Title:  Combinations of fungal cell wall degrading enzyme and fungal cell membrane affecting compound

United States Patent:  6,512,166

Issued:  January 28, 2003

Inventors:  Harman; Gary E. (Geneva, NY); Lorito; Matteo (Salerno, IT); Di Pietro; Antonio (Cordoba, ES); Hayes; Christopher K. (Geneva, NY); Scala; Felice (Sorrento, IT); Kubicek; Christian P. (Vienna, AT)

Assignee:  Cornell Research Foundation, Inc. (Ithaca, NY)

Appl. No.:  611504

Filed:  March 5, 1996

A system for inhibiting the germination or growth of a fungus comprises (a) fungal cell wall degrading chitinolytic or glucanolytic enzyme and (b) antifungal cell membrane affecting: compound. Exemplified antifungal fungal cell membrane affecting compounds include flusilazole, miconazole, osmotin, gramicidin, valinomycin, phospholipase B, and trichorzianines. The system components (a) and (b) may be supplemented with polyene macrolide antibiotic, antifungal epithiodiketopiperazine antibiotic (e.g., gliotoxin), fungal cell wall biosynthesis inhibitor (e.g., L-sorbose) and/or detergent. Embodiments include method of contacting a plant which expresses cell wall degrading enzyme with antifungal fungal cell membrane affecting compound.

DETAILED DESCRIPTION OF THE INVENTION

The fungal cell wall degrading chitinolytic and glucanolytic enzymes for use in the embodiments of the invention herein include, for example, chitinolytic enzymes and .beta.-1,3-glucanolytic enzymes for degrading cell walls of fungi where the cell walls contain, as a major structural component, chitin and .beta.-1,3-glucans.

These enzymes are found in fungi; bacteria and higher plants. They can be in natural form, i.e., not separated from the source, e.g., by utilizing source microorganisms in the system herein, or they may be in partially purified form, i.e, purified compared to natural form but with other protein present or they may be in biologically pure form or may be expressed by transgenic plant. Fungal cell wall degrading enzymes are readily obtained in biologically pure form from source fungal microorganisms by culturing the source microorganism, concentrating the culture filtrate, fractionating by gel filtration chromatography, concentrating and further purifying by chromatofocusing followed, if necessary, by isoelectrofocusing in a ROTOFOR.RTM. cell (BioRad, Richmond, Calif.). Fungal cell wall degrading enzymes are readily obtained in biologically pure form from bacteria and higher plants by processing comprising culturing, precipitating with NH₄ SO₄, dissolving and purifying by chromatography and/or isoelectric focusing.

The fungal cell wall degrading chitinolytic enzymes cleave chitin, and include, for example, antifungal endochitinases, chitin 1,4-.beta.-chitobiosidases and .beta.-N-acetylglucosaminidases. These can be obtained from fungi, for example, from the genera Trichoderma, Gliocladium, Lycoperdon and Calvatia; from bacteria, e.g., from the genera Streptomyces, Vibrio, Serratia and Bacillus; and from higher plants, e.g., Nicotiana, Cucumis and Phaesolus.

The endochitinases are enzymes that randomly cleave chitin. Endochitinase activity is readily measured by determining optical density at 510 nm as reduction of turbidity of a 1% suspension of moist purified colloidal chitin in 100 mM sodium acetate buffer, pH 5.5, or in 50 mM KHPO₄ buffer, pH 6.7, after 24 hours of incubation at 30^oC. For calculation of specific activity, one unit is defined as the amount of enzyme required to obtain a 5% turbidity reduction.

A very preferred endochitinase is coded for by gene of the genome of and is isolated and derived from Trichoderma harzianum strain P1 having accession No. ATCC 74058. The protein has a molecular weight of 36 kDa (as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis after the protein was prepared under reducing conditions, on direct comparison to migration of a 36 kDa protein) and an isoelectric point of 5.3.+-.0.2 as determined based on its elution profile from a chromatofocusing column, and a molecular weight of 40 kDa (as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis after the protein was prepared under reducing conditions, from a regression based on the log of molecular weight of standard proteins) and an isoelectric point of 3.9 as determined by isoelectric focusing electrophoresis from a regression of distance versus the isoelectric point of standard proteins. The specific activity of the purified endochitinase was determined to be 0.86 units/.mu.g protein with the turbidity reducing assay and 2.2 nkatal/.mu.g protein when nitrophenyl-.beta.-D-N,N',N"-triacetylchitotriose was used as a substrate. This enzyme and its production and purification to homogeneity are described in Harman et al U.S. Pat. No. 5,173,419, and also in Ser. No. 07/919,784, filed Jul. 27, 1992.

Another endochitinase is coded for by gene of the genome of and is isolated and derived from Gliocladium virens strain 41 having accession No. ATCC 20906 and has a molecular weight of 41 kDa (as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis after the protein was prepared under reducing conditions, from a regression based on the log of molecular weight of standard proteins) and an isoelectric point of 7.8 as determined by isoelectric focusing from a regression of distance versus the isoelectric point of standard proteins. The procedures used for molecular weight determination and isoelectric point determination are the same as those-described in detail in Ser. No. 07/919,784. The enzyme is active in citric acid/K₃ (PO₄) buffer over a pH range of 3.5 to 7.0 and shows a 90-100% activity between pH 4.0 and 6.0 and shows maximum activity at pH 4.5. The optimum temperature for endochitinase activity at pH 5.5 is between 30 and 37^oC., and activity drops off sharply at temperatures above 40^oC. This enzyme and its production and purification to homogeneity are described in. DiPietro, A., et al, Phytopathology 83, No. 3, 308-313 (1993). Furthermore, its purification to homogeneity is described in detail in Reference Example 1 hereinafter. The enzyme was purified to an activity 105-fold that of its activity in the culture filtrate.

Two endochitinases are coded for by gene of the genome of and are isolated and derived from Nicotiana tabacum cv. Havana 425 and these respectively have molecular weights of 32 kDa and 34 kDa. These endochitinases and their production and purification and obtaining of cDNA clone for endochitinase from Nicotiana tabacum cv. Havana 425 and transformation of plant to contain gene from Nicotiana tabacum cv. Havana 425 expressing endochitinase activity are described in Shinshi, H., et al, Proc. Natl. Acad. Sci. USA, 84, 89-93 (1/87) and Neuhaus, J. -M., et al, Plant Molecular Biology 16, 141-151 (1991).

The chitin 1,4-.beta.-chitobiosidases cleave dimeric units from chitin from one end. Chitin 1,4-.beta.-chitobiosidases are sometimes referred to for convenience hereinafter as chitobiosidases. Chitobiosidase activity is readily determined by measuring the release of p-nitrophenol from p-nitrophenyl-.beta.-D-N,N'-diacetylchitobiose, e.g., by the following procedure. A substrate solution is formed by dissolving 3 mg of substrate in 10 ml 50 mM KHPO₄ buffer, pH 6.7. Fifty .mu.l of substrate solution is added to a well in a microtiter plate (Corning). Thirty .mu.l of test solution is added, and incubation is carried out at 50^oC. for 15 minutes. Then the reaction is stopped by the addition of 50 .mu.l of 0.4 M Na₂ CO₃, and the optical density is read at 410 nm. An activity of one nanokatal (nkatal) corresponds to the release of 1 nmol nitrophenol per second.

A chitobiosidase is coded for by gene of the genome of and is isolated and derived from Trichoderma harzianum strain P1 having accession No. ATCC 74058 and in its most prevalent form has a molecular weight of 36 kDa (as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis after the protein was prepared under reducing conditions, on direct comparison to migration of a 36 kDa protein), and an isoelectric point of 4.4.+-.0.2 as determined based on its elution profile from a chromatofocusing column and a molecular weight of 40 kDa (as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis after the protein was prepared under reducing conditions, from a regression based on the log of the molecular weight of standard proteins), and an isoelectric point of 3.9 as determined by isoelectric focusing electrophoresis from a regression of distance versus isoelectric point of standard proteins. Conditions for molecular weight determination and isoelectric point determination are described in detail in Ser. No. 07/919,784. It has an optimum pH for activity of about 3 to 7. This chitobiosidase and its production and purification are described in Harman et al U.S. Pat. No. 5,173,419 where it is referred to as a chitobiase, and also in Ser. No. 07/919,784, filed Jul. 27, 1992, where it is referred to as a chitobiase and also as a chitobiosidase. The enzyme obtained in Ser. No. 07/919,784 has a specific activity of 127 nkatal/mg protein and is purified to greater than a 200-fold increase in specific activity compared to its activity in the culture filtrate. Ser. No. 07/919,784 refers to the presence also of a minor band at 36 kDa. It has since been discovered that the chitobiosidase from Trichoderma harzianum strain P1 (ATCC 74058) gives three closely spaced protein bands with molecular weights of 40 kDa (staining most intensely), 38 kDa (faintest stain) and 35 kDa (intermediate intensity stain), as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis after the protein was prepared under reducing conditions, from a regression based on the log of the molecular weight of standard proteins, and that the three bands represent different levels of N-glycosylation of the same protein.

Another chitobiosidase is coded for by gene of the genome of and is isolated and derived from Gliocladium virens strain 41 having accession No. ATCC 20906 and has a molecular weight of 38 kDa (as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis after the protein was prepared under reducing conditions, from a regression based on the log of molecular weight of standard proteins), and an isoelectric point of 4.95 (as determined by isoelectric focusing electrophoresis from a regression of distance versus isoelectric point of standard protein). The proteins used in the determination of molecular mass were 6 standard proteins obtained from Bio-Rad Laboratories, Hercules, Calif., and these proteins and their molecular weights in kDa are respectively hen egg white lysozyme, 14.4; soybean trypsin inhibitor, 21.5; bovine carbonic anhydrase, 31; hen egg white ovalbumin, 45; bovine serum albumin, 66.2; and rabbit muscle phosphorylase b, 97.4. The proteins used in the determination of isoelectric point were 12 standard proteins obtained from Sigma Chemical Company and are respectively amyloglucosidase, 3.6; methyl red dye, 3.8; soybean trypsin inhibitor, 4.6; .beta.-lactoglobulin, 5.1; bovine carbonic anhydrase B, 5.9; human carbonic anhydrase B, 6.6; horse myoglobin cyanocytic band, 6.8; horse myoglobin basic band, 7.2; L-lactic dehydrogenase from rabbit muscle acidic band, 8.3; L-lactic dehydrogenase from rabbit muscle middle band, 8.4; L-lactic dehydrogenase from rabbit muscle basic band, 8.6; and trypsinogen, 9.3. For the linear regressions, r² values ranged from 0.94 to 0.99. This enzyme and its production and purification to homogeneity are described in Reference Example 2 hereinafter.

Two chitibiosidases are coded for by gene of the genome of and are isolated and derived from. Streptomyces albidoflavus having accession no. NRRL B-16746. These respectively have molecular weights of 27 kDa and 34 kDa and have isoelectric points less than 3.0. The chitobiosidase activity was isolated as follows: The bacteria were grown on slants of trypticase soy agar (BBL, Cockeysville, Md.). Growth was transferred to a liquid medium (50 mM Tris, pH 9.0, 0.012% magnesium sulfate, 0.1% glucose, 0.1% calcium chloride, 0.05% manganese sulfate, 0.025% ferrous. sulfate, 0.00125% zinc sulfate, 0.5% crab shell chitin (Sigma Chemical Co., St. Louis)). The biomass was removed from the broth by centrifugation and filtration. The remaining liquid was brought to 95% saturation with ammonium sulfate, and the precipitate was collected by centrifugation at 6000 .times.g for 30 min. at 4^oC. The pellet was resuspended in dH₂ O, dialyzed against ice-cold dH₂ O to remove salt and centrifuged at 6000 .times.g for 10 min. at 4^oC. to remove insoluble particles. The culturing and purification up to this point is described in Broadway, R. M., et al, Lett. Appl. Microbiol. 20, 271-276 (1995). Isolation of chitobiosidase activity was obtained by isoelectric focusing separation as follows: The resulting liquid was applied in approximately equal amounts to compartments of a Rotofor Isoelectric Focusing apparatus (Bio-Rad). The first three fractions contain the chitobiosidase activity.

The .beta.-N-acetylglucosaminidases cleave monomeric units from chitin from one end. .beta.-N-Acetylglucosaminidases may be referred to for convenience hereinafter as glucosaminidases or as nagases. Glucosaminidase activity is readily determined by measuring the release of p-nitrophenol from p-nitrophenyl-.beta.-D-N-acetylglucosaminide, e.g., by the same procedure as described above for assaying for chitobiosidase activity except for the substitution of substrate. An activity of one nanokatal (nkatal) corresponds to the release of 1 nmol nitrophenol per second. Glucosaminidase activity is present in culture filtrates from Trichoderma harzianum strain P1 having accession No. ATCC 74058 and from Gliocladium virens strain 41 having accession No. ATCC 20906.

A nagase coded for by gene of the genome of and isolated and derived from Trichoderma harzianum strain P1 having accession No. ATCC 74058 has a molecular weight of 72 kDa (as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis after the protein was prepared under reducing conditions, from a regression. based on the log of molecular weight of standard proteins), and an isoelectric point of 4.6 (as determined by isoelectric focusing electrophoresis from a regression of distance versus isoelectric point of standard proteins). It has good activity over a pH range of 4 to 7 and optimal activity between pH 5.0 and 5.5, as determined in a 50 mM citric acid/potassium phosphate buffer mixture at pH levels ranging from 3.0 to 9.0. It has good activity over a temperature range of 25 to 85^oC. with optimal activity at 60 to 70^oC., as determined in 50 mM potassium phosphate buffer pH 6.7 at temperatures between 20^oC. and 100^oC. It is quite resistant to heat inactivation, retaining about 70, 25 and 10% of activity after 15 minutes at 80, 90 and 100^oC., respectively. This enzyme and its production and purification are described in Harman et al Ser. No. 08/049,390.

The fungal cell wall degrading glucanolytic enzymes include, for example, antifungal glucan 1,3-.beta.-glucosidases. The glucan 1,3-.beta.-glucosidases cleave 1,3-.beta.-glucans. The sources for these enzymes are typically the same as the sources for chitinolytic enzymes and are preferably microorganisms from the genera Trichoderma and Gliocladium. Glucan 1,3-.beta.-glucosidase activity is readily determined by measuring the amount of reducing sugar release from laminarin in a standard assay containing 250 .mu.l of enzyme solution and 250 .mu.l of a 0.1% solution of laminarin in 50 mM potassium phosphate buffer, pH 6.7, wherein incubation is carried out at 30^oC. for 1 hour whereupon 250 .mu.l of a copper reagent (prepared by dissolving 28 g Na₂ PO₄ and 40 g potassium sodium tatrate in 700 ml deionized water, adding 100 ml of 1N NaOH, then adding 80 ml of a 10% (w/v) solution of CuSO₄.5H₂ O with stirring, then adding 180 g Na₂ SO₄, when all the ingredients have dissolved, bringing to 1 L with deionized water, then allowing to stand for 2 days, then decanting and filtering) is added, and the admixture is covered with foil and heated for 20 minutes in a steam bath, whereupon, after cooling, 250 .mu.l of arsenomolybdate reagent (prepared by dissolving 25 g of (NH₄)₆ Mo₇ O₂4.4H₂ O in 450 ml deionized water, adding 21 ml concentrated H₂ SO₄ with mixing, then adding a solution containing 3 g Na₂ HAsO₄.7H₂ O in 25 ml distilled water and mixing, incubating at 37^oC. for 2 days and storing in a brown bottle until used) is added with mixing, followed by adding. of 5 ml deionized water, and reading color in a spectrophotometer at 510 nm, and wherein appropriate controls without either enzyme or substrate may be run simultaneously; the quantity of reducing sugar is calculated from glucose standards included in the assay. An activity of one nkatal corresponds to the release of 1 nmol glucose equivalent per second. Glucan 1,3-.beta.-glucosidase activity is present in culture filtrates from Trichoderma harzianum strain P1 having accession No. ATCC 74058 and from Gliocladium virens strain 41 having accession No. ATCC 20906.

A glucan 1,3-.beta.-glucosidase is coded for by gene of the genome of and is isolated and derived from Trichoderma harzianum strain P1 having accession No. ATCC-74058 and has a molecular weight of 78 kDa (as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis after the protein was prepared under reducing conditions, from a regression based on the log of molecular weight of standard proteins) and an isoelectric point of 6.2 as determined by isoelectric focusing electrophoresis from a regression of distance versus the isoelectric point of standard proteins. The procedures for molecular weight determination and for isoelectric point determination are the same as those described in Ser. No. 07/919,784. The enzyme has activity against .beta.-1,3 glucan laminarin between pH 4 and 7, with the strongest activity between 4.5 and 5.5. It releases glucose from laminarin at the same rate as reducing groups, which indicates that it is an exoglucanase cleaving monomeric glucose from the laminarin molecule. The enzyme is obtained and purified as generally described above with the medium for culturing of the microorganism being SMCS medium (the same medium used for production of endochitinasen from G. virens as described in Reference Example 1 hereinafter). After the chromatofocusing step, several peaks with glucan 1,3-.beta.-glucosidase activity are detected and fractions from major activity peaks are pooled, dialyzed, concentrated and applied to the Rotofor cell to obtain an electrophoretically pure exo-glucanase. The production and purification of the enzyme are described in detail in Reference Example 3 hereinafter. The enzyme was purified to a specific activity. about 35-fold that of its activity in the culture filtrate.

Purified cell wall degrading enzyme has been found to inhibit the germination or growth of a fungus at a concentration in solution, for example, of 50 ppm to 1000 ppm.

As indicated above, the antifungal fungal cell membrane affecting compounds of the embodiments of the invention are selected from. the group consisting of sterollsynthesis inhibiting fungicides, antifungal peptide antibiotics, zeamatin and proteins that are serologically related to zeamatin, and antifungal lipid lytic enzymes.

The sterol synthesis inhibiting fungicides include dimethylation synthesis step inhibitors which are pyridines and pyrimidines and azoles including imidazoles and triazoles. Pyridines and pyrimidines are useful for agricultural purposes and include, for example, triarimol, fenarimol, nuarimol, buthiobate and pyrifenox. Imidazoles useful for agricultural purposes include, for example, imazalil, prochloraz, and triflumidol. Imidazoles useful for medicinal purposes include, for example, miconazole, isoconazole, econazole, clotrimazole, bifonazole, butoconazole, ketoconazole, tioconazole, oxiconazole, fenticonazole, sulconazole and omoconazole. Triazoles useful for agricultural purposes include, for example, triadimefon, triadimenol, bitertanol, diclobutrazole, propiconazole, penconazole, diniconazole, flutriafol, flusilazole, hexaconazole, tebuconazole, myclobutanil, cyproconazole, furconazole and CGA 169374. Triazoles useful for medicinal purposes include, for example, vibunazole, terconazole, itraconazole, fluconazole and ICI 195-739.

The antifungal peptide antibiotics. include, for example, valinomycin, gramicidin and peptaibols including trichorzianines, trichotoxins, alamethicins, paracelsins, trichobrachin, and zervamicins. As indicated in Ghisalberti, E. L, et al, Soil Biol. Biochem., Vol. 23, No. 11, 1011-1020 (1991), the peptaibols are peptides containing aminoisokutyric acid and a C-terminal. aminoalcohol with the term "peptaibol" being a coined word from the underlined portions of the aforestated definition. Valinomycin and gramicidin are available from Sigma Chemical Company. Langs, D. A., Science, Vol. 241, 188-191 (July 1988) describes gramicidin and refers to it as forming ion channels in lipid membranes. Ghisalberti, E. L., et al, Soil Biol. Biochem., Vol. 23, No. 11, 1011-1020 in a review article on antifungal antibiotics produced by Trichoderma spp. describes antifungal peptaibols alamethicin 1, alamethicin 2, paracelsins a-d, trichobrachin, trichotoxin a40, trichotoxin a50, trichorzianine A IIIc, and trichorzianine B IIIc. Ghisalberti et al describes these as interacting with phospholipid membranes and inducing membrane permeability. Brewer, D., et al, Canadian J. Microbiol. 33, 619-625 (1987) describes alamethicins produced by Trichoderma spp. and the isolation of two of them, namely alamethicin 3 and alamethicin 6 and mentions and describes zervamicins produced by Emericellopsis spp. Argondelis, A. D., J. Antibiot. 27, 321-328 (1974) describes zervamicins I and II. Schirmbock, M., et al, Applied and Environmental Microbiology, Vol. 60, No. 12, 4364-4370 (12/94) describes trichorzanines A1 and B1 from T. harzianum rifae (ATCC 36042). Schirmboch et al describes these as forming voltage-gated ion channels in black lipid membranes.

We turn now to zeamatin and proteins that are serologically related to zeamatin, that is cross react with antizeamatin antibody under reducing conditions. Zeamatin and its purification are described in Roberts, W. K., et al, Journal of General Microbiology, 136, 1771-1778 (1990). Roberts et al indicates that zeamatin has a molecular mass of 22 kDa and suggests its antifungal properties are the result of forming transmembrane pores in fungal membranes. The proteins that are serologically related to zeamatin include, for example, osmotin, thaumatin, PR-R, PR-S, NP24 and 22 kDa proteins having similar N-terminal amino acid sequence to zeamatin isolated from sorghum, oats and wheat. These are found in plants in response to stress, e.g., salt stress, and cause cell membrane permeabilization. Singh, N. K., et al, Plant Physiol., 15, 529-536 (1987) describes the recovery of osmotin from Nicotiana tabacum var Wisconsin 38 and indicates it has a molecular mass of 26 kDa and an isoelectric point-greater than 8.2 and occurs in two forms, an aqueous soluble form (osmotin I) and a detergent soluble form (osmotin II). Thaumatin is described in Edens, L., et al, Gene 18, 1-12 (1982). PR-R and PR-S, i.e., pathogenesis-related protein R and pathogenesis-related protein S, are characterized in Kauffman, S., et al, Plant Mol. Biol 14, 381-390 (1990). PR-R is also described in Cornelissen, B. J. C., et al, Nature (London) 321, 531-532 (1986). NP 24 is described in King, Plant Mol. Biol. 10, 401-411 (1988). Proteins of approximately 22 kDa molecular mass (i.e., similar to zeamatin) and having similar N-terminal amino acid sequence to zeamatin, thaumatin, PR-R and osmotin I and which cross reacted with antizeamatin antiserum are described in Vigers, A. J., et al, Molecular Plant-Microbe Interactions, Vol. 4, No. 4, 315-323 (1991) which suggests the name permatins to describe "this family of membrane-permeabilizing antifungal proteins" and proposes the names sormatin (for the protein isolated from sorghum), avematin (for the protein isolated from oats) and trimatin (for the protein isolated from wheat). Vigers, A. J., et al, Plant Science, 83, 155-161 (1992) describes the serological relation of PR-S and osmotin to zeamatin.

The antifungal lipid lytic enzymes include phospholipases and lipases. Phospholipases include phospholipase A (present in honey bee venom) and phospholipase B (available from Sigma Chemical Company. Lipases include Type I lipase (from wheat germ), Type I-A lipase (insoluble enzyme from wheat germ attached to 4% beaded agarose), Type II lipase (from porcine pancreas), lipase from human pancreas, Type VI-S lipase from porcine pancreas, Type VII lipase (from Candida cylindracea), Type VII-A lipase (insoluble enzyme from Candida cylindracea), Type XI lipase (from Rhizopus arrhizus), Type XII lipase (from Chromobacterium vicosum) and Type XII lipase (from Pseudomonas spp.); all these specifically mentioned lipases are available from Sigma Chemical Company.

We turn now to optional adjuvant components of the systems herein.

The optional antifungal polyene macrolide antibiotic adjuvants are described in Martin, J.-F., Am. Rev. Microbiol. 31:13-38 (1977) which describes them as having a lactone ring of 26-38 atoms, a polyene chromophore consisting of a series of 4-7 alternating double bonds that form part of the macrolide ring and usually one aminosugar moiety. Polyene macrolide antibiotics include the following which are listed in Martin: acosin, amphotericin B, aureofungin, aytactin, candicidin, candihexin A, candihexin B, candihexin E, candihexin F, chainin, dermostatin, DJ-400 B₁, DJ-400 B₂, etuscomycin, eurocidin A, eurocidin B, filipin, flavofungin, fungichromin, hamycin, heptafungin A, levorcin, mycoheptin, nystatin, perimycin, pimaricin, rimocidin, tetrin A, tetrin B and trichomycin. These, when included, are included in the systems herein in a fungus inhibition improving amount. This can be the conventional antifungal amount (dosage).

The antifungal epithiodiketopiperizine antibiotics include, for example, gliotoxin, gliovirin, chaetomin and sporidesmin. Jones, R. W., et al, Journal of General Microbiology, 134, 2067-2075 (1988) states that these are characterized as low-M_r, non polar molecules with bridged polysulfide region which confers activity. and suggests that the primary mechanism of action of gliotoxin involves selective binding to cytoplasmic membrane thiol groups. Gliotoxin is available from Sigma Chemical Company. These, when included, are included in a fungus inhibition improving amount. For gliotoxin, this can range, for example, from 1 ng/ml to 5,000 ng/ml.

The optional fungal cell wall biosynthesis inhibitor adjuvants include chitin synthetase inhibitors and .beta.-1,3 glucan synthetase inhibitors.

The chitin synthetase inhibitors include, for. example, polyoxins A, B, D, E, F, G, H, J, K, L, M, N and O; kitazin P and nikkomycin Z. The isolations and characterization of polyoxin A and polyoxin B are described in Isano, K., et al, Biol. Chem. 29, 848 (1965). The isolations and characterizations of polyoxins D, E, F, G, H, J, K and L are described in Isono, K., et al, Agr. Biol. Chem. 30, 817 (1966) and 32, 792 (1968). The isolation and characterization of polyoxin M are described in Isono, K., et al, Tetrahedron Letters, 1970, 425. The isolation of polyoxins N and O are described in Japanese Kokai 72/23,596 (Chemical Abstracts 78:41566t (1973). Polyoxin B is available from Kaken Chemical Co., Ltd. Kitazin P is available from Kumiai Chemical Industry Co., Ltd. Antifungal usage of Polyoxin B and Kitazin P is mentioned in Watanabe, R., et al, Agric. Biol. Chem. 52(4), 895-901 (1988). Nikkomycin Z is available from Calbiochem and is mentioned in Roberts, W. K., et al, Journal of General Microbiology, 136, 1771-1778 (1990). The chitin synthetase inhibitors, when included, are included in the systems herein in a fungus inhibition improving amount which for these agents is a chitin synthesis inhibiting amount. An assay for chitin synthetase activity is-described in Cabib, E., et al, Chitin Synthase from Saccharomyces cerevesiae, pages 643-649, in Methods of Enzymology, Vol. 138, Ginsburg, V., editor, Academic Press, New York, 1987. Minimum inhibitory concentrations can be determined by including the inhibitor in the assay mixture of the assay for chitin synthetase activity. Minimum inhibitory concentration of polyoxin B against B. cinerea disclosed in Watanabe et al is 12.5 .mu.g/ml. Minimum inhibitory concentrations of kitazin P against B. cinerea disclosed in Watanabe et al is 500 .mu.g/ml.

A .beta.-1,3-glucan synthetase inhibitor is L-sorbose. The mechanism of action of L-sorbose is discussed in Mishra, N. C., et al, Proc. Nat. Acad. Sci. USA, Vol. 69, No. 2, pp. 313-317, 2/72. The .beta.-1,3-glucan synthetase inhibitors, when included, are included in the systems herein in a fungus inhibition improving amount. L-Sorbose may be included in compositions for systems herein in an amount ranging from 1 to 10%, for example 1% to 3%.

The optional detergent adjuvant component of the systems herein include, for example, non ionic detergents, e.g., sorbitan esters, polyoxyethylene fatty alkyl ethers, polyoxyethylene nonylphenol ethers, dialkyl sulfosuccinates, ethbxylated and propoxylated mono- or diglycerides, acetylated mono- or diglycerides, lactylated mono- or diglycerides, sugar esters, polysorbates and polyglycerol esters. The sorbitan esters include, for example, polyoxyethylene sorbitan monolaurate (Tween 21), polyoxyalkylene sorbitan monoleate (Tween 20), polyoxyalkylene sorbitan monooleate (Tween 81) and polyoxyalkylene sorbitan monopalmatate (Tween 40). An example of a polyoxyethylene fatty. alkyl ether is polyoxyethylene lauryl ether which is sold under the tradename Emulgen 120. An example of a polyoxyethylene nonylphenol ether is Emulgen 909. An example of a dialkyl sulfosuccinate is dioctyl sulfo succinate (Pelex OTP). The optional adjuvant detergent component can also be an anionic detergent, e.g., sodium lauryl sulfate, or a cationic detergent, e.g., trimethyl palmityl ammonium sulfate. The detergents, when included, are included in the systems herein in a fungus inhibition improving amount and this amount depends on the detergent included and can be as low, for example, as a concentration of 0.001% or as high, for example, as a concentration of 1%. Use of detergents in combination with antifungal agent is described in Watanabe, R., et al, Agric. Biol. Chem. 52 (4), 895-901 (1988) and minimum concentrations at which certain detergents inhibited mycelial growth of P. oryzae are described therein.

As indicated above, the weight ratio of fungal cell wall degrading enzyme to antifungal fungal cell membrane affecting compound is 0.005:1 to 500,000:1, in many cases 2:1 to 500,000:1. Preferred ratios are set forth in Table 1 below wherein "endochit." stands for endochitinase, and "chitobios" stands for chitobiosidase.

Compositions for use in the systems herein are readily formulated by admixing the fungal cell wall degrading enzymes and the antifungal fungal cell membrane affecting compounds with non-toxic carriers appropriate for the particular use for a composition, e.g., agriculturally acceptable carriers for agricultural uses and pharmaceutically acceptable carriers for medicinal uses. They may be formulated as liquids (solutions or suspensions) or as solids. Since the fungal cell wall degrading enzymes need free water for activity, water must be present at the time of function. This can be accomplished, for example, by applying the fungal cell wall degrading enzymes as aqueous solutions or by formulating the system components as dry powders and applying the powders with the enzyme becoming active once water becomes available, e.g., from rain. Water is a preferred vehicle where components are soluble in it. Osmotin, phospholipase B, trichorzianine A1 and trichorzianine B1 are readily dissolved in water. Organic solvents can also be used and may be required in some cases if a solution is desired. Guidance for forming solutions follows. Gramicidin dissolves in ethanol. Miconazole dissolves in 50% (v:v) ethanol. Flusilazole and valinomycin dissolve in acetone. Gliotoxin dissolves in methanol and ethanol. Suspension can also be employed.

We turn now to formulations for application of the optional adjuvant components. Most polyene macrolide antibiotics have poor water solubility and are therefore normally formulated as dispersions or suspensions for application or applied as a powder. The chitin synthetase inhibitors may also be applied as a powder. Polyoxin B may be dissolved in aqueous ethanol. Kitazin P may be dissolved in a small amount of dimethylformanide and then diluted with water. L-sorbose may be dissolved in water.

We turn now to the method of the invention of inhibiting the germination or growth of a fungus which comprises contacting such fungus or a locus to be protected from such fungus with an antifungal effective amount of a combination of a fungal cell wall degrading chitinolytic or glucanolytic enzyme in a concentration where said enzyme individually provides 2 to 50% inhibition of spore germination and antifungal fungal cell membrane affecting compound which is not chitinolytic or glucanolytic enzyme and which is not expressed by the same organism as the fungal cell wall degrading enzyme in nature in a concentration where said compound individually provides about 4 to 95% inhibition of spore germination, the total of the percentage inhibitions individually provided by the fungal cell wall degrading chitinolytic or glucanolytic enzyme and the antifungal fungal cell membrane affecting compound being less than 100%. The fungal cell wall degrading enzyme and the antifungal fungal cell membrane affecting compound are the same as those discussed in the description of the system of the invention. The effective concentrations are the same as those discussed in the description of the system of the invention. The antifungal effective amount is an amount which inhibits the germination or growth of the fungus that is treated in the method.

For medicinal purposes (i.e., human and veterinary therapy) all the active components can be administered in the same way as the antifungal fungal cell membrane affecting compound is applied when used as the only active ingredient, e.g., topically applied to the skin of a human or non-human animal. Administration can also be, at least in some instances, via parenteral injection, e.g., intraperitoneally; this administration route is particularly useful where the immune system has been compromised since immune-deficient humans and individuals will inactivate enzymatic proteins more slowly than normal individuals.

For agricultural purposes, application can be, for example, to the seed, foliage, roots or fruit of a plant to be protected, or to the soil surrounding said plant, or to the fungus thereon which is to be inhibited. Normally, application is topical. However, other administration strategies can be used.

The system and method described above in the detailed description section contemplate application of the fungal cell wall degrading enzyme and the antifungal fungal cell membrane application as part of the same composition or concurrently as part of separate compositions or separately at different times. When the fungal cell wall degrading enzyme and antifungal fungal cell membrane affecting compounds are applied separately at different times, the inhibition obtained is the same as when the two kinds of agents are applied in the same composition or in different compositions but concurrently, when the antifungal fungal cell membrane affecting compound is applied even as much as 8 hours after the fungal cell wall degrading enzyme. On the other hand, application of the antifungal cell membrane affecting compound before the cell wall degrading enzyme results in reduction in the percentage inhibition obtained compared to when the two kinds of agents are applied as part of the same composition or concurrently in separate compositions until the cell wall degrading enzyme and the antifungal fungal cell membrane affecting compound are both in contact with the fungus for at least about 16 hours. The presence of fungal cell wall degrading enzyme is necessary for about 4 to 8 hours for the highest level of synergistic antifungal effect to be obtained.

The system and method described above in the detailed description section inhibit the germination or growth of fungal species from genera including Fusarium, Gliociadium, Rhizoctonia, Trichoderma, Uncinula, Ustilago, Erysiphe, Botrytis, Saccharomyces, Sclerotium and Alternaria. The specific examples hereinafter show synergism for said system and method herein, in every instance where the antifungal fungal cell membrane affecting compound is used in a concentration less than that where it is substantially entirely effective alone, in application to Botrytis cinerea, and in application to Fusarium oxysporum, which were selected in the work supporting this invention as model test fungi. Botrytis cinerea is a fungus which is pathogenic to fruits including grapes, raspberries, and apples and to beans and other crops. Fusarium oxysporum is a fungus which is pathogenic, for example, to tomatoes, melons, garden peas, cowpeas, beans, soybeans, alfalfa, flax, cotton, carnations, and tobacco.

The synergistic interaction provided by the system and method described above in the detailed description section allows reduction of the quantity of antifungal fungal cell membrane affecting compound that is required for use for a particular inhibition of fungi as much 100- to 1000-fold and this reduction allows usage of fungicides which are otherwise too highly toxic or produce unacceptable side effects at fungicidal or fungistatic dosages, allows usage at dosages less than those which produce side effects and should reduce or at least delay the occurrence of natural resistance to important chemical fungicides.

We turn now to the embodiment herein directed to a method of protecting from a fungus a plant which expresses fungal cell wall degrading chitinolytic or glucanolytic enzyme at a level of 0.05 to 5% of total cellular protein, said method comprising contacting said plant with an antifungal effective amount of an antifungal fungal cell membrane affecting compound at a concentration where it individually provides about 4 to 95% inhibition of spore germination. The plant is a plant which is susceptible to the fungus being protected against which is transformed to contain gene which expresses fungal cell wall degrading chitinolytic or glucanolytic enzyme in the stated amount or which has been infected with transgenic endomorphic microorganisms producing said fungal cell wall degrading chitinolytic or glucanolytic enzyme, typically in the xylem, to produce enzyme internally in the plant in the stated amount. The fungi protected against can be, for example, from the genera of pathogenic fungi described above. Genes coding for fungal cell wall degrading chitinolytic or glucanolytic enzymes can be isolated from microorganisms or other organisms producing them. For example, the characterization and isolation of the gene coding for the aforedescribed endochitinase from Trichoderma harzianum strain P1 is described in Harman et al U.S. Pat. No. 5,378,821. Such gene can be inserted into the genome of a plant to be protected, for example, by Agrobacterium-mediated transformation, by biolistic transformation or by other methods known to those skilled in the art. Methods for use for transformation of plants to contain genes are described in Broglie, K., et al., Science 254,1194-1197 (1991); and in Neuhaus, J.-M., et al., Plant Molec. Biol. 16,141-151 (1991); and in Norelli, J. L., et al., J. Amer. Soc. Hort. Sci. 118,311-316 (1993) taken with Norelli, J. L., et al., Euphytica 77,123-128 (1993); these articles are incorporated herein by reference. The antifungal fungal cell membrane affecting compounds are those described above and the application of antifungal cell membrane affecting compound to the plant can be carried out as described above. Example XX hereinafter is directed to transforming the above described endochitinase encoding gene from Trichoderma harzianum strain P1 into tobacco plants using Agrobacterium to obtain the expression of active enzyme in different parts of the plant in an amount of 1-3% of the total cellular protein and application of antifungal fungal cell membrane affecting compound thereto so that it becomes systemic to act synergistically with the expressed endochitinase in providing fungal inhibition.

We turn now to the embodiment herein directed to a transgenic plant protected against pathogenic fungi which is a plant susceptible to fungal attack which has been transformed to contain gene which expresses fungal cell wall degrading chitinolytic or glucanolytic enzyme at a level of about 0.05 to 5% of total cellular protein and also which also has been transformed to contain gene which expresses protein antifungal cell membrane affecting compound or which has been infected with transgenic endomorphic microorganism producing said protein antifungal fungal cell membrane affecting compound typically in the xylem, in an amount to provide a concentration of said compound where it individually provides about 4 to 95% inhibition of spore germination. The fungi protected against can be, for example, from the genera of pathogenic fungi described above. The transformation to contain gene which expresses fungal cell wall degrading chitinolytic or glucanolytic enzyme in the named amounts is described above. Genes coding for protein antifungal fungal cell membrane affecting compound are described in Kumar, V., Plant Molec. Biol. 18,621-622 (1992) and in Watanabe, Y., et al., FEMS Microbiology Letters 124,29-34 (1994). Such genes can be inserted into the genome of a plant, for example, as described. in the paragraph directly above. Exemplary of this embodiment is a crop plant, e.g., a tobacco plant, transformed to contain gene from Nicotiana tabacum coding for osmotin and which has been transformed to contain the above described endochitinase encoding gene from Trichoderma harzianum strain P1 so the expressed osmotin and expressed endochitinase interact synergistically in providing fungal inhibition.

Claim 1 of 17 Claims

What is claimed is:

1. A transgenic plant transformed with a DNA molecule encoding a fungal cell wall degrading enzyme, wherein the enzyme is selected from the group consisting of Trichoderma endochitinase and Trichoderma .beta.-N-acetylglucosaminidase and the transgenic plant is more fungal resistant than an untransformed form of the plant.
 

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