MXPA99010078A - A novel strain of bacillus - Google Patents

Abstract

The present invention relates to a novel antibiotic-producing and metabolite-producing Bacillus subtilis strain that exhibits insecticidal, antifungal and antibacterial activity. The supernatant of this novel strain contains effective insecticidal, antifungal and antibacterial agents. Also included in the invention is a solvent extractable, small molecular weight (<10,000 daltons) corn rootworm-active metabolite produced in the supernatant. Also included in the invention are methods of protecting or treating plants from fungal and bacterial infections and corn rootworm infestations comprising the step of applying to the plant an effective amount of the antibiotic/metabolite-producing novel Bacillus subtilis strain, the antibiotic/metabolite produced by the novel Bacillus subtilis strain or a combination thereof, optionally further comprising another antiobiotic-producing bacterial s train and/or a chemical pesticide. The invention also includes methods of preventing or treating fungal and bacterial infections using whole broth cultures or supernatants obtained from cultures of the novel Bacillus subtilis strain alone or in combination with chemical pesticides and/or other biocontrol agents. The invention also includes novel antifungal and antibacterial compounds designated agrastatins and a novel combination comprising an A-type iturin, a plipastatin, a surfactin and an agrastatin. Methods of treating or protecting plants from fungal and bacterial infections and corn rootworm infestations comprising administering the novel agrastatins and the novel combination comprising an A-type iturin, a plipastatin, a surfactin and an agrastatin are provided.

Description

NOVEDOSA BACILLS STRAW FOR CONTROLLING PLANT DISEASES AND CORN ROOT WORM DESCRIPTION OF THE INVENTION The present invention relates to the field of biopesticides. More particularly, it refers to the novel Bacill us subtilis strain, AQ713, which can inhibit a wide variety of diseases of fungal and bacterial plants and also has activity against the corn rootworm. The invention also relates to fungicidal, bactericidal and insecticidal compositions comprising this novel Bacillus strain and the antibiotics and metabolites produced through this strain either alone, or in combination with other chemical and biological pesticides. This application is a continuation in part of Series No. 08 / 853,753, filed May 9, 1997. For several years, it has been known that various microorganisms exhibit biological activity in order to be useful for controlling plant diseases. Although progress has been made in the field to identify and develop biological pesticides to control various plant diseases of agronomic and horticultural importance, most of the pesticides in use remain synthetic compounds. Many of these chemical fungicides are classified as carcinogens by the EPA, are toxic to wildlife and other non-target species.

In addition, pathogens can develop resistance to chemical pesticides (see, for example Sch Inn et al., P 244, ADVANCES IN PLANT PATHOLOGY: PHYTOPHPHTHORA INFESTANS, THE CAUSE OF LATE BLIGHT OF POTATO (Academic Press, San Diego 1991). $ 250-300 million of chemical pesticides are used to control corn rootworm pests.Many of these chemical pesticides are toxic to humans, wildlife and other non-target species.It has also been found in groundwater. New chemical insecticides cost $ 100 million to develop Biological control offers an attractive alternative to synthetic chemical fungicides Biopesticides (living organisms and naturally produced compounds produced through these organisms) can be safer, more biodegradable and less expensive to develop. The classification programs have identified certain strains of Bacill us spp. (Bacillus spp.) Includes B subtil is, B. cereus, B. mycoides, B. thuringiensis) that exhibit antifungal activity. (See, for example, Stabb et al (1990) Applied Environ, Microbiol 60: 4404-4412). These strains have been shown to produce zwittermicin-A and / or canosamine (Milner et al. (1996) Appl. Environ. Microb. 62: 3061-3066), two antibiotic agents that are effective against excessive wetting disease arising from the soil. caused by Phytophthora medicaginis, P. nicotianae, P. aph to nidermature or Scl erotinia minor (See Stabb et al., supra). Zwittermicin-A is an acid-soluble, water-soluble linear aminopolyol molecule (see, He et al, (1994) Tetra Left, 35 (16) 2499-2502, U.S. Patent No. 5,049,379 to Handelsman et al. describes how zwittermicin-A produces the disease of excessive wetting in alfalfa and soybean When the seed was coated with B. cereus ATCC 53522, the pathogenic activity of the root fungus was inhibited.Similarly, the application of formulations to ba. The spores of certain strains of B. cereus to soybeans or to the surrounding soil of the seeds have been shown to improve the yield of soybean at these field sites (See, Osburne et al (1995) Am. Phytopa thol. Soc. (6): 551-556.) Methods for applying biopesticides are well known in the art and include, for example, wettable powders, dry flowable products, microencapsulation of effective agents, liquid or solid formulations of antibiotic fractions of suitable cultures (See p or example, U.S. Patent No. 5,061,495 to Rossall or U.S. Patent No. 5,049,379 to Handelsman).

Smith et al. (1993) Plant Disease 77 (2) 139-142 report that the activity of the fungus that develops in the soil Pythium aphanidermatum, which causes the lack of cotton formation capacity of the cucumber, can be suppressed using the B. cereus strain of production of zwittermicin U 85. Leifert et al. (1995) J. Appl. Bacteriol. 78: 97-108 reports the production of anti-Botrytis and anti-Alternaria antibiotics through two strains of Bacillus, B. subtilis CL27 and B. pumilis CL 45. Complete broth and cell-free filtrates were active against Botrytis and Alternating in in vitro tests and were active against Botrytis in small tests in plants in vivo of A tilbe. Leifert et al. (1997) U.S. Patent No. 5,597,565 disclose that B. subtilis, B. pumilis, and B. polymyr.a are particularly effective in inhibiting fungi that cause post harvest diseases. They also describe the presence of antibiotics produced in the cell-free culture filtrate and their activity at different pH values, but do not identify these compounds. Rossall (1994) U.S. Patent No. 5,344,647 discloses Bacillus subtilis strains with broad antifungal activity. Sholberg et al. (1995) Can. J. Microbiol. 41 247-252, Swinburne et al. (1975) Trans. Brit. Mycol. Soc. 65 211-217, Singh and Deverall (1984) Trans. Br. Mycol. Soc. 83 487-490, and Ferreira, et al. (1991) Phytopathology 81: 283 287. Baker et al. (1983) Phytopathology 73: 1148-1152 describe the use of Bacillus spp. and Bacillus subtilis as a biocontrol agent for fungal plant pathogens. Baker et al. (1983) Phytopathology 73: 1148-1152 also report a Bacillus subtilis antifungal for use in plant pathogens. Pusey et al. (1988) Plant Dis. 72: 622-626, Pusey and Robins (U.S. Patent No. 5,047,239), and McKeen et al. (1986) Phytopathology 76: 136-139 describe control of fruit rot after harvest using B. subtilis. McKeen et al, supra, have shown that antibiotics similar to low molecular weight utichrin cyclic polypeptides contribute to this fungal activity of B. subtilis. Liu et al. (1995) U.S. Patent No. 5,403,583 describe a Bacillus megaterium, ATCC 55000 and a method for controlling the fungal plant pathogen, Rhizactonia solani. Islam and Nandi (1985) Journal of Plant Diseases and Protection 92 (3): 241-246 describe a Bacillus megaterium with antagonism to Drechslera oryzae, the causal agent of rice brown spot. The same authors, Islam and Nandi (1985) Journal of Plant Diseases and Protection 92 (3) 233-240 also describe the in-vitro antagonism of B. megaterium against Drechslera oryzae, Alternaria alternata and Fusarinm roseum. The most active antibiotic was highly soluble in water and methanol with a peak V at 255 nm and a backing at 260 nm, which proved to be a lipopeptide similar to polyoxin. Cook ((1987) Proceedings Belt wide Cot ton Production -Mechaniza tíon Research Conference, Cotton Council, Memphis, pp. 43-45) describe the use of a suspension of Bacillus mega terium to reduce the number of cotton plants killed by Phymatotrichum omnivorum, a root cause of cotton root rot. The production of antibiotic B. mega teri um has been registered by Berdy (CRC Handbook of Antibiotic Compounds, Vols. I-XIV, (CRC Press, Inc. Boca Raton, FL 1980-87) who reports the production of toxic peptide antibodies in lower mammals such as ansamitocin -PDM-O, bacimetrine, megacine, pentapeptide, homopeptides Bacilli is known to produce secondary antifungal and antibacterial metabolites (Korzybski et al. (1978)). Researchers at the University of Wisconsin and Cornell have identified a novel fungal compound, zwittermicin. A, produced by Bacill us sp. (He et al. (1994) Tetra Left. 35 (16): 2499-2502) A second fungal metabolite produced by the same strain was recently identified as the known amino sugar, canosamine (Milner ev al. (1996) Appl. Environ Microb. 62: 3061-3065). Another group of metabolites of Ba cillus previously described are the cyclic lipopeptides of the class iturin, some of which are potent fungal agents. These agents consist of a cyclic octapeptide with seven amino acids and a β-amino acid with an aliphatic side chain. There are several groups of iturins that differ in order and content of the amino acid sequence. These are shown in Table 1 below. Generally, a group of molecules related to differences in length and branching of the aliphatic amino acid residue is produced. When tested against Sa ccharomyces cerevesiae, it was found that micosubtilin is the most active agent (LC50 = 10 μg / mL) followed by iturin-A and bacilomycin L (both having LC50 = 30 μg / mL) (Beeson et al. 1979) J. An tibiotics 32 (8): 828-833). The mode of action of these cyclic lipopeptides has been reported due to the interaction with fungal membranes creating transmembrane channels that allow the release of vital ions. (Latoud et al (1986) Biochem Biophys, Acta 856: 526-535). Iturin-C is inactive against fungi including Penicilliam chrysogenum (Peypoux et al. (1978) Tetrahedron 34: 1147-1152). Table 1 Structures of the iturin family of antibiotics R = CH3, CH (CH3) 2, CH3CH2CH CH, A research group at USDA has investigated the structure / activity relationship of the iturins by synthesizing a number of analogs that differ in amino acid chain length. The researchers reported that the activity of the iturines increased with the length of the side chain of the fatty acid and the terminal branch in the order iso > normal > anteiso (Bland et al. (1995) Proc. Plant Growth Regulation Soc. Am. 22nd: 105-107). They also established that the "quantities of iturines obtained from natural production are inadequate to be commercially viable", based on their work with a number of Bacillus strains of iturin production.

Other groups of cyclic lipopeptides isolated from ß. cereus are the plipastatins. These compounds are a family of acylated decapeptides, the structures of which are shown in Figure 1 (Nishikiori et al (1986) J. An tibiotics 39 (6): 755-761). These compounds were originally isolated as inhibitors of phospholipase A? pancreatic pig (Umezawa et al. (1986) J. An tibiotics 39 (6): 737-74), but was later found to inhibit some pathogenic fungi of plants including Botrytis, Pyricularia and Al ternaría (Yamada et al. 1990) Nippon Noya your Gakkaishi 15 (1): 95-96). Yamada also reported a synergistic effect observed between iturin A and the plipastatins, both produced by the same B strain. subtil is. Work has been done on fermentation improvements to increase the production of iturines in both liquid state fermentations (Phae and Shoda (1991) J. Ferment, Bioeng 71: 118-121); Ohno et al. (1993) J. Ferment. Bioeng. 75: 463-465) as in solid state. (Ohno et al (1992) Biotech, Left, 1 (9): 817-822, Ohno et al (1995) J. Fermen, Bioeng 5: 517-519). There is a report of synergy between the closely related surfactins, which by themselves are inactive, and the iturins produced by the same strain B. Subtilis (Hiraoka et al. (1992) J. Gen. Appl. Mi crobiol. 38: 635- 640). The nucleotide sequence for the gene that co-regulates the biosynthesis of iturin A and surfactin has been published (Huang et al (1993) J. Ferment, Bioeng, 76 (6): 445-450). Field work on the strains that produce íturin has been concentrated in soil treatment to control Rhi? Octonia (Asaka and Shoda (1996) Appl. Environ.Microbiol. 62: 4081-4085) and no field applications have been reported Foliar of iturines. Another cyclic lipopeptide compound produced by B. subtil is is surfactma, which possesses an exceptional activity of surfactant (Kanmuma et al (1969) Agrie, Biol. Chem. 33: 973-976). The surfaetma contains a β-hydroxy fatty acid of C14 or C15 linked through a lactone ring to a heptapeptide portion with a LLDLLDL sequence (Apma et al (1968) Biochem. Biophys. Res. Commun. 31: 488-494 Sandrin et al. ((1990) Biotechnol, Appl. Biochem. 12: 370-375) found that the B. subtilis strains produce both surfactin and urine A, the bacillomicmas F and L and micosubtilma.The novel microorganism AQ713 discovered by the inventors, it was previously thought to be a Ba cillus mega terium c pa and now it was identified as a c-l of Ba cillus subt ilis, and produces A, plipastatm, and surfaetmas .The production of this combination of lipopeptides a In addition, the inventors have discovered that AQ713 also produces a newly described group of compounds designated as "agrastatins." The combination of these three previously known compounds with the novel grastatinas is also novel. A commonly used biopesticide is the gram positive bacterium Bacillus thuringiensis. The strains B. Thuringiensis pesticides are known to produce crystal proteins during sporulation, which are specifically toxic to certain orders and species of insects and nematodes. (See, for example, US Patents Nos. 4,999,192 and 5,208,017). The proteinaceous endotoxins produced by B. thuringiensis also act as insecticidal agents against the rootworm of corn and other beetles, (for example, Pa ten te North American No. 5,187,091; Johnson, T.J. et al. (1993), J. Economic In tomology 86: 330-333). The B exotoxins. thuringiensis have been shown to be effective as purified crystals, washed cell pellets and expressed proteins. Warren et al. (WO 96/10083), describe non-endotoxin proteins produced during the vegetative stage of Bacillus cereus and B. thuringiensis. These vegetative proteins, called Vipl and Vip2, have a potent activity against the corn rootworm (north and west) (Estruch et al. (1997), Na ture Biotechnology 15: 137-141 and Mullins et al. (1997)). , Appl. Environ Microbiol., 63, (in press) A thermostable B metabolite, Thuringiensis, termed beta-exotoxin, has also been shown to have pesticidal properties Burgjeron and Biache (1979), Entomophaga 11: 279-284 report a beta-exotoxin which is active against the Colorado potato beetle (Leptinotarsa decemlineata) .In addition, the known B. thuringiensis beta-exotoxins exhibit non-specific pesticidal activity, killing not only nematodes, but also flies, worms, lice and worms. The root of corn, sigma-exotoxin has a structure similar to beta-hexotoxin, and is active against Colorado potato beetle (Argauer et al. (1991) J. Entomol. Sci 26: 206-213). alpha-exotoxin is toxic with tra the larvae of Musca domestica (Cluthy (1980) FEMS Microbiol. Let t. 8: 1-7). The gamma-exotoxins are several protiolitic enzymes, chitinases and proteases. The toxic effects of gamma-exotoxins are only expressed in combination with beta-exotoxin or delta-endotoxin. Forsberg et al. (1976) "Ba cill us thuringiensis: Its effects in Environmental Quality", National Research Council of Canada. Stonard et al (1994) ACS Symposium Series 551: 25 reported a water-soluble secondary metabolite active against the corn rootworm in the supernatant of a Bacillus cereus strain. There are no documented strains of Bacillus both with fungal activity and for the corn rootworm. There are no known metabolites produced by Ba cil lus subtilis that are of a molecular weight less than 10,000 and that can be attracted in a non-polar solvent. A novel antibiotic-producing and metabolite-producing strain of Ba cil lus subtilis, previously identified as Bacillus mega terium, which exhibits broad fungal and bacterial activity and also exhibits activity against the corn rootworm, is provided. A novel metabolite is also provided from novel Bacill us subtilis with activity in the corn rootworm. A method is also provided for treating or protecting plants from fungal and bacterial infections comprising the step of applying an effective amount of Ba cil lus subtil is antibiotic products. The Bacillus subtilis antibiotic product can be provided as a suspension on a whole broth culture or as a supernatant containing antibiotic obtained from a whole broth culture of the Bacillus antibiotic production strain. A method for treating or protecting roots of maize rootworm pest plants is also provided, comprising the step of applying an effective amount of novel metabolite production Bacillus subtilis. The Bacill us subtilis of novel metabolite production can be provided as a suspension in the whole broth culture or as a supernatant containing metabolite or a purified metabolite obtained from a culture of whole broth of the microorganism. Also provided are novel compounds, agrastatms, produced by Id novel strain AQ713 and a novel combination of compounds comprising inturma A, a plipastatm, a surfactin and an agrastatm. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the structure of the plipastatm antibiotics. Figure 2 shows the HTLC chromatogram of metabolites A0713. The present invention provides a novel strain, AQ713, from Bacill us subtilis, previously identified as a Bacillus mega terium, or mutants thereof with a broad antifungal and antibacterial activity and the novel combination of antifungal activity and against the corn rootworm. This novel strain is designated Romeo AQ713 and was deposited with NRRL on March 7, 1997 under the provisions of the Budapest Treaty in International Collection of the Deposit of Microorganisms for the Purpose of Patent Procedure under Accession No. B21661. The invention also includes methods for preventing and treating fungal and bacterial diseases in plants using such bacterial strains or supernates containing antibiotic or pure antibiotics obtained from such bacterial strains. The invention also includes methods for treating plant roots or soil to control the development of larvae of the corn rootworm with a bacterial suspension of AQ713 or a supernatant containing metabolite from a culture of AQ713 or purified metabolites from the strain A0713 The invention also includes a solvent extractable metabolite with activity in the corn rootworm with a molecular weight of less than 10,000 daltons. The invention also includes novel compounds, agrastatins, produced by the novel microorganism. Also included is a novel combination comprising an iturin of type A, a plipastatin, a surfactin and an agrastatin. Definitions As used herein, "biological control" is defined as the control of a pathogen or insect through the use of a second organism. The known mechanisms of biological control include enteric bacteria that control root rot by competing fungi for space on the root surface. Bacterial toxins, such as antibiotics, have also been used to control pathogens. The toxin can be isolated and applied directly to the plant or the bacterial species can be administered as they produce the toxin in si tu.

The term "fungus" or "fungi" includes a wide variety of organisms that carry nucleane spores that lack chlorophyll. Examples of fungi include yeasts, mulches, mold, rust and mushrooms. The term "bacteria" includes any prokaryotic organism that contains a different nucleus. "Fungicide" represents the ability of a substance to increase mortality or inhibit the growth rate of fungi. "Antibiotics" includes any substance that is capable of killing or inhibiting a microorganism. Antibiotics can be produced by a microorganism or through a synthetic process or semi-synthetic processes. Therefore, the term includes a substance that inhibits or kills fungi, for example, zwittermicma-A or canosamma. "Antifungal" includes any substance that is rapacious to kill or inhibit the growth of fungi. The term "cultivate" refers to the propagation of organisms on or in a medium of various types. "Whole broth culture" refers to a liquid culture containing broth cells and media. "Supernatant" refers to the liquid broth that remains when the cells developed in the broth are removed by centrifugation, filtration, sedimentation or other means known in the art.

An "effective amount" is a quantity sufficient to effect beneficial or desired results. An effective care can be administered in one or more administrations. In terms of treatment and protection, an "effective amount" is that amount sufficient to mitigate, stabilize, reverse, reduce or delay the progression of disease states by fungi or bacteria. As used herein, the term "insect" includes all organisms in the "Insect" class. "Preadult" insects refer to any form of an organism before the adult stage, including, for example, eggs, larvae and nymphs. "Insecticide" refers to the ability of a substance to increase mortality or inhibit the scale of insect growth. "Nematicide" refers to the ability of a substance to increase mortality or inhibit the growth rate of nematodes. "Pesticide" refers to the ability of a substance to increase mortality or inhibit the rate of development of insects, nematodes and mites. "Positive control" means a compound that is known to have pesticidal activity. "Positive controls" include, but are not limited to, commercially available chemical pesticides. The term "negative control" means a compound known to have no pesticidal activity. Examples of negative controls are water or ethyl acetate. The term "solvent" includes any liquid that holds another substance in solution. "Extractable solvent" refers to any compound that dissolves in a solvent and can then be isolated from the solvent. Examples of solvents include but are not limited to organic solvents such as ethyl acetate. The term "metabolite" refers to any compound, substance or byproduct of a fermentation of a microorganism having pesticidal activity. The antibiotic as defined above is a metabolite specifically active against a microorganism. The term "agrastatins" refers to a group of novel compounds that have the following structures: where Ri is an aliphatic side chain rf-cta or branched, C8-C20; X is either Ala or Val; R. is an acetate or an ester derivative; and Glx is Gln or Glu. These compounds have antibacterial and antifungal activity as well as activity against the corn rootworm. A novel metabolite and an antibiotic production strain of Bacill us subtilis, previously identified as Bacillus mega terium, which has a broad antifungal and antibacterial activity and which also annihilates or prevents the growth of larvae of the rootworm, is described. of corn. In another aspect, the present invention provides a method for treating or protecting plants from fungal and bacterial infections, which comprises applying an effective amount of a supernatant obtained from a whole-broth culture of Bacillus subtil is AQ713 within the present invention, The supernatant can be obtained as is well known in the art by centrifugation, filtration, sedimentation and the like. In another aspect, the invention encompasses a method for treating or protecting plants from fungal or bacterial infections comprising applying an effective amount of the whole broth of the novel Bacillus subtilis strain. In yet another aspect, the invention encompasses a method for treating or protecting plants from fungal and bacterial diseases which comprises applying an effective amount of the antibiotic produced by the novel strain of Bacillus subtilis. In another aspect, the present invention provides a method for treating or protecting roots of maize rootworm pest plants, comprising applying an effective amount of a supernatant obtained from an entire broth culture of Bacill us subtilis AQ713 within the present invention. The supernatant can be obtained as is well known in the art by centrifugation, filtration, sedimentation and the like. In another aspect, the invention encompasses a method for treating or protecting maize rootworm pest plants comprising applying an effective amount of the whole broth of the novel Bacill us subtil is strain. In a further aspect, the invention encompasses a method for treating or protecting plant roots of corn rootworm pests comprising applying an effective amount of the metabolite produced by the novel strain of Bacillus subtilis. In order to achieve a good dispersion and adhesion of the compositions within the present invention it may be advantageous to formulate the culture of whole broth, the supernatant and / or metabolite / antibiotic with components that aid dispersion and adhesion. Suitable formulations will be well known to those skilled in the art. Compositions within the present invention can be formulated as wettable powders, granules, and the like, or they can be microencapsulated in a suitable medium and the like. Examples of other formulations include, but are not limited to, soluble powders, wettable granules, dry flowable products, aqueous flowable products, dispersible or wettable granules, emulsifiable concentrates, and aqueous suspensions. Other suitable formulations will be well known to those skilled in the art. In still another aspect of the present invention, a novel group of compounds designated as "agrastatin". These compounds exhibit antibacterial and antifungal activity in addition to an activity < ontra the worm of the root of corn. In yet another aspect of the present invention, there is provided a novel composition comprising a type A canal, a plipastatm, a surfactin and an agrastatm. In another aspect of the present invention, there are provided methods for treating or protecting plants of fungal and bacterial diseases, which comprises applying an effective amount of a novel combination of compounds comprising a type A protein, a pastatin, a surfactant and a agrastatin All patents and publications cited herein are incorporated herein by reference in their entirety.

The following examples are provided to illustrate the invention. These examples are not constructed as limitations.

EXAMPLES Example 1 Characterization of Strain AQ713 The isolate was identified with base < -n the use of the Biolog microplate panel (Biolog, Inc., Hayward, CA) as described in Bochner (1989) Na tur-e 339: 157-158. The Biolog microplate is composed of pre-filled and dry panel cavities with 95 different plates of carbon substrates available for gram-positive and gram-negative bacteria. The isolate was grown in a liquid medium at 28 ° C and after 24 hours a washed cell suspension (0.85? Saline composition) was inoculated into each panel cavity of a GP Microplate (Biolog, Ene.). After 24 hours at 28 ° C, carbon utilization reactions were analyzed. The substrate utilization profiles were then compared with the Gram Positive Biology Database (release 3.50) and very similar species were isolated. The results of Biolog gave an index of similarity of 0.883 to Bacillus mega terium. A more extensive characterization of AQ713 was conducted through the American Type Culture Collection, Rockville, Md. Isolated submitted as: Unknown; Strain AQ713 Isolated identified as: Using available physiological and biochemical data, these strains are too similar to Bacill us subtil is.

Cell morphology: The mobile cells were found individually, with an endospore formed in the central or subterminal region. The cells are uniformly stained with Gram positive. Colony morphology: The colonies are opaque and irregular with convex elevation, a rigid, inactive surface, and a margin of erose. Characterization Data of the AQ 713 Strain: Comments: Using the available physiological and biochemical data, this strain closely resembles Bacill us subtilis Key Characterization Results Re erence: Gordon, R.E., W.C. Haynes and C.H.N. Pang. 1973. The Genus Bacillus. Handbook No. 427. U.S. Department of Agriculture, Washington, D.C. Example 2 Activity of AQ 713 Against the Corn Rootworm Bacillus samples were developed in a culture medium of Ba cillus subtilis. Medium 2 contained 57. of peptone, 57 of dextrose, 3% of yeast extract, 3% of malt extract, 1.5% of proflo cottonseed extract (59% protein, 4.26% fat, 6.737. ash, 3.19% fiber and trace amounts of gossypol, the rest being water), 10% soybean meal, and 0.5% MgS04x7HO. Medium 3 contained the same ingredients, except with 207 peptone and 3.4? KH2P04 and 4.3% K2HP0. One-day-old striped cultures were used to inoculate 250 ml of labeled agitation flasks. The flasks were shaken at 200 rpm at 29 ° C for 5 days. To aid the insecticidal activity, 35 ml of the culture broth was centrifuged at 5200 rpm for 20 minutes and the supernatant was used in the microassay described below. Tests were carried out on 96-well microplates. Each cavity contained a solid agar substrate, a test organism and either a positive control, a negative control or a supernatant obtained as described in Example 1 from the novel Bacillus strain. To analyze the insecticidal activity, an agar substrate was prepared for the cavities of the microplate according to Marrone et al (1985), J. Econ. In tomol. 78: 290-293. To analyze the nematicidal activity, flat agar (1.5%) was used in the cavities as well. A solution of 1 ppm of Avidcy (avermectin) was used as a positive control. Deionized water was used as a negative control. Two replicates of the test or control sample were used for each test. 40 μL of the supernatant sample or the whole broth developed in medium 1, 2 or 3 were filled in each sample well. The plates were then placed in a smoke cover to dry for approximately 2-3 hours until dried the agar solution. The test organisms were either preadult maize rootworms (Diabrotica undecimpuneza ta), preadult German cockroaches (Bla tella germanica), preadult beetworms (Spodoptera exigua), preadult flies (Drosophila melanogos ter), or strain N2 of the nematode Caenorhabditis elegans. The test organisms were diluted in 0.1% agar at a concentration of approximately 5 organisms per 25 μL of agar in each cavity. The microplate was sealed with an air-tight substance such as Mylar (-), and each cavity was ventilated with a pin press. Plates were incubated at 27 ° C for up to 7 days. After incubation, the cavities were classified observing infant mortality or the degree of larval development. In the sample cavities containing all the dead larvae or with growth prevented, they were given a classification of 1, the cavities containing some dead larvae and others severely impeded to grow, they were given a classification of 2, live larvae but without letting grows were classified as 3 and the sample cavities that do not contain any dead larvae were given a classification of 4. The classifications were averaged between replicas within each sample. The results are summarized in Tables 2 and 3. Table 2: Classification of AQ713 Against Complete Insect Pest Broth C. Control Worm Worm Control elegans beet root positive fruit Negative of corn Medium 2 NT 1. 0 4. 0 4. 0 1. 0 4. 0 Medium 3 NT 2. 0 4. 0 4. 0 1. 0 4. 0 NT = not tested Table 3: Classification of AQ713 Against Insect Pest Supernatant ('. MO.SM Worm Susano Spoon' ti ... 'ontrol ..ntroi ii / jn.': German root beet osi io .-. jdtivo del maíz fruta Midió 2 4.0 3.0 4.0 4.0 4.0 1.0 4.0 Medium 2 4.0 4.0 4.0 4.0 4.0] .0 4.0 These tests show that AQ713 was active in both media as a full broth culture, with the best activity in medium 2. The supernatant was only active when AQ713 was developed in medium 2. Example 3 Chemical Properties of Active Metabolite AQ713 Corn Rootworm Twenty 50 ml of AQ713 were grown in the middle. To each culture was added 50 ml of ethyl acetate and the mixture was stirred in a separatory funnel for 2 minutes.

The aqueous layer was removed and the organic layer was collected in a bottle containing magnesium sulfate. The organic filtrate was then filtered in a round bottom flask and the solvent was stirred in a rotary evaporator. For the bioassay, the dried organic extract is redissolved in 2. 5 mL of acetone. An aliquot of 40 μL was removed and diluted with 800 uL with 70% acetone / water. This is a 10X concentration of the organic extract. Dilutions in series were performed to obtain samples in the neonate corn rootworm with the recorded mortality percentage of neonatal larvae. One per well in a microtiter plate as prepared above) after 7 days. The results are presented in Table 4. Table 4: Activity of Ethyl Acetate Extract of AQ713 Against the Corn Rootworm Sample% Mortality AQ713 Organic extract 10X 89 Organic extract 5X 93 Organic extract IX 65 Whole broth 100 70% water / acetone 27 water 59 The results show that AQ713 produces a solvent extractable metabolite that annihilates corn rootworms. To determine the molecular weight scale of the active metabolite, 50 ml of AQ713 culture was grown in the medium. One mi was placed in a centrifuge tube and rotated at 12,000 rpm for 15 minutes. The supernatant was removed, 500 microliters of supernatant was placed on the top of a centricon filter of 10,000 dantons molecular weight. This was centrifuged according to the manufacturer's instructions (12,000 rpm for 35 minutes). The filtrate was collected and the retentate recovered via centrifugation and the filter was washed. Samples of the filtered and retentate supernatant were tested against the larvae of the neonatal corn rootworm (96-well plates with insect diet, Marrone et al., Supra as above, 40uL of the sample per well and 8 cavities for each sample, one larva / cavity). The results of the test are shown in Table 5. Table 5: Molecular Weight Cut of AQ713 Percent Mortality Against Corn Root Worm AQ713: Supernatant 43 Filtration 63 Retentate 17 The results show that the supernatant and the filtrate were active, in this way the molecular weight of the metabolite is less than 10,000 daltons. Example 4 Chemical property of metabolite AQ713 against plant pathogens. 50 ml of AQ713 were grown in the medium. To each culture was added 50 ml of ethyl acetate and the mixture was stirred in a separatory funnel for 2 minutes.

The aqueous layer was removed and the organic layer was collected in a bottle containing magnesium sulfate. The organic filtrate was then filtered in a round bottom flask and the solvent was stirred in a rotary evaporator. For the bioassay, the dried organic extract was redissolved in 2.5 ml of acetone. An aliquot of 40 uL was removed and diluted with 80 uL with 707 acetone / water. This is a 10X concentration of the organic extract. A 96-well plate assay (described below) of plant pathogen with Pythium ul timun and Botrytis cinerea was conducted to determine the activity of the organic extract. The whole broth gave 100?; of control (classification of 1), but the organic extract 10X did not give any control of the pathogens of two plants (classification of). This indicates that the active antibiotics, unlike the active metabolites of the corn rootworm produced through AQ713 are not extractable in an organic solvent such as ethyl acetate. The additional test provided the isolation of the novel compound, agrastatin A. A butanol extract was made from the fermentation broth by first extracting the broth twice with an equal volume of ethyl acetate and separating the layers. The aqueous fraction was then extracted twice with an equal volume of butanol. The butanol extracts were combined and the solvent was removed with a rotary evaporator. A powder was obtained through freeze drying resulting in the extract. The powder was dissolved in 80% acetonitrile / water and sound was applied. The solution was applied to a C-18 solid phase extraction cartridge (SPE) that had been activated with methanol and equilibrated with 80 acetonitrile / water. The SPE cartridge was eluted with 80 ?. of ACN / water and this eluent was collected and the solvents were removed. The eluent was purified by HPLC. A C-18 HPLC column (1 cm X 25 cm) (UV detection at 210 nm) with acetonitrile plus 0.05% TFA / water plus 0.05% TFA solvent gradient was used as follows: 0-20 minutes, 33% ACN; 20-30 minutes, 40% ACN; 30-45 minutes, 45-55 ?, ACN; and 45-63 minutes, 55% ACN. An HPLC chromatogram of AQ713 shows the presence of iturins, iturin-type compounds (plipastatins and agrastatins) and sufactins. See Figure 1.

The intussines A2, A3, A, A7 and A6 were identified through the combination of the NMR data and LC mass spectrometry data and comparison with literature values. The surfactins were identified by comparison with the surfactin standards compared by HPLC and LC mass spectroscopy. The compounds of the iturin type were determined as a mixture of plipastatins and the novel agrastatins through a combination of amino acid analysis and LC mass spectrometry. Extensive NMR data were also collected for one of the novel compounds (peak HPLC 20), designated as agrastatm A. It was found that agrastatm A contains the following amino acids: Thr; 3 Glu; Pro; To; Val; 2 Tyr; and Orn. This development differs from plipastatin A by the presence of Val and the loss of lie. The molecular weight of agrastatin A was determined as 1448, which corresponds to the following structure: The straight chain nature of the fatty acid portion was confirmed by XH NMR. The position of the amino acids in the cyclic peptide was determined through detailed analysis of the TOCSY and ROESY datasets. Mass spectroscopy and the amino acid analysis of agrastatin B (peak HPLC 26) suggest that its structure is similar to plipastatm B2 with the substitution of the Ala with Val residue. The structure is shown below: CH3CH2CH (CH2) 10-CH-CH2-CO-Glu-Orn-Tyr.Thr-Glu-Val-ProGln-tyr-Va! CH3 OH Example 5 Activity of AQ713 Against Plant Pathogens in In vitro cultures (96-well plate) To determine whether AQ713 is effective against fungi, Phytophthora infestans, Pythium ulumum, Botrytis cinerea, Rhizoctonia solani, Al ternarla solani, were performed the following experiments. 96-well plates (flat bottom, 400 microliters per well, Nunc brand) were filled with agar medium (potato dextrose agar) (PDA, Difco). Cultures Phytoph thora infestans were grown for three days in the liquid YPG-1 medium (0.4 g yeast, 0.1 'KH2P0, 0.5% MgSO4 X 7 H20, 1.5% glucose). For the other fungi, spores were scraped from the surface of petri dishes and aliquots of 0.1-0.2 mL of deionized water and the spore suspension (concentration of approximately 2 X 106 spores / mL) of pathogen was spread on the agar. AQ713 was grown for 72 hours in medium 2 or 3 as described in Example 2. To obtain the supernatants, the whole broth culture was centrifuged at 5200 rpm for 20 minutes. Pathogens of the fungal plant were placed in pipettes in the 96-well plates (8 cavities / pathogen). The presence or absence of fungal development was recorded for each of the 8 cavities. Approximately 40 uL of the AQ713 supernatant or 20 uL of the whole broth were added to each well. A classification of "1" represents a complete inhibition of fungal growth. A classification of "4" means no inhibition of fungal growth. The results are shown in Table 6. Table 6 In Vitro Inhibition of Fungal Growth (96-well plate) Supernatant AQ713 Medium 2 Medium 3 Classification Classification Phytoph thora infestans 1 1 Pythium ul timum 1 1 Botrytis cinerea 1 1 Rhi zoctonia solani 4 1 Al ternarla brassica 1 1 Whole broth of AQ713 Col letotrichum cocodes 1 NT Al ternarla brassicicola 1 NT Botrytis cinerea 1 NT Cladospori um cucumerinum 1 NT Monil inia fructicola 1 NT Venturia pyrina 1 NT Al ternarla solani 1 NT NT Not tested The results show that AQ713 had a broad fungicidal spectrum in vi tro and that both the whole broth and the supernatant are highly active. The supernatant was active on Rhizoctonia solani in medium 3 but not in medium 2. Example 6 Activity of A 713 Against Plant Pathogens in Culture in vitro (zone assay) To determine the activity of AQ713 in an agar diffusion assay (zone)spores of plant pathogens were spread on the surface of potato dextrose agar in 10 cm petri dishes. Cavities of 7.0 mm were removed from the agar, if 100 uL of the sample of the Aq713 supernatant developed in medium 2 was placed in the cavity. The supernatant was prepared by centrifuging at 4200 rpm for 40 minutes. The supernatant was then rotated again at 4200 rpm for another 40 minutes. Typical results consisted of an area without growth and / or reduced growth of the pathogen around the cavity. The size of the area in millimeters was measured and recorded. The results are shown in the Table 7.

Table 7: In Vitro Inhibition of Fungal Plant Pathogen Growth (Zone Test) When terry Botrytis Monil inia Supernatant AQ713 brassicicola cinérea fructicola Zone size (mm) 16 23! 4 Whole broth of AQ713 22 15 18 Example 7 Activity of AQ713 Against Bacterial Plant Pathogens A standard agar diffusion assay was performed as in Example 6. A layer of each bacterial pathogen was spread on the surface of a petri dish. 100 uL of the entire medium of AQ713 developed in medium 2 were placed in each cavity. The size of the zone was measured in millimeters. Table 8: In Vitro Inhibition of Bacterial Plant Pathogens (Zone Test) AQ713 Complete Broth Inhibition Zone (mm) Acidovorax avenae subs. Ci trull i 18 Pseudomonas syríngae pv. Toma to 11 Xanthomonas campestris pv. Campestris 18 Erwinia carotovora subs. Carotovora 11 Clavibacter míchiganense subs. Michiganense 22 AQ713 was active against all pathogen species of bacterial plants tested in vi tro.

Example 8 Activity of A 713 against Plant Pathogens in Plant Tests The activity of AQ713 against gray mold, Botrytis cinerea, on bean and geranium larvae, Alternarla solani on tomato crops and mild mold of Bremia la ctucae lettuce was tested. For to . solani, tomato plantings were sprinkled in the stage of leaf 2-3 planted in 6 packages to operate with whole broth of AQ713 (medium 2). After spraying, the plantings were allowed to dry (approximately 1.5 hours). The sows were then sprayed with 5.0 x 104 spores / ml. The plantings were covered with a plastic dome and kept at 28 ° C in a Percival incubator. Water was used without any AQ713, with and without spores of the pathogen as a negative control and a positive pathogen control. After four days the test was ready. In the control of A. Solani of water, there were uniform lesions on all the leaves and the cotyledons were detached and were severely infected (classification of 5 = complete infection, without control). The plants treated with AQ713 had few light lesions on the true leaves. The cotyledons were joined but with small lesions (classification of 1) The negative control was not infected.

A second test was performed using separate tomato plantings (broken stems at ground level) were placed in water filled containers placed under domes and stored as previously done. The plants were sprayed as previously done and the symptoms of A were recorded. Solani after four days. No symptoms were seen in the negative control. In the positive control, there were uniform lesions on the sowings. Treatment with AQ713 was classified as 1 (few or no lesions). Two days later, the plants in the positive control were destroyed, but the seedlings treated with AQ713 remained virtually clean and looked the same as the negative controls (plants sprayed with water). For the test in Botrytis cinerea, the first true leaves of a bean plant were cured by compressing the mouth of a 13 X 100 culture tube on each leaf. Each leaf received two wounds / leaf. The leaves were sprayed with whole broth of AQ713 (medium 2) or only water or only pathogen. When they dried, they were again sprayed with B spores. Cinérea (0.8 X 10 ° spores / ml). Plates were placed on flat roofs covered with plastic domes and stored at 18-20 ° C in a Percival incubator. Five days later, the positive control (pathogen only) rotted in an area approximately 25 mm in diameter. The negative control (water only) did not rot. The AQ713 showed no infection in 7 of the 8 circles where the leaves were wounded. The only one that was infected had a slight infection in two places around the circle. For the Bremia test, lettuce seeds were planted in a layer of sterilized culture mixture containing peat, perlite and vermiculite in a small clean plastic plant condominium with a height and width of approximately 8 centimeters. After the lettuce germinates (one week), the lettuce plantings are sprayed with a broth of AQ713 or sample of supernatant. The plants were allowed to dry and then a suspension of soft mold spores was sprayed from the infected lettuce plantings on the planting. The plastic covers were placed on the plants and incubated at 18-20 ° C in a Percival incubator. A week later the test was evaluated. AQ713 did not avoid the soft mold of Bremia in the lettuce fields. Example 9 Efficiency of AQ713 Against Plant Diseases (Greenhouse Test) Soft Grape Mold AQ713 was developed in a soy-based medium in a 400 liter thermidor for 48 hours. Grape plants (Chardonnay cultivation) were sprayed with a manual sprinkler to operate with the entire 400-liter fermentation broth diluted with sterile water at concentrations of 0.5X and 0.25X.

When the foliage dried, the plants were sprayed a second time. After drying, the plants were inoculated with the pathogen that causes soft mold on the grape, Plasmopara vi ticola. 3 plants were treated for each dose. Each plant was evaluated estimating the percentage of disease control based on a scale of 0 to 100% control. A 100% control is a plant without visible lesions. A chemical fungicide, metalaxyl, was used for comparison. The results were as follows: AQ713 whole broth 0.5X 97.7% control Whole broth of AQ713 0.25X 1007, control Metalaxyl 30 ppm 1007 control Metalaxyl 10 ppm 98.3% control Metalaxyl 1 ppm 807 control The results show that AQ713 carried out the control of soft mold in grapes as well as the chemical fungicide. Example 10 Efficiency of AQ713 Against Pumpkin Powder Mold AQ713 was developed in a soy-based medium in a 400 liter fermentor for 48 hours. Squash plants (Crookneck and Acorn) were sprinkled with a manual sprinkler to operate the whole broth from the 400-liter fermentation operation and a sample diluted with sterile water at 0.5X concentration. After drying, the plants were inoculated with the pumpkin powder mold pathogen, sphaerotheca ful ginea. Two plants were treated for each dose. The spray-dried powder of the whole broth was also tested. The 400-liter fermentation broth was sprayed to remove the water. Dry powder spray solutions of 10% and 2.5% were sprinkled on the plants to operate as previously done. The incidence of powdery mildew disease was classified on a base of 0 to 5. The number 5 is rated as 100% disease, while the 0 rating is no disease. The results are shown below in Table 9. Table 9 The whole broth of AQ713 and the dry powder by spray provided absolutely complete control of pumpkin powder. Example 11 Efficiency of AQ713 in Late Pest, Gray Mulch, Grape Powder Mold, Cereal Powder Mold, Pod Plague and Greenhouse Rice Rescue AQ713 was developed in a soy-based medium for 72 hours in a flask of agitation of 250 mL. The disease, the cause pathogen and the host are listed in Table 10 below. This whole broth culture was tested on plants as shown in Table 11 below. Table 10 Each broth was sprayed to operate at a concentration of IX on the sample plants with a manual sprinkler, followed by drying and then sprinkling a second time. Three plants were treated for each disease and treatment. After drying, the plants were inoculated with the pathogens, each plant was evaluated estimating the percentage of disease control based on a scale of 0 to 100% control. 100% control refers to a plant that has no visible injuries. Chemical fungicides were used for comparison. The disease index is the severity of the disease in the untreated control. Table 11 AQ713 showed activity that was equivalent to chemical fungicides in all tested pathogens. Example 12 Efficiency of AQ713 Against Moderate Brassica Mold AQ713 of the bacillus strain was developed in a 10 liter thermidor in a soy-based medium for 48 hours. The culture of whole broth at a resistance of IX was sprayed on plants with cauliflower spots and brucellae of 3 weeks of age in the total cotyledonous stage with an artist air brush activated by compressed air. 3 replicas of 15-25 sowings / container were sprayed by treatment. Quadris ™ an azoxystrobin fungicide from Zeneca was also sprayed on the plants (three per treatment) at speeds of 250 ppm and 125 ppm. A waiting suspension of moderate mold, Peronospora parasí tica, at 1-5 X 104 spores / mL was sprayed on the Brassi ca plants after they were spray dried. The plants were kept at 15-17 ° C for 24 hours for infection, after the sowings were incubated at 20-24 ° C for 6 days. The vessels were returned at 15-17 ° C overnight to allow sporulation of the pathogen until the test was evaluated. Each plant was evaluated estimating the percentage of disease control based on a scale of 0 to 100% control. A 100% control is a plant that did not show sporulation lesions. The results averaged through recipients per replicate are shown below in Table 12. Table 12 NT = Not Tested AQ713 effectively controlled the soft mold during Example 13 Synergism of AQ713 and a Commercial Fungicide AQ713 was developed in a ten liter thermidor in a soy-based medium for 72 hours. The bacterial culture was diluted with sterile water at concentrations of 0.5X and 0.25X. The culture at concentrations of IX, 0.5X and 0.25X was sprinkled on pepper plants of three weeks of age with an artistic air brush activated by compressed air. Three plants were sprayed by treatment. Quadris ™, an azoxystrobin fungicide from Zeneca, was also sprayed on the plants (three per treatment) at concentrations of 500 ppm, 250 ppm and 125 ppm. In addition, combinations of Quadris plus the whole broth culture of AQ713 in a ratio of 1: 1 were sprayed on the pepper plants (3 per treatment). Treatments with and without Quadris are presented in Table 13 below. A spore suspension of Botrytis cinerea, gray mulch, at 1 x 105 spores / mL was sprayed on the pepper plants after drying the sprays with AQ713 and Quadris. The plants were kept at 20-22 ° C for 3 days until the test was evaluated. The incidence of gray mulch disease was evaluated on a scale of 0 to 5. The classification of 5 indicates 100% of disease, while the classification of 0 indicates no disease. The results are shown in Table 13 below.

Table 13 The results clearly show that the combinations of Quadris and AQ713 gray mulch disease control are significantly better than Quadris or AQ713.

Claims (31)

1. CLAIMS 1. A pure culture, isolated from the strain Baus s ubtil is AQ713, NRRL Accession No. B21661 and its mutants.
2. 2. The metabolite produced through the strain Ba cill us subtilis according to claim 1, characterized in that it exhibits activity against the corn rootworm, is a removable solvent and has a molecular weight of less than 10,000 daltons.
3. 3. The supernatant obtained from a culture of the strain Bacillus subtilis AQ713 according to claim 1, characterized in that it exhibits antifungal and antibacterial activity and activity against the corn rootworm.
4. 4. The composition characterized in that it comprises the culture of whole broth of the strain Bacillus subtypes AQ713 according to claim 1, and a chemical fungicide.
5. 5. The composition characterized in that it comprises the culture of whole broth of the strain Bacillus subtil is AQ713 according to claim 1 and a biological or chemical pesticide.
6. 6. The composition according to claim 5, characterized in that it also comprises a chemical fungicide.
7. 7. The composition characterized in that it comprises a metabolite according to claim 2 and a chemical fungicide.
8. 8. The composition characterized in that it comprises the metabolite of claim 2 and a biological or chemical pesticide.
9. 9. The composition according to claim 8, characterized in that it also comprises a chemical fungicide.
10. 10. The composition characterized in that it comprises the supernatant of claim 3 and a chemical fungicide.
11. 11. The composition characterized in that it comprises the supernatant according to claim 3 and a biological or chemical pesticide.
12. 12. The composition according to claim 11, characterized in that it also comprises a chemical pesticide.
13. 13. The method for protecting or treating plants and fruits of fungal and bacterial infections and pests by corn rootworms, characterized in that it comprises applying an effective amount of the strain Bacil lus subti l is in accordance with claim 1.
14. 14. The method for protecting or treating plants and fruits of fungal and bacterial infections and pests of the corn rootworm characterized in that it comprises applying an effective amount of the metabolite according to claim 2.
15. 15. The method for protecting and treating plants and fruit of fungal and bacterial infections and pests by corn rootworm characterized in that it comprises applying an effective amount of the supernatant in accordance with claim 3.
16. 16. The method for protecting or treating plants and fruits from fungal and bacterial infections and of corn rootworm pests characterized because it comprises applying an effective amount of the corn ion according to claims 4, 5, 6, 7, 8, 9, 10, 11 or 12.
17. 17. The method according to the claim 13, 14 or 15, characterized in that the infections are caused by at least one microorganism selected from the group consisting of Phytoph thora infes tans, Rhizoctonia solani, Pythium ul timum, Botrytis cinerea, Al ternarla solani, Colletotrichum cocodes, Al ternarla brassicicola, Cladosporium cucumerinum, Monilinia fructicola, Venturia pyrina, Acidovorax avenae, Pseudomonas syringae, Xanthomonas campestris, Erwinia carotovora, Clavibacter michiganense, Plasmopara vi tícola, Sphaerotheca fuliginea, Uncinula neca tor, and Peronospora parasí tica.
18. 18. The method according to claim 16, characterized in that the infections are caused by at least one microorganism selected from the group consisting of Phytophthora infestans, Rhizoctonia solani, Pythium ul timum, Botrytis cinerea, Al ternarla solani, Colletotrichum cocodes, Al ternarla brassicicola, Cladosporium cucumerinum, Monilinia fructicola, Ven turia pyrina,
19. Acidovorax avenae, Pseudomonas syringae, Xanthomonas campestris, Erwinia carotovora, Clavibacter michiganense, Plasmopara vi tícola, Sphaerotheca fuliginea, Uncinula neca tor, and Peronospora parasí tica. 19. The method according to claim 13, characterized in that the strain Ba cill us subtilis AQ713 is applied as a culture of whole broth.
20. 20. The method of compliance with the claim 13, acterized in that the strain of Ba cill us subtil is AQ713 is applied as a supernatant.
21. 21. The method according to the claim 19, acterized in that the strain of Ba cill us subtil is AQ713 is applied as wettable powders, granules, flowable products or microencapsulations.
22. 22. The method of compliance with the claim 20, acterized in that the strain of Bacill us subtil is AQ713 is applied as wettable powders, granules, flowable products or microencapsulations.
23. 23. The method according to claim 13, 14 or 15, acterized in that the roots of plants or the soil around the roots are treated.
24. 24. The method according to claim 16, acterized in that the roots of the plants or the soil around the roots are treated.
25. 25. A compound having the formula: R? -CH-CH2-CO-Glx-Orn-Tyr-thr-Glx-X-Pro-Glx-Tyr-Val 0R2 O acterized in that Rx is a branched or straight aliphatic side n of C8-C20; X is Val, R2 is an acetate or an ester derivative and Glx is Gln or Glu.
26. 26. A compound acterized in that it has the formula: CH3 (CH2) i2-CH-CH2-CO.Glu-Orn-Tyr-Thr-Glu-Ala-Pro-Gln-Tyr-Val OH O
27. 27. A compound acterized in that it has the formula CH3CH2CH (CH2)? O -CH-CH2-CO-Glu-Orn-Tyr.Thr-Glu-Val-Pro-Gln-Tyr-Val
28. 28. A compound acterized in that it comprises type A iturin, a plipastatin, a surfactant and an agrastatin.
29. 29. The method for protecting or treating plants and fruits from fungal and bacterial infections and corn rootworm pests, acterized in that it comprises applying an effective amount of the composition according to claims 25, 26, 27 or 28.
30. 30. The The method according to claim 29, acterized in that the infections are caused by at least one microorganism selected from the group consisting of Phytophthora infestans, Rhizoctonia solani, Pythium ul timum, Botrytis cinerea, Al ternarla solani, Colletotrichum cocodes, Al ternarla brassicicola, Cladosporium. cucumerinum, Monilinia fructicola, Ven turia pyrina, Acidovorax avenae, Pseudomonas syringae, Xanthomonas campestris, Erwinia carotovora, Clavibacter michiganense, Plasmopara vi ticola, Sphaerotheca fuliginea, Uncinula neca tor, and Peronospora parasitica.
31. 31. The method for protecting or treating pest plants by corn rootworms acterized in that it comprises applying an effective amount of the composition according to claim 25, 26, 27 or 28 to the roots of the plants or to the surrounding soil. of the roots. SUMMARY The present invention relates to a novel Bacillus subtilis strain of antibiotic production and metabolite production exhibiting insecticidal, antifungal and antibacterial activity. The supernatant of this novel strain contains effective insecticidal, antifungal and antibacterial agents. Also included in the invention is a removable solvent, an active metabolite in the corn rootworm with a small molecular weight (<10,000 daltons) produced in the supernatant. Also included in the invention are methods for protecting or treating plants from fungal and bacterial infections and pests by corn rootworm, which comprises the step of applying to the plant an effective amount of the Bacillus subtilis strain of novelty. antibiotic / metabolite production, the antibiotic / metabolite produced by the novel strain of Bacill us subtilis or a combination thereof, optionally further comprises another bacterial strain of antibiotic production and / or a chemical pesticide. The invention also includes methods for preventing or preventing fungal and bacterial infections using whole broth cultures or supernatants obtained from cultures of the novel strain of Bacillus subtilis alone or in combination with chemical pesticides and / or other biocontrol agents. The invention also includes novel antifungal and antibacterial compounds designated as agrastatins and a novel composition comprising an iturin of type A, a plipastatin, a surfactin and an agrastatin. Methods for treating or protecting plants from fungal or bacterial infections and pests by corn rootworms comprising administering the novel agrastatins and the novel combination comprising an iturin of type A, a plipastatin, a surfactin and an agrastatin are also provided.