipopeptide. In another embodiment, the physical membrane disrupting agent is not an amphiphilic cyclic lipopeptide.

[0012] In some embodiments, the physical membrane disrupting agent may be an antimicrobial peptide. In another embodiment, the antimicrobial peptide may be a cyclotide, melittin, gramicidin S, or Gramicidin D. In another embodiment, the antimicrobial peptide may be pantocin A, pantocin B, polyoxin, nikkomycin, rhizocitin, bacilysin, blasticidin, mildiomycin, PEP6, PAF26, BPC194, PEP3, PEP11, BP76, CAMEL, Iseganan, D4E1, TYP, ESF12, ESF1 , Pexiganan, MSI-99, MB-39, Pen4-1, D32R, cecropin A, B, tachyplesin, heliomicin/drosomycin, sacrotoxin IA, mussel defensin, magainin, esculentin-1, Rs-AFP2, Alf-AFP, Spil, DRR230-a, BSD1, WT1, Dm- AMP 1 , Mj-AMPl, Pn-AMP, hordonthionin, alpha thionin, AFP, SB-37, Shiva- 1, SB37, MB-39, MsrAl , MSI-99, Myp30, or Rev4. In another embodiment, the physical membrane disrupting agent may be an antibiotic non-ribosomal peptide.

[0013] In another embodiment, the antibiotic non-ribosomal peptide may be an echinocandin. In another embodiment, the echinocandin is caspofungin, micafungin, or anidulafungin. In another embodiment, the physical membrane disrupting agent may be an antimicrobial peptide. In another embodiment, the antimicrobial peptides may be a non- ribosomally synthesized peptide or a ribosomally synthesized peptide (RAMP). In another embodiment, the antimicrobial peptide may be gramicidin, bacitracin, polymyxin B, melittin, cecropin, or vancomycin. In another embodiment, the antimicrobial peptide may be Gramicidin S or Gramicidin D. In another embodiment, the physical membrane disrupting agent may be a cyclotide.

[0014] In some embodiments, the synergistic fungicidal combination may comprise of at least one physical membrane disrupting agent and one or more lipopeptides. In another embodiment the synergistic fungicidal combination may comprise at least one physical membrane disrupting agent and two or more lipopeptides. The lipopeptides may be one or more compounds from one or more of the following families of compounds: surfactin-type compounds, iturin-type compounds and/or fengycin-type compounds. In one instance, the lipopeptides are comprised of one or more iturins and/or one or more fengycin-type compounds and/or surfactin. In one embodiment, the lipopeptide component is comprised of iturins (A, B and/or C), bacillomycin, surfactin, fusaricidin, and/or fengycin-type compounds, either individually or in combination. In one particular embodiment, the lipopeptides are semi-purified or purified from a fermentation product of a lipopeptide-producing Bacillus species bacteria.

[0015] The weight to weight ratio of the physical membrane disrupting agent and the polyene fungicide component (or, alternatively, the lipopeptide component {e.g., a lipopeptide- containing fermentation broth, a crude extract containing lipopeptides; a purified or semi-purified lipopeptide extract; or chemically synthesized or derivatized pure lipopeptide(s))) is from about 500: 1 to 1 :500. In one embodiment, the weight to weight ratio of natamycin or a derivative thereof or nystatin or a derivative thereof to a physical membrane disrupting agent is about 500: 1 to about 1 :500. In another embodiment, the weight ratio of the lipopeptide component comprised of one or more compounds from one or more of the following families of compounds, surfactin- type compounds, iturin-type compounds, fengycin-type compounds, and/or fusaricidins to a physical membrane disrupting agent is about 500: 1 to about 1 :500. In some embodiments, the weight to weight ratio of any of the above-described combinations of polyene fungicide and physical membrane disrupting agent is about 100: 1 to about 1 : 100; in others it is about 10: 1 to about 1 : 10; in still other it is about 5: 1 to about 1 :5; in yet others is it about 2: 1 to about 1 :2; and in yet others it is 1 : 1. In some embodiments, the weight to weight ratio of any of the above- described combinations of lipopeptide and physical membrane disrupting agent is about 100: 1 to about 1 : 100; in others it is about 10: 1 to about 1 : 10; in still other it is about 5: 1 to about 1 :5; in yet others is it about 2: 1 to about 1 :2; and in yet others it is 1 : 1. [0016] In one embodiment, the polyene fungicide component is natamycin or a derivative thereof or nystatin or a derivative thereof. In another instance of this embodiment, the derivative of natamycin or nystatin has equal or better fungicidal activity compared to the parent compound.

[0017] In another embodiment, the lipopeptide component of the above-referenced compositions includes one or more of the following: iturin, bacillomycin, mycosubtilin, esperin, lichenysin, pumilacidin, surfactin, fengycin A, fengycin B, plipastatin A, plipastatin B, and/or agrastatin. In one instance of the aforementioned embodiment, the lipopeptide component includes one or more of the following: iturin, surfactin, fengycin and/or plipastatin. In another instance of the aforementioned embodiment, surfactin is excluded from the composition.

[0018] Compositions of the present invention are useful in various fungal control applications. The above-described compositions may be used to control fungal phytopathogens, post-harvest fungal pathogens, fungal pathogens of food or feed and human fungal pathogens.

[0019] In one embodiment, any of the above-described compositions are used to control target pathogens such as Fusarium species, Botrytis species, Verticillium species, Rhizoctonia species, Trichoderma species and Pythium species by applying the composition to plants, the area surrounding plants, or edible cultivated mushrooms, mushroom spawn or mushroom compost. In one embodiment, the polyene fungicide component of compositions of the present invention used to control such pathogens is natamycin or a derivative thereof. In another it is nystatin or a derivative thereof.

[0020] In another embodiment, compositions of the present invention are used to control post-harvest pathogens such as Penicillium, Geotrichum, Aspergillus niger, and

Colletotrichum species. In one embodiment, the polyene fungicide component of compositions used to control such pathogens is natamycin or a derivative thereof. In another embodiment, the polyene fungicide is nystatin or a derivative thereof.

[0021] In yet another embodiment, compositions of the present invention are used to control fungal pathogens that occur in food or feed, such as Penicillium species, Aspergillus species and Fusarium species. In one embodiment, the polyene fungicide component of compositions used to control such pathogens is natamycin or a derivative thereof. In another embodiment, the polyene fungicide is nystatin or a derivative thereof. In yet another embodiment, the lipopeptide component is a purified extract of one or more lipopeptides. In yet another embodiment, the physical membrane disrupting agent component is a peptide such as a cyclotide, melittin, gramicidin S, or gramicidin (gramicidin D).

[0022] In still another embodiment, the compositions of the present invention are used to treat or prevent a fungal infection in a subject by administering to the subject a composition comprising a polyene fungicide (or lipoprotein) and at least one physical membrane disrupting agent. In one embodiment, the fungal infection is caused by Candida and the polyene fungicide used in the composition is nystatin or a derivative thereof. In another embodiment, the fungal infection is caused by Candida and the polyene fungicide used in the composition is natamycin or a derivative thereof. In another embodiment, the fungal infection is caused by Fusarium or Aspergillus and may be a corneal infection. In such an embodiment, the polyene fungicide component used in the composition is natamycin or a derivative thereof. In another embodiment where the composition is used to treat a corneal infection, the polyene fungicide component is nystatin or a derivative thereof. In yet another embodiment, the lipopeptide component of the composition is a purified extract of one or more lipopeptides. In yet another embodiment, the physical membrane disrupting agent component is a peptide such as a cyclotide, melittin, gramicidin S, or gramicidin D.

[0023] The present invention also includes a method for producing a fungicidal composition by making a combination of one or more polyene fungicides or one or more lipopeptides with a physical membrane disrupting agent, testing the combination for synergistic efficacy against target fungi and producing a fungicidal composition comprising the combination and a carrier. In one embodiment, the one or more lipopeptides are part of or are an extract of a fermentation product from a Bacillus species bacteria, such as those described above and herein. In another instance of this embodiment, prior to making the combination, a lipopeptide- producing bacteria is selected, such as a Bacillus species strain or Paenibacillus species strain, and a fermentation product containing lipopeptides is produced using this lipopeptide-producing bacteria, and such fermentation product or an extract thereof is used to make the combination. In one embodiment, such fermentation product would include one or more of the following lipopeptides: surfactin-type compounds, fengycin-type compounds, iturin-type compounds and/or fusaricidin. In a more particular embodiment, such fermentation product would include one or more of the following lipopeptides: surfactin, plipastatin, fengycin, iturin and/or bacillomycin. In another embodiment, each of the polyene fungicide component or lipopeptide component would be screened for fungicidal activity against the target pathogen prior to making the combination and only the polyene fungicide or lipopeptide components with at least some fungicidal activity would be used to make the combination. In other embodiments, target fungi are phytopathogens, such as Fusarium, Botrytis and Verticillium; post-harvest pathogens, such as Penicillium and Geotrichum; fungal pathogens of food or feed, such as Aspergillus, Fusarium and Penicillium; and human fungal pathogens, such as Candida, Apsergillus and Fusarium. Saccharomyces cerevisiae may also be used as a target pathogen in the above method as a model for Candida. In one embodiment, the polyene fungicide component of the tested combination is natamycin or a derivative thereof or nystatin and a derivative thereof. In another embodiment, the tested lipopeptides are one or more of surfactin-type compounds, iturin-type compounds, fengycin-type compounds and/or fusaricidins. In another embodiment, the physical membrane disrupting agent component is a peptide such as a cyclotide, melittin, gramicidin S or gramicidin D.

[0024] In one embodiment, the synergistic fungicidal composition may comprise a polyene fungicide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is an antimicrobial peptide. In another embodiment, the synergistic fungicidal composition may comprise a polyene fungicide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a cyclotide.

[0025] In one embodiment, the synergistic fungicidal composition may comprise a lipopeptide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is an antimicrobial peptide. In another embodiment, the synergistic fungicidal composition may comprise a lipopeptide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a cyclotide. In another embodiment, the lipopeptide may be an amphiphilic cyclic lipopeptide.

[0026] In one embodiment, the synergistic fungicidal composition may comprise an antimicrobial peptide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a cyclotide. In another embodiment, the synergistic fungicidal composition may comprise an antimicrobial peptide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a lipopeptide. In another embodiment, the synergistic fungicidal composition may comprise an antimicrobial peptide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a polyene fungicide.

[0027] In one embodiment, the synergistic fungicidal composition may comprise a cyclotide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a polyene fungicide. In another embodiment, the synergistic fungicidal composition may a comprise cyclotide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a lipopeptide. In another embodiment, the lipopeptide may be an amphiphilic cyclic lipopeptide. In another embodiment, the synergistic fungicidal composition may comprise a cyclotide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is an antimicrobial peptide.

[0028] In one embodiment, a method for controlling fungal pathogens may comprise administering a synergistic fungicidal composition. In another embodiment, a method for controlling fungal phytopathogens may comprise administering a synergistic fungicidal composition. In another embodiment, a method for controlling post-harvest fungal pathogens may comprise administering a synergistic fungicidal composition. In another embodiment, a method for controlling fungal pathogens of food or feed may comprise administering a synergistic fungicidal composition. In another embodiment, a method for controlling human fungal pathogens may comprise administering a synergistic fungicidal composition. In another embodiment, the fungal pathogen may be a Fusarium species, Botrytis species, Verticillium species, Penicillium species, Aspergillus species and Fusarium species, Rhizoctonia species, Trichoderma species, or Pythium species. In another embodiment, the administration may comprise applying the composition to plants, the area surrounding plants, or edible cultivated mushrooms, mushroom spawn or mushroom compost. In another embodiment, the fungal pathogen may be a Penicillium, Geotrichum, Aspergillus niger, and Colletotrichum species. In another embodiment, the post-harvest pathogens may be Botrytis cinerea, pathogenic

Colletotrichum species, Erwinia carotovora subsp. carotovora, Geotrichum candidum, Geotrichum citri-aurantii, Helminthosporium solani, Monilinia fructicola (brown rot),

Penicillium species, Rhizopus species, Aspergillus niger, Thielaviopsis basicola, mucor rot of fruit, and Alternaria rots. In another embodiment, the Colletotrichum species may be

Colletotrichum acutatum, Colletotrichum coccodes, Colleotrichum musa, or Colletotrichum capsici. In another embodiment, the Penicillium species may be Penicillium digitatum or Penicillium expansum.

[0029] In one embodiment, a method for treating or preventing a fungal infection in a subject by administering to the subject a composition administering a synergistic fungicidal composition. In a further embodiment, the fungal infection may be caused by a Candida, Fusarium, or Aspergillus species. In a further embodiment, the infection may be a corneal infection. In a further embodiment, the administration may be oral or topical.

[0030] In one embodiment, the synergistic fungicidal composition may be formulated as a cream or eye drops.

[0031] In one embodiment, a method for controlling fungal pathogens may comprise applying to a locus in need of treatment an effective amount of synergistic fungicidal composition. In a further embodiment, the locus may be post-harvest food and the fungal pathogen may be a postharvest pathogen. In a further embodiment, the postharvest pathogen may be Penicillium species or Geotrichum species. In a further embodiment, the locus may be a plant, root, seed or soil surrounding the plant and the fungal pathogen may be a fungal phytopathogen. In a further embodiment, the locus may be food or feed. In a further embodiment, the locus may be a human or an animal and wherein the fungal pathogen is Candida. In a further embodiment, the locus may be a human or an animal and wherein the fungal pathogen may be Aspergillus species or Fusarium species.

[0032] In one embodiment, the synergistic fungicide composition may comprise a polyene fungicide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is not a lipopeptide. In a further embodiment, the synergistic fungicide composition may comprise a lipopeptide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is not a polyene fungicide. In one embodiment, the synergistic fungicidal composition may comprise a polyene fungicide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a cyclotide or an antimicrobial peptide. In another embodiment, the synergistic fungicidal composition may comprise a lipopeptide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a cyclotide or an antimicrobial peptide. In another embodiment, the synergistic fungicidal composition may comprise an antimicrobial peptide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a cyclotide, a lipopeptide, or a polyene fungicide. In another embodiment, the synergistic fungicidal composition may comprise a cyclotide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is an antimicrobial peptide, a lipopeptide, or a polyene fungicide. In a further embodiment, the synergistic fungicidal composition may comprise a cyclotide and an antimicrobial peptide.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0033] The present invention provides a low-toxicity, low resistance-inducing, increased-efficacy fungicidal composition that comprises components less toxic than many traditional synthetic fungicides and applied at rates lower than either compound individually. The fungicidal composition may comprise a polyene fungicide with a physical membrane disrupting agent other than a lipopeptide. The invention also pertains to synergistic fungicidal compositions comprising a lipopeptide with physical membrane disrupting agents other than polyene fungicides, to improve the fungicidal activity of both components. The combination of a polyene fungicide with a physical membrane disrupting agent or a lipopeptide with a physical membrane disrupting agent improves the fungicidal activity of both components in a synergistic, rather than an additive manner.

[0034] Without wishing to be bound by any theory, inventors hypothesize that these combinations act in a synergistic fungicidal manner because each type of compound disrupts fungal cell membranes, albeit via a different mode of action. Various mechanisms of action for the lipopeptides have been proposed, all of which depend on the fact that the hydrocarbon tail of the molecule can insert itself readily into the fungal membranes where it forms associations with the hydrophilic fatty acid chains of the phospholipids. By contrast, for example, the polyene fungicides bind to ergosterol, the principal sterol in fungal membranes, thereby perturbing membrane function to the point of causing leakage of cellular contents. Because they act at the fungal cell membranes, the additive effects would not be expected to be synergistic.

[0035] The inventors determined that when a polyene fungicide was combined with a physical membrane disrupting agent the combination displayed an unexpected synergistic fungicidal activity. For example, a composition may comprise a polyene fungicide and a physical membrane disrupting agent, wherein the physical membrane disrupting agent is an antimicrobial peptide. A composition may comprise a polyene fungicide and a physical membrane disrupting agent, wherein the physical membrane disrupting agent is not a lipopeptide. A composition may comprise a polyene fungicide and a physical membrane disrupting agent, wherein the physical membrane disrupting agent is a cyclotide. The synergistic effect from the combination of the fungicide with a physical membrane disrupting agent is unexpected because the polyene fungicide and the antimicrobial peptide and/or cyclopeptide may have different mechanisms of action. For example, antimicrobial peptides may form pores in the membrane of their target. Paredes-Gamero, et al. (2012) Biochim Biophys Acta. 1820(7): 1062-72; Lam, et al. (2006) J Phys Chem B. 110(42): 21282-6; Yeaman & Yount (2003) Pharmacological Reviews 55: 27-55. Similarly, cyclotides are also suggested to induce disruption of membranes by a pore formation mechanism. Henriques & Craik (2012) ACS Chem. Biol. 7(4): 626-636.

[0036] The inventors also determined that when a lipopeptide was combined with a physical membrane disrupting agent the combination displayed an unexpected synergistic fungicidal activity. For example, a composition may comprise a lipopeptide and a physical membrane disrupting agent, wherein the physical membrane disrupting agent is an antimicrobial peptide. A composition may comprise a lipopeptide and a physical membrane disrupting agent, wherein the physical membrane disrupting agent is a cyclotide. A composition may comprise a lipopeptide and a physical membrane disrupting agent, wherein the physical membrane disrupting agent is not a polyene fungicide. The synergistic effect from the combination of the lipopeptide with a physical membrane disrupting agent was unexpected because the lipopeptide and the antimicrobial peptide and/or cyclopeptide may have different mechanisms of action. As discussed above, both antimicrobial peptides and cyclotides may form pores in the membrane of their target.

Definitions

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs. [0038] As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise.

[0039] "Antibiotic," as used herein, refers broadly to agents that have the ability to kill or slow the growth of microbial pathogens, including but not limited to fungi, oomycetes and/or bacteria.

[0040] "Crude extract," as used herein, refers broadly to organic extracts of fermentation broth, including but not limited to ethyl acetate extracts, in which the extract is enriched for lipopeptides. One method to obtain a crude extract of lipopeptides from a bacterial culture is described in Example 1. Strains and methods for obtaining an extract of lipopeptides are also described herein.

[0041] "Fermentation broth," as used herein, refers broadly to the culture medium resulting after fermentation of a microorganism and encompasses the microorganism and its component parts, unused raw substrates, and metabolites produced by the microorganism during fermentation, among other things.

[0042] "Fermentation product," as used herein, refers broadly to fermentation broth and/or fermentation solids.

[0043] "Fermentation solid," as used herein, refers broadly to concentrated and/or dried fermentation broth.

[0044] "Fungicidal," as used herein refers broadly to the ability of a substance to increase mortality or inhibit the growth rate of fungi.

[0045] "Lipopeptides," as used herein, refers broadly to lipopeptides that are part of a fermentation product and to lipopeptides that are purified to at least some extent, whether chemically synthesized or biologically produced. Lipopeptides include but are not limited to amphiphilic cyclic lipopeptides.

[0046] "Physical membrane disrupting agent," as used herein, refers broadly to compounds that disrupt a fungal cell membrane by physically preventing a fungal cell membrane from forming or by physically disrupting or disturbing an existing fungal cell membrane.

Examples of physical membrane disrupting agents are provided herein. Such physical disruption may occur by binding of the compound to a component of the fungal cell membrane, including but not limited to a phospholipid or sterol. Disruption of the cell membrane by inhibition of sterol synthesis (e.g. , the mode of action of the azole compounds) is not a physical disruption of the cell membrane, as that term is used herein. [0047] "Purified lipopeptides," as used herein, refers broadly to lipopeptides that are isolated from fermentation broth that are about 91% to about 100% pure. Lipopeptides of the present invention may be either purified or semi -purified.

[0048] "Semi-purified lipopeptides," as used herein, refers broadly to lipopeptides isolated from fermentation broth that are about 50% to about 90% pure.

[0049] "Synergistically effective amount," refers broadly to a quantity of a combination of a fungicide and a physical membrane disrupting agent that is statistically significantly more effective against fungi than the physical membrane disrupting agent or the fungicide only.

Physical Membrane Disrupting Agent

[0050] The physical membrane disrupting agent can be an antimicrobial peptide, cyclotide, polyene fungicide, or lipopeptide. The physical membrane disrupting agent may be a lipopeptide including, but not limited to a fusaricidin. The physical membrane disrupting agent may not an azole. In some instances, the physical membrane disrupting agent may not be a lipopeptide. In some instances, the physical membrane disrupting agent may not be a polyene fungicide.

Polyene Fungicides

[0051] Polyene fungicides of the present invention are antifungal antibiotics with a macrocyclic lactone ring having (i) a rigid lipophilic polyene portion and a flexible, hydrophilic hydroxylated portion and (ii) the ability to bind to a sterol in the cell membrane of most fungi, principally ergosterol. The macrocyclic lactone ring may have 12-40 carbons, 6-14 hydroxyl groups and may or may not be linked to a carbohydrate. The ring may be linked to one or more sugars including but not limited to a simple sugar with five or more carbon units, a deoxy sugar, amino sugars and the like, which contain substituent groups attached to the ring including oxygenated linkages. Polyene fungicides of the present invention may be obtained from a species of Streptomyces bacteria. Such fungicides include natamycin, nystatin, amphotericin B, aureofungin, filipin and lucensomycin as well as derivatives thereof. Examples of derivatives include the amphotericin B derivatives described in U.S. Patent No. 5,606,038, for example, or the nystatin derivatives/analogues including but not limited to S44HP, NYST1068, and the octaene nystatin described in Bruheim, et al, ANTIMICROBIAL AGENTS AND

CHEMOTHERAPY, Nov. 2004, pages 4120-4129. Derivatives are naturally occurring analogs of a parent molecule or synthetic or semi-synthetic compounds derivatized from a parent molecule that retain at least some fungicidal activity compared to the parent molecule. The derivatives have at least the same or greater fungicidal activity compared to the parent molecule. Derivatives include salts and solvates and other modified forms that have enhanced solubility compared to the parent molecule.

[0052] Antibiotic non-ribosomal peptides (NRPs) of the present invention are cell membrane or cell wall disrupting non-ribosomal peptides, excluding enzymes. Such antibiotic NRPs are synthesized by large enzymatic complexes called nonribosomal peptide synthetases, rather than by ribosomes. A database of nonribosomal peptides called Norine is provided on the internet and described in Caboche, S., et al., "NORINE: A Database of Nonribosomal Peptides," Nucleic Acids Research, 36:D326-D331, (2008). Antibiotic NRPs of the present invention disrupt cell membranes, including organelle membranes, or cell walls. Membranes or walls may be disrupted through various means, including inhibition of synthesis of components of cell membranes or walls; physical disruption of the components of the cell membrane, including but not limited to through permeabilization of phospholipid membranes, either by membrane solubilization or osmotic perturbation; or binding to small molecules in the cell membrane.

[0053] Cell-wall disrupting antifungal NRPs of the present invention include echinocandins, which are semi- synthetic amphiphilic lipopeptides composed of a cyclic hexapeptide core linked to a variably configured lipid side chain. Echinocandins inhibit synthesis of l,3- -glucan, a predominant polysaccharide component of the ascomycete cell wall that maintains the osmotic integrity of the cell and is involved in cell division and growth.

Echinocandins include but are not limited to caspofungin, micafungin and anidulafungin.

Lipopeptides

[0054] Lipopeptides, including but not limited to amphiphilic cyclic peptides obtainable from various bacteria, including Bacillus sp., Paenibacillus sp., and Streptomyces sp. As used herein the term "lipopeptides" may refer to amphiphilic cyclic peptides.

[0055] Amphiphilic cyclic lipopeptides are composed of six to ten a-amino acids linked to a β-amino or β-hydroxy fatty acid, including but not limited to fengycin-type compounds, iturin-type compounds, surfactin-type compounds and fusaricidins. The iturin-type compounds are composed of seven amino acids and are linked to a β-amino fatty acid. The length of the fatty acid chain may vary from C14 to CI 7. These compounds are obtainable from various species of Bacillus, including subtilis and amyloliquefaciens . The iturins and their variants are described in Ongena, et al., "Bacillus Lipopeptides: Versatile Weapons for Plant Disease Biocontrol," Trends in Microbiology, 16(3): 115-125, (2007). Iturin-type compounds of the present invention include one or more of the following compounds: bacillomycin D, bacillyomycin F, bacillomycin L, bacillomycin LC (also known as bacillopeptin), mycosubtilin, iturin A, iturin AL, and iturin C (with the latter three compounds referred to herein, collectively, as iturins). [0056] Fengycin-type compounds are composed of ten amino acids linked to a β- hydroxy fatty acid with a chain that varies in length from C14 to CI 8. These compounds are obtainable from various species of Bacillus, including subtilis, amyloliquefaciens, cereus and thuringiensis and from Streptomyces sp. The fengycin-type compounds are described in Ongena, supra. Fengycin-type compounds suitable for the compositions described herein include fengycin A, fengycin B, plipastatin A, plipastatin B, the plipastatins from a Streptomyces sp. described in Kimura, et al., "SNA 60-367 - New Peptide Enzyme Inhibitors against Aromatase," Journal of Antibiotics, 50(6): 529-531 , (1997), and agrastatins, as described in U.S. Patent No. 6,291 ,426 (with the latter four listings referred to herein, collectively, as plipastatins).

[0057] Surfactin-type compounds are composed of seven amino acids linked to a β- hydroxy fatty acid with a chain that varies in length from CI 3 to C 16. These compounds are obtainable from various species of Bacillus, including subtilis, amyloliquefaciens, coagulans, pumilus and licheniformis. The surfactin family of compounds is described in Ongena, supra. Surfactin-type compounds of the present invention include one or more of the following compounds: esperin, lichenysin, pumilacidin and surfactin.

[0058] Fusaricidins are composed of six amino acids linked to a 15-guanidino-3- hydroxypentadecanoic acid. Fusaricidins are obtainable from Paenibacillus sp., including polymyxa. The fusaricidin family of compounds is described in Choi, S-K, et al., "Identification and Functional Analysis of the Fusaricidin Biosynthetic Gene of Paenibacillus polymyxa E681," Biochemical and Biophysical Research Communications, 365:89-95, (2008). Fusaricidins of the present invention include one or more of the following compounds: fusaricidins A-D and fusaricidins LI-F03, LI-F04, LI-F05, LI-F06, LI-F07 and LI-F08.

[0059] Certain bacteria produce one or more lipopeptides, and combinations of various lipopeptides are known to have synergistic fungicidal activity. The lipopeptide component of the composition may comprise a combination of lipopeptides from at least two of the following lipopeptide classes: surfactin-type compounds, iturin-type compounds, and fengycin-type compounds. The combination may comprise two or more of the following compounds: iturin As, plipastatins A and B, fengycins A and B and surfactin. The combination may comprise one or more of the following compounds: iturin As, plipastatins A and B, fengycins A and B, surfactin and agrastatin. Compositions comprising a polyene fungicide and a lipopeptide are also described in U.S. Patent Publication No. 2012/0302492.

Antimicrobial Peptides

[0060] The physical membrane disrupting agent may be an antimicrobial peptide. See Wang & Wang (2004) Nucleic Acids Research 32: D590-D592. The antimicrobial peptides may be a non-ribosomally synthesized peptide or ribosomally synthesized peptides (RAMP). Non- ribosomally synthesized peptides are found in bacteria and fungi. These antimicrobial peptides are assembled by peptide synthetases as opposed to ribosomal-supported synthesis. Gramicidin (e.g. , Gramicidin S, Gramicidin D), bacitracin, polymyxin B, melittin, cecropin, and vancomycin are examples of non-ribosomally synthesized antimicrobial peptides. Zeitler, et al. (2013) PLOS One 8(8): e71687. Further Pantocin A, pantocin B, polyoxins, nikkomycins, rhizocitin, bacilysin, blasticidin, and mildiomycin are all antimicrobial peptides active against plant pathogens that may be used in the claimed compositions or methods. PEP6, PAF26, BPC194, PEP3, PEP11, BP76, CAMEL, Iseganan, D4E1, TYP, ESF12, ESF1, Pexiganan, MSI-99, MB- 39, Pen4-1, and D32R are all synthetic antimicrobial peptides with activity against plant pathogens that may be used in the claimed compositions or methods. Additionally, cecropin A, B, tachyplesin, heliomicin/drosomycin, sacrotoxin IA, mussel defensin, magainin, esculentin-1 , Rs-AFP2, Alf-AFP, Spil , DRR230-a, BSD1, WT1, Dm- AMP 1 , Mj-AMPl , Pn-AMP, hordonthionin, alpha thionin, AFP, SB-37, Shiva- 1 , SB37, MB-39, MsrAl , MSI-99, Myp30, Rev4, and D4E1 have all be expressed in transgenic plants that confer at least partial resistance to pathogens and may be used in the compositions and methods described herein. Montesinos (2007) FEMS Microbiol Lett 270: 1-11.

Cyclotides

[0061] The physical membrane disrupting agent may be a cyclotide. Cyclotides are small disulfide rich peptides isolated from plants. Cyclotides generally contain 28-37 amino acids and are characterized by their head-to-tail cyclised peptide backbone and the interlocking arrangement of their three disulfide bonds. These combined features have been termed the cyclic cystine knot (CCK) motif. Craik, et al. (2012) Methods Enzymol. 516: 37-62; Plan, et al. (2010) Biopolvmers 94(5): 647-58; Smith, et al. (2011) Expert Opinion 21(11): 1657-1672.

Lipopeptides Produced by Bacteria

[0062] Lipopeptides of the present invention are produced by one or more bacteria, including but not limited to those described above, or are chemically synthesized. Methods of culturing bacteria are well known in the art. Conventional large-scale microbial culture processes include submerged fermentation, solid state fermentation, or liquid surface culture. For Bacillus, towards the end of fermentation, as nutrients are depleted, cells begin the transition from growth phase to sporulation phase, such that the final product of fermentation is largely spores, metabolites and residual fermentation medium. Sporulation is part of the natural life cycle of many Bacilli and is generally initiated by the cell in response to nutrient limitation. For this invention, fermentation is configured to obtain high levels of lipopeptides and to promote sporulation. [0063] The bacterial cells, spores and metabolites in culture media resulting from fermentation (i.e. , fermentation broth) may be used directly or concentrated (to make a fermentation solid) by conventional industrial methods, including but not limited to

centrifugation, tangential-flow filtration, depth filtration, and evaporation. The concentrated fermentation solid is washed, for example, via a diafiltration process, to remove residual fermentation broth and metabolites.

[0064] The fermentation broth or fermentation solids can be dried with or without the addition of carriers using conventional drying processes or methods including but not limited to spray drying, freeze drying, tray drying, fluidized-bed drying, drum drying, or evaporation. The resulting dry fermentation solids may be further processed, including but not limited to by milling or granulation, to achieve a specific particle size or physical format. Carriers may also be added post-drying, as appropriate for the desired method of use.

[0065] Bacterially produced lipopeptides may be separated from bacterial cells or further purified from other bacterial components and, from each other. The term "cell-free preparation" refers to fermentation broth from which cells have been removed or substantially removed through means well known to those of skill in the art. Some methods of creating cell- free preparations are described below. Cell-free preparations of fermentation broth can be obtained by any means known in the art, including but not limited to extraction, centrifugation and/or filtration of fermentation broth. Those of skill in the art will appreciate that so-called cell- free preparations may not be devoid of cells but rather are largely cell-free or substantially cell- free, depending on the technique used (e.g. , speed of centrifugation) to remove the cells. The resulting cell-free preparation may be dried and/or formulated with components that aid in its particular application. Concentration methods and drying techniques described above for fermentation broth are also applicable to cell-free preparations.

[0066] After a cell-free preparation is made by centrifugation of fermentation broth, the metabolites may be purified by size exclusion filtration including but not limited to the Sephadex resins including LH-20, G10, and G15 and G25 that group metabolites into different fractions based on molecular weight cut-off, including but not limited to molecular weight of less than about 2000 daltons, less than about 1500 daltons, less than about 1000 daltons and so on, as the lipopeptides are between 800 daltons and 1600 daltons.

[0067] Lipopeptides of the present invention may be obtained from Bacillus subtilis QST713 or a fermentation product of Bacillus subtilis QST713 is used as the lipopeptide- containing component of the composition. Bacillus subtilis QST713, its mutants, its

supernatants, and its lipopeptide metabolites, and methods for their use to control plant pathogens and insects are fully described in U.S. Patent Nos. 6,060,051 ; 6,103,228; 6,291,426; 6,417,163 and 6,638,910. In these patents, the strain is referred to as AQ713. Bacillus subtilis QST713 has been deposited with the NRRL on May 7, 1997, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure under Accession Number B-21661. Any references in this specification to QST713 refer to Bacillus subtilis QST713. Particular variants of Bacillus subtilis QST713 {e.g. , Bacillus subtilis AQ30002 and AQ30004, deposited as Accession Numbers NRRL B-50421 and NRRL B-50455) that would also be suitable for the present invention are described in U.S. Patent Publication No. 2012/0231951.

[0068] Other Bacillus strains capable of producing lipopeptides may be used as a source of lipopeptides for the present invention. For example, Bacillus amyloliquefaciens strain D747 (available as BACSTAR^® from Etec Crop Solutions, NZ and also available as DOUBLE NICKEL™ from Certis, US); Bacillus subtilis MB 1600 (available as SUBTILEX^® from Becker Underwood, US EPA Reg. No. 71840-8); Bacillus subtilis Y1336 (available as BIOBAC^® WP from Bion-Tech, Taiwan, registered as a biological fungicide in Taiwan under Registration Nos. 4764, 5454, 5096 and 5277); Bacillus amyloliquefaciens, in particular strain FZB42 (available as RHIZO VITAL^® from ABiTEP, DE); and Bacillus subtilis var. amyloliquefaciens FZB24 is available from Novozymes Biologicals Inc. (Salem, Virginia) or Syngenta Crop Protection, LLC (Greensboro, North Carolina) as the fungicide TAEGRO^® or TAEGRO^® ECO (EPA Registration No. 70127-5), are all Bacillus strains capable of producing lipopeptides that may be used as a source of lipopeptides for the present invention.

[0069] A mutant of FZB24 that was assigned Accession No. NRRL B-50349 by the Agricultural Research Service Culture Collection is also described in U.S. Patent Publication No. 2011/0230345. Bacillus amyloliquefaciens FZB42 is available from ABiTEP GMBH, Germany, as the plant strengthening product RHIZO VITAL^®; FZB42 is also described in European Patent Publication No. EP2179652 and also in Chen, et al., "Comparative Analysis of the Complete Genome Sequence of the Plant Growth-Promoting Bacterium Bacillus amyloliquefaciens FZB42," Nature Biotechnology Volume 25, Number 9 (September 2007). Mutants of FZB42 are described in International Publication No. WO 2012/130221 , including Bacillus

amyloliquefaciens ABI01 , which was assigned Accession No. DSM 10-1092 by the DSMZ - German Collection of Microorganisms and Cell Cultures.

[0070] As described above, fermentation broth or extracts from fermentation broth may be used as the lipopeptide-containing component of the synergistic fungicidal combination of the present invention. Methods for obtaining lipopeptides from a fermentation broth of QST713 are described in the examples. Obtaining lipopeptides from fermentation broth of Bacillus bacteria, in general, and analyzing fermentation broths for presence of lipopeptides is well known to those of skill in the art, such that other bacterial strains suitable for the present invention could be readily identified by the skilled artisan. Bacillus strains that produce various lipopeptides are described in the Ongena Trends in Microbiology (2007) Vol. 16, No. 3. Other articles describe lipopeptide -producing Bacillus strains and methods for extracting lipopeptides from fermentation broths of such strains: see, e.g. , Alvarez, et al. Journal of Applied

Microbiology (2011) 112: 159-174; Ongena, et al . Applied Microbiology Biotechnology (2005) 69: 29-38; Wang, et al. Eur. Food Res. Technol. (2010) 231 : 189-196.

[0071] The combinations described herein may comprise polyene fungicides with physical membrane disrupting agents other than a lipopeptide; and a lipopeptide with physical membrane disrupting agents other than polyene fungicides, to improve the fungicidal activity of both components.

Ratio Between Fungicide and Physical Membrane Disrupting Agent

[0072] The polyene fungicide and the physical membrane disrupting agent may be applied at a 1 : 1 ratio (w/w). The polyene fungicide to physical membrane disrupting agent component ratio may be about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 10: 1 , about 15: 1, about 20: 1, or about 50: 1. The polyene fungicide to physical membrane disrupting agent component ratio may be about 1 :2, about 1 :3, about 1 :4, about 1 :5, about 1 : 10, about 1 : 15, about 1 :20, or about 1 :50.

[0073] The polyene fungicide and a physical membrane disrupting agent may be provided at various weight to weight (w/w) ratios in the composition. The weight to weight ratio of the polyene fungicide and the physical membrane disrupting agent component may be from about 500: 1 to about 1 :500. The weight to weight ratio of the polyene fungicide and the physical membrane disrupting agent component may be from about 100: 1 to about 1 : 100. The weight to weight ratio of the polyene fungicide and the physical membrane disrupting agent component is from about 10: 1 to about 1 : 10. The weight to weight ratio of the polyene fungicide and the physical membrane disrupting agent component may be from about 5: 1 to about 1 :5. The weight to weight ratio of the polyene fungicide and the physical membrane disrupting agent component may be from about 2: 1 to about 1 :2. The weight to weight ratio of the polyene fungicide and the physical membrane disrupting agent component may be about 1 : 1.

[0074] The lipopeptide-containing component {e.g. , purified or semi-purified lipopeptides, crude extracts, fermentation products or chemically synthesized or derivatized products) and the physical membrane disrupting agent and may be applied at a 1 : 1 ratio (w/w). The lipopeptide-containing component to physical membrane disrupting agent ratio is about 2: 1, about 3: 1, about 4: 1, about 5: 1 , about 10: 1, about 15: 1, about 20: 1, or about 50: 1. The lipopeptide-containing component to physical membrane disrupting agent ratio is about 1 :2, about 1 :3, about 1 :4, about 1 :5, about 1 : 10, about 1 : 15, about 1 :20, or about 1 :50.

[0075] The lipopeptide-containing component including one or more lipopeptides and physical membrane disrupting agent may be provided at various weight to weight (w/w) ratios in the composition. The weight to weight ratio of the lipopeptide-containing component to physical membrane disrupting agent is from about 500: 1 to about 1 :500. The weight to weight ratio of the lipopeptide-containing component and physical membrane disrupting agent is from about 100: 1 to about 1 : 100. The weight to weight ratio of the lipopeptide-containing component and the physical membrane disrupting agent is from about 10: 1 to about 1 : 10. The weight to weight ratio of the lipopeptide-containing component and the physical membrane disrupting agent is from about 5: 1 to about 1 :5. The weight to weight ratio of the lipopeptide-containing component and the physical membrane disrupting agent is from about 2: 1 to about 1 :2. The weight to weight ratio of the lipopeptide-containing component and the physical membrane disrupting agent is about 1 : 1.

Methods of Use

[0076] The composition may be applied to a plant or to an edible cultivated mushroom. In plant applications, compositions may be applied to any part of a plant, including its root, foliage, or fruit, or to the area surrounding the plant, including the soil surrounding the plant. In mushroom applications, compositions may be applied to mushroom or to mushroom spawn or to mushroom compost. Application may occur before, at and/or post-planting.

Exemplary target pathogens are Fusarium sp., Botrytis sp., Verticillium sp., Rhizoctonia sp., Trichoderma sp. (green mold), Pythium sp. and Phytophthora sp. Determining application rates for the present compositions is well known to those of skill in the art. A composition may comprise 0.005 g/L to 100 g/L each of the polyene fungicide and the physical membrane disrupting agent. The compositions described herein may be applied to a plant, plant part or to the area surrounding the plant or to an edible cultivated mushroom, mushroom spawn or mushroom compost.

[0077] Compositions used for pharmaceutical or veterinary applications are combined with pharmaceutically acceptable carriers that vary based on the mode of administration.

[0078] Compositions of the present invention may be applied to a locus in need of treatment in an amount effective to control a pathogen. The term "control," as used herein, means to kill or inhibit the growth of the pathogen. The pathogen may be a fungus. In another the pathogen is an oomycete.

[0079] The locus may be a post-harvest food and post-harvest non-edible plant materials, including but not limited to feedstock for biofuel. Post-harvest food, as used herein, refers to fruit, vegetables, grains, oilseeds, and any other edible plants and nuts after harvest from the field but before packaging. Exemplary post-harvest pathogens include, but are not limited to: Botrytis cinerea (gray mold), pathogenic Colletotrichum species including but not limited to Colletotrichum acutatum, Colletotrichum coccodes, Colleotrichum musa, and Colletotrichum capsici, Erwinia carotovora subsp. carotovora, Geotrichum candidum, Geotrichum citri-aurantii (sour rot), Helminthosporium solani (silver scurf of potato), Monilinia fructicola (brown rot), Penicillium sp., including digitatum (green mold of citrus) and expansum (blue mold of pome fruits), Rhizopus sp. (Rhizopus rot or leak), Aspergillus niger (black mold), Thielaviopsis basicola (black root rot of carrot), mucor rot of fruit {e.g. , Mucor piriformis on pear), and Alternaria rots (mostly affecting carrots, broccoli, potatoes, peppers, apples, kiwis, pears, quinces and tomatoes). Determining application rates for the present compositions for post-harvest food and post-harvest non-edible plant materials is well known to those of skill in the art. The composition described herein may be applied to post-harvest food and post-harvest non-edible plant materials, including but not limited to feedstock for biofuel. In some embodiments, the polyene fungicide component is natamycin or a derivative thereof. The compositions described herein may be applied before or during packaging of the product. The compositions described herein may be applied after harvest. The compositions described herein may be applied after washing on the product or after exposure to rain.

[0080] The compositions described herein may be used for the treatment of plants or plant parts (which includes seeds and plants emerging from the seed), harvested fruits and vegetables with the composition described herein is carried out directly or by action on their surroundings, habitat or storage space using customary treatment methods, for example dipping, spraying, atomizing, irrigating, evaporating, dusting, fogging, broadcasting, foaming, painting, spreading-on, watering (drenching), drip irrigating.

[0081] The locus can be food or feed. The term food, as used herein includes processed foods, including but not limited to dairy products, breads, tortillas, deli meats, and bakery products; semi-processed or minimally processed foods, including but not limited to meat and cut fruit and vegetables; and packaged foods, including but not limited to packaged lettuce, spinach and other vegetables. The term feed includes processed animal feedstuffs and silage. In such applications the target pathogens are spoilage-inducing and mycotoxin-producing pathogens. Exemplary pathogens are Aspergillus sp., Penicillium sp. and Fusarium sp.

Determining application rates for the present compositions is well known to those of skill in the art. Dose levels and recommended methods of application are described in detail in numerous references, including Davidson, P. Michael, et al. [Eds.], Antimicrobials in Food, 3^rd ed. CRC Press 2005, Chapter 8, pages 275-289. The composition of the present invention may be applied to food or feed. In such embodiments, the polyene fungicide component is natamycin or a derivative thereof.

[0082] The compositions can be applied in a variety of ways: as an aqueous suspension (including but not limited to mixed into a brine) that is sprayed on the product or that the product is dipped into, or in powdered form (along with an anticaking agent including but not limited to cellulose) sprinkled on or mixed into the product.

[0083] The locus may be a human or animal. In human and veterinary applications, the compositions are applied topically to the skin and mucosal membranes to control Candida albicans and/or Fusarium sp. The compositions may be used to prevent or treat vaginal infections, especially those caused by Candida albicans but also those caused by C. glabrata, C. parapsilosis, C. guilliermondii, and C. tropicalis. The composition may be applied to treat Candida the polyene fungicide component is nystatin. In humans, the compositions of the present invention may also be applied to nails, scalp and skin to control dermatophytes, including but not limited to Trichophyton, Epidermophyton and Microsporum, which are responsible for a variety of disease manifestations that localize to keratinized structures of the body {e.g. , skin, nail, and hair). In humans, the compositions may be administered as lozenges, to treat oral candidiasis or orally to treat or prevent intestinal candidiasis. Compositions of the present invention may also be used to treat Aspergillus and Fusarium corneal infections. The compositions can also be used to treat corneal infections. In such embodiments,the polyene fungicide component of the composition is natamycin or a derivative thereof. Determining appropriate administration rates for the present compositions would be well within the knowledge of one of skill in the art. Further, the synergistic compositions described herein may be used in a method for treating or preventing a fungal infection in a subject comprising administering to the subject a synergistic fungicidal composition described herein.

[0084] Natamycin may be administered as a topical 0.15-0.30% solution or intracameral administration (20-30 μg/mL). The compositions described herein may be applied topically to the skin and mucosal membranes through cream or eye cream/ointment.

[0085] The locus in need of treatment may be identified before the compositions are applied or administered.

Formulations

[0086] The compositions may include one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers may vary depending on their suitability for various dosage forms. The compositions may include one or more pharmaceutically acceptable carriers as a topical composition. Topical composition can be in the form of a cream, gel, oil, spray, powder, paste, clay or any other form, way or method known in the art for administering the composition to the skin or a subject whether human or animal. The acceptable carrier contained in the topical composition may be varied depending on the type of the formulation. For example, the formulation of ointment, pastes, creams or gels may comprise animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silica, talc, zinc oxide or mixtures of these substances. All of these pharmaceutical carriers and formulations are well known to those of ordinary skill in the art. See, e.g. , WADE & WALLER, HANDBOOK OF PHARMACEUTICAL EXCIPIENTS (2nd ed. 1994).

[0087] In addition to those described above, any appropriate fillers and excipients may be utilized in preparing the compositions of the present invention so long as they are consistent with the objectives described herein. For example, binders are substances used to cause adhesion of powder particles in granulations. Such compounds appropriate for use in the present invention include, by way of example and without limitation, acacia, compressible sugar, gelatin, sucrose and its derivatives, maltodextrin, cellulosic polymers, including but not limited to ethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl cellulose, carboxymethylcellulose sodium and methylcellulose, acrylic polymers, including but not limited to insoluble acrylate ammoniomethacrylate copolymer, poly acrylate or polymethacrylic copolymer, povidones, copovidones, polyvinylalcohols, alginic acid, sodium alginate, starch, pregelatinized starch, guar gum, polyethylene glycol and others known to those of ordinary skill in the art.

[0088] Diluents also may be included in the compositions of the present invention in order to enhance the granulation of the compositions. Diluents can include, by way of example and without limitation, microcrystalline cellulose, sucrose, dicalcium phosphate, starches, lactose and polyols of less than 13 carbon atoms, including but not limited to mannitol, xylitol, sorbitol, maltitol and pharmaceutically acceptable amino acids, including but not limited to glycin, and their mixtures.

[0089] Lubricants are substances used in composition formulations that reduce friction during composition compression. Lubricants that may be used in the present invention include, by way of example and without limitation, stearic acid, calcium stearate, magnesium stearate, zinc stearate, talc, mineral and vegetable oils, benzoic acid, poly(ethylene glycol), glyceryl behenate, stearyl fumarate, and others known to those of ordinary skill in the art.

[0090] Glidants improve the flow of powder blends during manufacturing and minimize composition weight variation. Glidants that may be used in the present invention include, by way of example and without limitation, silicon dioxide, colloidal or fumed silica, magnesium stearate, calcium stearate, stearic acid, cornstarch, talc and others known to those of ordinary skill in the art. [0091] Colorants also may be included in the compositions of the present invention. As used herein, the term "colorant" includes compounds used to impart color to pharmaceutical preparations. Such compounds include, by way of example and without limitation, FD&C Red No. 3, FD&C Red No. 20, FD&C Yellow No. 6, FD&C Blue No. 2, D&C Green No. 5, FD&C Orange No. 5, D&C Red No. 8, caramel, and ferric oxide, red and others known to those of ordinary skill in the art. Coloring agents also can include pigments, dyes, tints, titanium dioxide, natural coloring agents, including but not limited to grape skin extract, beet red powder, beta carotene, annato, carmine, turmeric, paprika and others known to those of ordinary skill in the art.

[0092] The compositions can include a solubilizer to ensure good solubilization and/or dissolution of the active ingredients including but not limited to the fungicide and/or lipopeptide. A solubilizer can also be added to increase the solubility of the fungicide and/or lipopeptide and/or other components, including but not limited to surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion.

Compositions

[0093] Compositions of the present invention may include carriers, which are inert formulation ingredients added to compositions comprising a lipopeptide-containing fermentation product, cell-free preparations of lipopeptides or purified, semi-purified or crude extracts of lipopeptides to improve recovery, efficacy, or physical properties and/or to aid in packaging and administration. Such carriers may be added individually or in combination.

[0094] The compositions of the present invention may be used for various purposes, including protection of crops and of post-harvest fruits, vegetables and plants; as preservatives for cosmetics, processed foods, animal feed, or timber; and for pharmaceutical and veterinary applications. Depending on the particular application, the compositions will be formulated with appropriate carriers to aid in their application or administration. The carriers may be anti-caking agents, anti-oxidation agents, bulking agents, and/or protectants. Examples of useful carriers include polysaccharides (starches, maltodextrins, methylcelluloses, proteins, including but not limited to whey protein, peptides, gums), sugars (lactose, trehalose, sucrose), lipids (lecithin, vegetable oils, mineral oils), salts (sodium chloride, calcium carbonate, sodium citrate), silicates (clays, amorphous silica, fumed/precipitated silicas, silicate salts), waxes, oils, alcohol and surfactants.

[0095] The compositions of the present invention further comprising a formulation inert or other formulation ingredient, including but not limited to polysaccharides, including but not limited to starches, maltodextrins, and methylcelluloses; proteins, including but not limited to whey protein, peptides, and gums; sugars, including but not limited to lactose, trehalose, and sucrose; lipids including but not limited to lecithin, vegetable oils, and mineral oils; salts including but not limited to sodium chloride, calcium carbonate, and sodium citrate; and silicates including but not limited to clays, amorphous silica, fumed/precipitated silicas, and silicate salts. The compositions of the present invention may also comprise a carrier, including but not limited to water or a mineral or organic material. The composition may be used for seed treatment or as a root dip, the carrier is a binder or sticker that facilitates adherence of the composition to the seed or root. The compositions can be used as a seed treatment the formulation ingredient is a colorant. In other compositions, the formulation may further comprising a preservative.

[0096] Compositions of the present invention may further comprise formulation inerts added to compositions comprising cells, cell-free preparations or metabolites to improve efficacy, stability, and usability and/or to facilitate processing, packaging and end-use application. Such formulation inerts and ingredients may include carriers, stabilization agents, nutrients, or physical property modifying agents, which may be added individually or in combination. The carriers may include liquid materials including but not limited to water, oil, and other solvents and solid materials including but not limited to minerals, polymers, or polymer complexes derived biologically or by chemical synthesis. The carrier is a binder or adhesive that facilitates adherence of the composition to a plant part, including but not limited to a seed or root. See, for example, Taylor, et al. "Concepts and Technologies of Selected Seed Treatments" Annu. Rev. Phytopathol. 28: 321-339 (1990). The stabilization agents include but are not limited to anti-caking agents, anti-oxidation agents, desiccants, protectants or preservatives. The nutrients may be carbon, nitrogen, and phosphors sources including but not limited to sugars,

polysaccharides, oil, proteins, amino acids, fatty acids and phosphates. The physical property modifiers may be bulking agents, wetting agents, thickeners, pH modifiers, rheology modifiers, dispersants, adjuvants, surfactants, antifreeze agents, or colorants.

[0097] The composition as described herein may additionally comprising at least one auxiliary including but not limited to extenders, solvents, spontaneity promoters, carriers, emulsifiers, dispersants, frost protectants, thickeners and adjuvants. Those compositions may be referred to as formulations.

[0098] Examples of typical formulations include water-soluble liquids (SL), emulsifiable concentrates (EC), emulsions in water (EW), suspension concentrates (SC, SE, FS, OD), water-dispersible granules (WG), granules (GR) and capsule concentrates (CS); these and other possible types of formulation are described, for example, by Crop Life International and in Pesticide Specifications, Manual on Development and Use of FAO and WHO Specifications for Pesticides, FAO Plant Production and Protection Papers - 173, prepared by the FAO/WHO Joint Meeting on Pesticide Specifications, 2004, ISBN: 9251048576. The formulations may comprise active agrochemical compounds other than one or more active compounds of the invention.

[0099] The formulations or application forms may comprise auxiliaries, including but not limited to extenders, solvents, spontaneity promoters, carriers, emulsifiers, dispersants, frost protectants, biocides, thickeners and/or other auxiliaries, including but not limited to adjuvants, for example. An adjuvant in this context is a component which enhances the biological effect of the formulation, without the component itself having a biological effect. Examples of adjuvants are agents which promote the retention, spreading, attachment to the leaf surface, or penetration.

[00100] These formulations are produced in a known manner, for example by mixing the active compounds with auxiliaries including but not limited to, for example, extenders, solvents and/or solid carriers and/or further auxiliaries, including but not limited to surfactants. The formulations are prepared either in suitable plants or else before or during the application.

[00101] Suitable for use as auxiliaries are substances which are suitable for imparting to the formulation of the active compound or the application forms prepared from these formulations (including but not limited to, e.g. , usable crop protection agents, including but not limited to spray liquors or seed dressings) particular properties including but not limited to certain physical, technical and/or biological properties.

[00102] Stabilizers, including but not limited to low-temperature stabilizers, preservatives, antioxidants, light stabilizers or other agents which improve chemical and/or physical stability may also be present. Additionally present may be foam-formers or defoamers.

[00103] Furthermore, the formulations and application forms derived from them may also comprise, as additional auxiliaries, stickers including but not limited to

carboxymethylcellulose, natural and synthetic polymers in powder, granule or latex form, including but not limited to gum arabic, polyvinyl alcohol, polyvinyl acetate, and also natural phospholipids, including but not limited to cephalins and lecithins, and synthetic phospholipids.

[00104] Further possible auxiliaries include mineral and vegetable oils. Examples of such additives include fragrances, protective colloids, binders, adhesives, thickeners, thixotropic substances, penetrants, retention promoters, stabilizers, sequestrants, complexing agents, humectants and spreaders. Generally speaking, the active compounds may be combined with any solid or liquid additive commonly used for formulation purposes.

[00105] Suitable retention promoters include all those substances which reduce the dynamic surface tension, including but not limited to dioctyl sulphosuccinate, or increase the viscoelasticity, including but not limited to hydroxypropylguar polymers, for example.

[00106] Suitable penetrants in the present context include all those substances which are typically used in order to enhance the penetration of active agrochemical compounds into plants. Penetrants in this context are defined in that, from the (generally aqueous) application liquor and/or from the spray coating, they are able to penetrate the cuticle of the plant and thereby increase the mobility of the active compounds in the cuticle. This property can be determined using the method described in the literature (Baur, et al., 1997, Pesticide Science 51, 131-152). Examples include alcohol alkoxylates including but not limited to coconut fatty ethoxylate (10) or isotridecyl ethoxylate (12), fatty acid esters including but not limited to rapeseed or soybean oil methyl esters, fatty amine alkoxylates including but not limited to tallowamine ethoxylate (15), or ammonium and/or phosphonium salts including but not limited to ammonium sulphate or diammonium hydrogen phosphate, for example.

[00107] Suitable carriers for animal feed additives are set forth in the American Feed Control Officials, Inc.'s Official Publication, which publishes annually. See, for example Official Publication of American Feed Control Officials, Sharon Krebs, editor, 2006 edition, ISBN 1-878341-18-9.

[00108] All publications, patents and patent applications, including any drawings and appendices therein, are incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

[00109] The following examples are given for purely illustrative and non-limiting purposes of the present invention.

EXAMPLES

Example 1: Synergistic Effect of Semi-Purified and Purified Plipastatins, Iturins and Surfactins and A Physical Membrane Disrupting Agent against Penicillium expansum

[00110] Combinations of (i) crude extracts of lipopeptides from Bacillus subtilis QST713 and a physical membrane disrupting agent and (ii) semi-purified and purified lipopeptides obtained from Bacillus subtilis QST713 and a physical membrane disrupting agent, may be tested against Penicillium expansum as a target pathogen. Iturin-type compounds, including iturin As, and fengycin-type compounds, including plipastatins (A, B and agrastatins) and fengycins, may be precipitated and extracted from B. subtilis QST713 fermentation broth using acidification followed by extraction with an organic solvent. These lipopeptides may be further purified and separated through reversed-phase chromatography to obtain fengycin-type compounds that may be about 83% pure and iturins that may be about 88% pure. Surfactins may be extracted using an organic solvent and then further purified using size exclusion column chromatography to 95% purity. Purity of each lipopeptide class may be determined using a high performance liquid chromatography trace. [00111] Final concentration of the crude lipopeptide extract in each well may be 2.23 ppm; final concentration of each semi-purified or purified lipopeptide extract of iturin-type compounds, surfactin or fengycin-type compounds in each well may be 2.23 ppm, and final concentration of the physical disrupting agent in each well may be 2.23ppm. Thus, the weight to weight ratio of each component in the wells containing combinations may be 1:1. The results should show synergy between the various combinations.

Example 2: Fungal Growth Assay

[00112] A liquid fungal growth assay can be performed in 96-well plates to determine whether the combination of a lipopeptide with a physical membrane disrupting agent, wherein the physical membrane disrupting agent is not a polyene fungicide or combination of a polyene fungicide with a physical membrane disrupting agent, wherein the physical membrane disrupting agent is not a lipopeptide is synergistic in inhibiting fungal growth. All procedures will be conducted aseptically. For each 96 well plate, three separate additions will be made to each well. First, 100 μL· of potato dextrose broth with 100 ppm chloramphenicol will be added. Then 25 μL· of each sample (the components individual and the combination) will be supplemented. Finally, 50 of DI H₂0 will be added to the blank plate (the plate intended to be left blank for an OD reference) or 50 μL· of 10⁵ fungal inoculum will be added to each well in its respective plate(s). The plates will be then covered with lids and placed at 20 °C for 3 days of incubation.

Rating Plates and Interpreting Data

[00113] After the incubation period, the growth of each well in each plate will be rated by optical density at 600 nm using a PerkinElmer Victor plate-reader using the setting "MIC Settings @ 600 (0.1s)" that can be found in the WALLAC^® VICTOR^® software. The results will be also roughly assessed visually as cloudy wells (partial growth) appearing in the dilution series where the sample fell off in activity.

[00114] After an OD will be obtained for each well, the relative growth will be determined by the following calculation: [(Treatment w/Bacteria)-(Treatment w/o

Bacteria)/(Diluent Control w/Bacteria)-(Diluent Control w/o Bacteria)] x 100. The positive control wells for bacterial growth will be considered acceptable if the average and standard deviation of the OD readings at 600 nm will be well above the range of the non-inoculated OD reference wells. (There must be a large difference between the un-inoculated diluent control and the inoculated diluent control wells' readings to ensure that good growth will be obtained). [00115] This percent growth will be then graphed against the sample for the dilutions tested and the last dilution providing at least 80% of growth inhibition will be taken as the minimum inhibitory concentration (MIC).

Synergy will be then determined by the following Gowing's Equation:

Exp = X + [Y * (100 - X)]/100

If Eob » Εχρ, then synergy exists

[00116] The following ratios can be tested starting with 1.12ppm, 0.16ppm and 0.08ppm: N1 :L1, N1 :L5, N1 :L10, N1 :L100, N1 :L500, L1 :N5, L1 :N10, L1 :N100, or Ll :N500. Post- harvest (e.g. , Penicillium expansum only), human pathogenic fungi (e.g. , Aspergillus niger, Candida albicans and Trichophyton tonsurans) may be tested. The compositions described herein may exhibit a synergistic fungicidal activity as shown by Gowing's Equation. Further, the fungicidal activity of the compositions described herein may be measured using this

methodology.

Claims

1. A composition comprising a synergistic fungicidal combination of a lipopeptide and a physical membrane disrupting agent, wherein the physical membrane disrupting agent is not a polyene fungicide.

2. The composition of Claim 1 , wherein the lipopeptide is an amphiphilic cyclic lipopeptide.

3. The composition of Claim 1, wherein the lipopeptide is an iturin- type compound, surfactin-type compound, fengycin-type compound, fusaricidin, or a combination thereof.

4. The composition of Claim 1 wherein the lipopeptide is an iturin-type compound.

5. The composition of Claim 4, wherein the iturin-type compound is bacillomycin D, bacillomycin F, bacillomycin L, bacillomycin LC (bacillopeptin), mycosubtilin, iturin A, iturin AL, or iturin C.

6. The composition of Claim 1 wherein the lipopeptide is a fengycin-type compound.

7. The composition of Claim 6, wherein the fengycin-type compound is fengycin A, fengycin B, plipastatin A, plipastatin B, or agrastatins.

8. The composition of Claim 1, wherein the lipopeptide is a surfactin-type compound.

9. The composition of Claim 8, wherein the surfactin-type compound is esperin, lichenysin, pumilacidin, or surfactin.

10. The composition of any one of Claims 1-9, wherein said composition comprises at least two lipopeptides.

11. The composition of Claim 10, wherein the lipopeptides are iturin-type

compounds, surfactin-type compounds, fengycin-type compounds, or combinations thereof.

12. The composition of Claim 1, wherein the lipopeptide is part of or an extract of a fungicidal lipopeptide-containing fermentation product.

13. The composition of Claim 12, wherein the lipopeptide-containing fermentation product is from a Bacillus species bacteria.

14. The composition of Claim 13, wherein the Bacillus species bacteria is Bacillus subtilis or Bacillus amyloliquefaciens.

15. The composition of Claim 14, wherein the Bacillus subtilis is Bacillus subtilis QST713 and its variants; Bacillus amyloliquefaciens, strain D747, Bacillus subtilis MBI600, Bacillus subtilis Y1336, Bacillus amyloliquefaciens strain FZB42, or Bacillus subtilis var.

amyloliquefaciens FZB24.

16. The composition of Claim 1 , wherein the physical membrane disrupting agent is an antimicrobial peptide.

17. The composition of Claim 16, wherein the antimicrobial peptide is a cyclotide, melittin, gramicidin S, Gramicidin D, bacitracin, polymyxin B, melittin, cecropin, or vancomycin.

18. The composition of Claim 16, wherein the antimicrobial peptide is pantocin A, pantocin B, polyoxin, nikkomycin, rhizocitin, bacilysin, blasticidin, mildiomycin, PEP6, PAF26, BPC194, PEP3, PEP11, BP76, CAMEL, Iseganan, D4E1, TYP, ESF12, ESF1, Pexiganan, MSI- 99, MB-39, Pen4-1, D32R, cecropin A, B, tachyplesin, heliomicin/drosomycin, sacrotoxin IA, mussel defensin, magainin, esculentin- 1 , Rs-AFP2, Alf-AFP, Spil, DRR230-a, BSD1, WT1, Dm- AMP 1, Mj-AMPl, Pn-AMP, hordonthionin, alpha thionin, AFP, SB-37, Shiva- 1, SB37, MB-39, MsrAl, MSI-99, Myp30, or Rev4.

19. The composition of Claim 16, wherein said antimicrobial peptide is a non- ribosomally synthesized peptide or a ribosomally synthesized peptide (RAMP).

20. The composition of Claim 1 , wherein said physical membrane disrupting agent is an antibiotic non-ribosomal peptide.

21. The composition of Claim 20, wherein said antibiotic non-ribosomal peptide is an echinocandin.

22. The composition of Claim 20, wherein said echinocandin is caspofungin, micafungin, or anidulafungin.

23. The composition of Claim 1, wherein said physical membrane disrupting agent is a cyclotide.

24. The composition of any one of Claims 1-23, wherein the physical membrane disrupting agent is not an azole.

25. The composition of any one of Claims 1-24, wherein the weight to weight ratio of the physical membrane disrupting agent and the lipopeptide is from about 500:1 to about 1:500, about 100:1 to about 1:100; about 10:1 to about 1:10; about 5:1 to about 1:5; about 2:1 to about 1 :2; or about 1 :1.

26. The composition of Claim 25, wherein the weight to weight ratio of the physical membrane disrupting agent and the lipopeptide is from about 100:1 to about 1:100; about 10:1 to about 1:10; about 5: 1 to about 1 :5; about 2:1 to about 1 :2; or 1:1.

27. The composition of Claim 26, wherein the weight to weight ratio of the physical membrane disrupting agent and the lipopeptide is about 1:1.

28. A composition comprising a synergistic fungicidal combination of a polyene fungicide and a physical membrane disrupting agent, wherein the physical membrane disrupting agent is not a lipopeptide.

29. The composition of Claim 28, wherein the polyene fungicide is natamycin, nystatin, amphotericin B, aureofungin, filipin and lucenosomycin and/or derivatives of each of these polyene fungicides.

30. The composition of Claim 29, wherein the polyene fungicide is natamycin or a derivative thereof.

31. The composition of Claim 29, wherein the polyene fungicide is nystatin or a derivative thereof.

32. The composition of Claim 28, wherein said physical membrane disrupting agent is an antimicrobial peptide.

33. The composition of Claim 32, wherein said antimicrobial peptides is a non- ribosomally synthesized peptide or a ribosomally synthesized peptide (RAMP).

34. The composition of Claim 32, wherein said antimicrobial peptide is gramicidin, gramicidin S, bacitracin, polymyxin B, melittin, cecropin, or vancomycin.

35. The composition of Claim 32, wherein the antimicrobial peptide is pantocin A, pantocin B, polyoxin, nikkomycin, rhizocitin, bacilysin, blasticidin, mildiomycin, PEP6, PAF26, BPC194, PEP3, PEPl l, BP76, CAMEL, Iseganan, D4E1, TYP, ESF12, ESFl, Pexiganan, MSI- 99, MB-39, Pen4-1, D32R, cecropin A, B, tachyplesin, heliomicin/drosomycin, sacrotoxin IA, mussel defensin, magainin, esculentin- 1 , Rs-AFP2, Alf-AFP, Spil, DRR230-a, BSD1, WT1, Dm- AMP 1, Mj-AMPl, Pn-AMP, hordonthionin, alpha thionin, AFP, SB-37, Shiva- 1, SB37, MB-39, MsrAl, MSI-99, Myp30, or Rev4.

36. The composition of Claim 28, wherein said physical membrane disrupting agent is a cyclotide.

37. The composition of any one of Claims 28-36, wherein the weight to weight ratio of the physical membrane disrupting agent and the at least one polyene fungicide is from about 500:1 to about 1:500, about 100:1 to about 1:100; about 10: 1 to about 1 :10; about 5:1 to about 1:5; about 2:1 to about 1:2; or about 1 :1.

38. The composition of Claim 37, wherein the weight to weight ratio of the physical membrane disrupting agent and the at least one polyene fungicide is from about 100:1 to about 1 :100; about 10: 1 to about 1 :10; about 5:1 to about 1:5; about 2:1 to about 1 :2; or 1 :1.

39. The composition of Claim 38, wherein the weight to weight ratio of the physical membrane disrupting agent and the at least one polyene fungicide is about 1:1.

40. The composition of any one of Claims 28-39, wherein the polyene fungicide is not natamycin.

41. The composition of any one of Claims 28-39, wherein the physical membrane disrupting agent is not azole.

42. A composition comprising a synergistic fungicidal combination of a polyene fungicide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is an antimicrobial peptide.

43. A composition comprising a synergistic fungicidal combination of a polyene fungicide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a cyclotide.

44. A composition comprising a synergistic fungicidal combination of a lipopeptide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is an antimicrobial peptide.

45. A composition comprising a synergistic fungicidal combination of a lipopeptide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a cyclotide.

46. A composition comprising a synergistic fungicidal combination of an

antimicrobial peptide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a cyclotide.

47. A composition comprising a synergistic fungicidal combination of an

antimicrobial peptide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a lipopeptide.

48. A composition comprising a synergistic fungicidal combination of an

antimicrobial peptide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a polyene fungicide.

49. A composition comprising a synergistic fungicidal combination of a cyclotide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a polyene fungicide.

50. A composition comprising a synergistic fungicidal combination of a cyclotide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is a lipopeptide.

51. A composition comprising a synergistic fungicidal combination of a cyclotide and a physical membrane disrupting agent, wherein said physical membrane disrupting agent is an antimicrobial peptide.

52. A method for controlling fungal pathogens comprising administering a composition of any one of Claims 1-51.

53. A method for controlling fungal phytopathogens comprising administering a composition of any one of Claims 1-51.

54. A method for controlling post-harvest fungal pathogens comprising administering a composition of any one of Claims 1-51.

55. A method for controlling fungal pathogens of food or feed comprising

administering a composition of any one of Claims 1-51.

56. A method for controlling human fungal pathogens comprising administering a composition of any one of Claims 1-51.

57. The method of any one of Claims 52-56, wherein the fungal pathogen is a Fusarium species, Botrytis species, Verticillium species, Penicillium species, Aspergillus species and Fusarium species, Rhizoctonia species, Trichoderma species, or Pythium species.

58. The method of any one of Claims 52-56, wherein the administration comprises applying the composition to plants, the area surrounding plants, or edible cultivated mushrooms, mushroom spawn or mushroom compost.

59. The method of any one of Claims 52-56, wherein the fungal pathogen is a Penicillium, Geotrichum, Aspergillus niger, or Colletotrichum species.

60. The method of Claim 59, wherein the post-harvest pathogens is Botrytis cinerea, pathogenic Colletotrichum species, Erwinia carotovora subsp. carotovora, Geotrichum candidum, Geotrichum citri-aurantii, Helminthosporium solani, Monilinia jructicola (brown rot), Penicillium species, Rhizopus species, Aspergillus niger, Thielaviopsis basicola, mucor rot of fruit, and Alternaria rots.

61. The method of Claim 60, wherein the Colletotrichum species is Colletotrichum acutatum, Colletotrichum coccodes, Colleotrichum musa, or Colletotrichum capsici.

62. The method of Claim 60, wherein the Penicillium species is Penicillium digitatum or Penicillium expansum.

63. A method for treating or preventing a fungal infection in a subject comprising administering to the subject a composition of any one of Claims 1-51.

64. The method of Claim 63, wherein said fungal infection is caused by a Candida, Fusarium, or Aspergillus species.

65. The method of Claim 63 or 64, wherein said infection is a corneal infection.

66. The method of Claim 63 or 64, wherein said administration is oral or topical.

67. A composition of any one of Claims 1-51, wherein said composition is formulated as a cream or eye drops.

68. A method for controlling fungal pathogens comprising applying to a locus in need of treatment an effective amount of any of the compositions of any one of Claims 1-51.

69. The method of Claim 68, wherein the locus is post-harvest food and the fungal pathogen is a postharvest pathogen.

70. The method of Claim 68, wherein the postharvest pathogen is Penicillium species or Geotrichum species.

71. The method of Claim 68, wherein the locus is a plant, root, seed or soil surrounding the plant and the fungal pathogen is a fungal phytopathogen.

72. The method of Claim 68, wherein the locus is food or feed.

73. The method of Claim 68, wherein the locus is a human or an animal and wherein the fungal pathogen is Candida.

74. The method of Claim 68, wherein the locus is a human or an animal and wherein the fungal pathogen is Aspergillus species or Fusarium species.

75. A method for producing a fungicidal composition comprising making a combination a fungicide with a physical membrane disrupting agent, testing the combination for synergistic efficacy against target fungi and producing a fungicidal composition comprising the combination and a carrier.