Abstract:
The invention relates to two lipopeptides a1 and a2 produced by  Bacillus subtilis  and their use as an anitfungal agent against  Aspergillus flavus.  Both peptides are cyclic, acidic and have broad range of antifungal and antimicrobial activity. Both peptides belong to the Bacillomycin D family. A method and composition for controlling aflatoxin contamination in plants susceptible to alflatoxin-producing fungi, like  Aspergillus flavus  or  Aspergillus parasiticus  is also disclosed.

Description:
RELATED CASES 
     This application is based on patent applications Ser. No. 60/080,879, filed Apr. 7, 1998, entitled: “Antifungal Peptide”, PROV and Ser. No. 60/087,535, filed Jun. 1, 1998, entitled: “Purification of a Small Peptide with Antifungal Activity Against  Aspergillus Flavus ”, PROV. This application claims the benefit of the filing dates of both of the above-identified patent applications, which are incorporated herein by reference in their entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SUPPORTED RESEARCH 
     This invention was made with a U.S. federally supported grant USDA-58-6435-3-122 awarded by the United States Department of Agriculture. The government has certain rights in the invention. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to the control of pathogenic microorganisms, particularly pathogenic fungi and certain bacteria. The invention also relates to lipopeptides having such antipathogenic activity i.e. high antifungal particularly against the Aspergillus genus and antibacterial activity. The invention also relates to the plants which have been treated with the lipopeptides or the microorganisms that synthesize and produce the lipopeptides. The invention also relates to other aspects further described herein. 
     An important objective of the invention is to address and to contribute to solve the aflatoxin problem that is caused by the aflatoxin-producing fungi,  Aspergillus flavus  and  Aspergillus parasiticus.    
     2. Description of the Related Art 
     Bacillus ssp is known to produce a variety of peptide antibiotics that are antibacterial and/or antifungal. Although the peptides antibiotics are composed of amino acids, they often differed from gene-encoded polypeptides in their structure and mechanism of biosynthesis. Some are gene-encoded and synthesized ribosomally, but these often undergo posttranslational processing and modifications. Antibiotics produced non ribosomally are composed of 2 to 20 amino acids organized in a linear, cyclic or branched cyclic structure.  Bacillus subtilis  produced gene-encoded antibiotics and a variety of small antibiotic peptides with a molecular weight less than 2000 daltons, synthesized non-ribosomally. Subtilin is one gene-encoded lantibiotic peptide synthesized by  B. subtilis  as a prepropeptide that undergoes posttranslational processing (1). Among the antibiotics synthesized non ribosomally are two family: the lipopeptides including iturin, surfactin, fengycin, plistatin and the small hydrophilic di- and tripeptides. Iturin is a group of cyclic lipopeptides produced by  Bacillus subtilis  including iturin A, C, D and E (2,3), bacillomycin D, F and L (4), Bacillopeptin (5) and mycosubtlin (6). All contains a β-amino fatty acid linked by amide bonds to the constituent amino acid residues of the iturin group. Iturin lipopeptide share a common sequence [β-hydroxy fatty acid-Asx-Tyr-Asx] and show variation at the other four positions. Surfactin is also a cyclic lipopeptide containing seven residues of D- and L-amino acids and one residue of a β-hydroxy fatty acid (7) with an amino acid sequence completely different from the iturin group. It is a powerful surfactant and has been described as an antifungal agent. Fengycin (8) and plipastatin (9) are lipopeptide with ten amino acid and a lipid attached to the N-terminal end of the molecule. They differed from iturin and surfactin by the presence of unusual amino acid such as ornithine and allo-threonine. 
     Table 1 shows the amino acid residues of the iturins and the chemical structure of Bacillomycin D. 
     
       
         
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE I 
               
             
             
               
                   
               
               
                   
                 Amino acid residues 
                   
               
             
          
           
               
                 Antibiotic 
                 L 
                 D 
                 D 
                 L 
                 L 
                 D 
                 L 
                 Major β amino acid 
               
               
                   
               
               
                 Iturin A 
                 Asn 
                 Tyr 
                 Asn 
                 Gln 
                 Pro 
                 Asn 
                 Ser 
                 n-C 14 , i-C 15 , a-C 15   
               
               
                 Iturin C 
                 Asp 
                 Tyr 
                 Asn 
                 Gln 
                 Pro 
                 Asn 
                 Ser 
                 n-C 14 , i-C 15 , a-C 15   
               
               
                 Bacillopeptin 
                 Asn 
                 Tyr 
                 Asn 
                 Ser 
                 Glu 
                 Ser 
                 Thr 
                 n-C 14 , i-C 15 , i-C 16   
               
               
                 Bacillomycin D 
                 Asn 
                 Tyr 
                 Asn 
                 Pro 
                 Glu 
                 Ser 
                 Tbr 
                 n-C 14 , i-C 15 , a-C 15   
               
               
                 Bacillomycin F 
                 Asn 
                 Tyr 
                 Asn 
                 Gln 
                 Pro 
                 Asn 
                 Thr 
                 i-C 16 , i-C 17 , a-C 17   
               
               
                 Bacillomycin L 
                 Asp 
                 Tyr 
                 Asn 
                 Ser 
                 Gln 
                 Ser 
                 Thr 
                 n-C 14 , i-C 15 , a-C 15   
               
               
                 Mycosubtilin 
                 Asn 
                 Tyr 
                 Asn 
                 Gln 
                 Pro 
                 Ser 
                 Asn 
                 i-C 16 , a-C 17   
               
               
                   
               
               
                 Primary structure of iturins.  
               
               
                 Bacillomycin D  
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
             
          
         
       
     
     Iturin exhibit a restricted antibacterial activity and a broad range of antifungal activity against fungi and yeast but none of them has been shown to have antifungal activity against  A. flavus  although iturin A has already been patented for control of aflatoxin (10). Culture filtrate from  B. subtilis  were described in 1948 as antifungal against important dermatophytes and systemic fungi, and the undescribed antibiotic was named “Bacillomycin” (11). Since then 3 class of bacillomycin D (12), F (13) and L (14), have been characterized according to their amino acid sequence. Among each class, different antibiotics have been reported such as bacillomycin Fa, Fb, Fc, and Lc. Bacillomycin Fb and Fc differ from bacillomycin Fa by the presence of one or two carboxyl group respectively instead of carboxamide groups and bacillomycin Lc from bacillomycin L only by the sequence positions of a side chain amide and a carboxylic acid (15). An exhaustive study of the literature show that bacillomycin F has been tested against a wide range of fungi and bacteria (Table II). 
     
       
         
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE II 
               
             
             
               
                   
                   
               
               
                   
                 MIC (ug/ml) 
               
             
          
           
               
                   
                 Bacillomycin 
                 Bacillomycin 
                 Bacillomycin 
               
               
                 Test Organisms 
                 Fb 
                 Fc 
                 Fa 
               
               
                   
               
               
                 
                   Aspergillus niger 
                 
                 25 
                 30 
                 40 
               
               
                 
                   Botrytis cinerea 
                 
                   
                   
                 20 
               
               
                 
                   Cladosporium cladosporioides 
                 
                 25 
                 40 
               
               
                 
                   Fusarium oxysporum 
                 
                 &gt;200    
                 &gt;200    
                 &gt;320    
               
               
                 
                   Mycosphaerella pinodes 
                 
                 100  
                 30 
                 10 
               
               
                 
                   Neurospora crassa 
                 
                   
                   
                 80 
               
               
                 
                   Penicillium chysogenum 
                 
                   
                   
                 20 
               
               
                 
                   Pleospora herbarum 
                 
                   
                   
                 10 
               
               
                 
                   Rhodotorula pillimanae 
                 
                   
                   
                 80 
               
               
                 
                   Sclerotina fructigena 
                 
                   
                   
                 40 
               
               
                 
                   Sclerotina sclerotiorum 
                 
                   
                   
                 50 
               
               
                 
                   Stemphylium radicinum 
                 
                   
                   
                 320  
               
               
                 
                   Trichophyton mentagrophytes 
                 
                   
                   
                 20 
               
               
                 
                   Candida albicans 
                 
                 &gt;200    
                 &gt;200    
                 40 
               
               
                 
                   Candida tropicalis 
                 
                 &gt;200    
                 &gt;200    
                 40 
               
               
                 
                   Saccharomyces cerevisiae 
                 
                 25 
                 30 
                 10 
               
               
                 
                   Azotobacter vinelandii 
                 
                   
                   
                 &gt;400    
               
               
                 
                   Brucella brochiseptica 
                 
                   
                   
                 &gt;400    
               
               
                   Escherichia coli  K12 
                   
                   
                 &gt;400    
               
               
                   Streptomyces albus  G 
                   
                   
                 &gt;400    
               
               
                 
                   Bacillus cereus 
                 
                   
                   
                 &gt;400    
               
               
                 
                   Micrococcus luteus 
                 
                   
                   
                 200  
               
               
                 
                   Sarcinia lutea 
                 
                   
                   
                 &gt;400    
               
               
                 
                   Staphylococcus aureus 
                 
                   
                   
                 &gt;400    
               
               
                 
                   Kluyveromyces bulgaris 
                 
                 75 
                 40 
               
               
                   
               
             
          
         
       
     
     Bacillomycin F showed a strong antifungal activity against various yeasts, fungi and phytopathogenic fungi but a weak antibacterial activity (16). Bacillomycin Lc has been described as a new antibiotic of the bacillomycin family with antifungal activity against phytopathogenic fungi such as  Ophiostoma ulmi, Verticillium dahliae, Ceratocystis fagacearum  and  Cryphonectria parasitica  the causal agents of Dutch elm disease, Verticillium wilt of maples, oak wilt and chestnut blight respectively. Bacillomycin D has been reported to be antifungal against  Absydia corymbifera, Aspergillus Niger, Candida Albicans, Fusarium oxysporum, Kluyveromyces bulgaris  and  Saccharomyces cerevisae  (17). 
     Mycosubtlin, iturin A and bacillomycin L inhibited the growth of  Micrococcus luteus,  their activity are different upon  Micrococcus luteus  protoplast. Mycosubtlin and Iturin A are able to lyse  Micrococcus luteus  protoplast and bacillomycin L has no effect (18). Even a slight modification of the molecule as the methylation of the aspartyl residues of bacillomycin L gives a strong lytic activity while natural bacillomycin L has no lytic activity (19). The phenolic group of the tyrosine residue has been shown to be essential for the antifungal activity, when the tyrosine residue was substituted no activity was observed (20). 
     Aflatoxin Problem 
     The invention relates and contributes to solve an important scientific problem of serious economic and business consequences in the United States and in the world. To date, there is no acceptable and effective way and means to control aflatoxin on plants susceptible to aflatoxin-producing fungi. There are no known aflatoxin-resistant plants, genetically transformed or otherwise. 
     Drought stress and high temperatures at critical times during kernel or seed development and/or insect injury of crops can contribute heavily to aflatoxin contamination of corn by the aflatoxin-producing fungi,  A. flavus  and  A. parasiticus.  The aflatoxin-producing fungi,  A. flavus  and  A. parasiticus,  present health hazards to humans and animals through the toxic and carcinogenic properties of their secondary products. Even a very low level of aflatoxin contamination can lead to severe economic losses in the peanut industry. To control this problem, breeding peanut varieties resistant to Aspergillus sp. or which support less aflatoxin production has been attempted with limited success. 
     No commercial corn hybrids with high resistance to either infection by  A. flavus  or to aflatoxin accumulation are currently available. Aflatoxin detected at levels of 20 parts per billion (ppb) (established by the FDA) or above can make the crop unsalable. Aflatoxin is considered to be toxic to livestock being fed contaminated grain and to be a carcinogen, correlated with liver cancer, in certain human populations around the world. Cottonseed grown in the Yuma Valley, Ariz., corn grown in certain states of the southern U.S. and peanuts grown in certain regions of Georgia are chronically contaminated with aflatoxins resulting in direct losses by farmers amounting to tens of millions of dollars annually. Corn grown in the mid western states are contaminated with aflatoxin on a sporadic basis. Exact economic losses due to sporadic or limited aflatoxin outbreaks are difficult to determine, particularly in terms of detrimental impact at each of the various steps in the marketplace (including foreign markets). However, during drought years (1983 and 1988) in the mid west corn belt, which led to large aflatoxin outbreaks, losses were estimated to total in the hundreds of millions of dollars. 
     A major outbreak of aflatoxin in Southern Grown Corn, occurred recently in 1998. Aflatoxin contamination of corn in Arkansas, Georgia, Louisiana, Mississippi, Missouri, South Carolina, Tennessee, and Texas in 1998 was one of the worst ever recorded. A potentially record number of grain-load rejections, due to aflatoxin levels in excess of FDA limits, occurred in some southern states. The aflatoxin outbreak in 1998 has additional significance since corn production in certain southern states has probably tripled in the past three years. It is certainly no coincidence that continuous days of 90(+) F temperatures and low or no rainfall dominated weather conditions for significant portions of the 1998 growing season in Southern states. The literature indicates that these conditions are highly conducive, even necessary for large aflatoxin outbreaks in susceptible crops. Insect injury is often suggested to be the major mode of entry by aflatoxin producing fungi. However, insect injury apparently does not account for the enormity of the aflatoxin outbreak experienced this past year in the South. Thus, controlling insects (e.g. cultivation of transgenic corn containing Bt, an insect toxin), although an essential part of good management practices, would not necessarily have been effective in reducing aflatoxin levels in Southern-grown corn in 1998. Some observations suggest that the fungus gained entry by some other, little understood means associated with high temperature and drought conditions. This highlights the need for extensive research on drought and temperature effects on plant health and physiology and fungal virulence and to find a solution to these problems. 
     In accordance with the invention, a method, microorganisms of the Bacillus family, peptides synthesized by the Bacillus, especially by  B. subtilis  have been discovered that contribute an important technical advance to the resolution of the aflatoxin problem. 
     SUMMARY OF THE INVENTION 
     The invention provides microorganisms of the Bacillus ssp, especially  B. subtilis  preferably  B. subtilis  AU 195 which is effective to control plant pathogens both fungi and bacteria. An important embodiment provides the control of aflatoxin in plants susceptible to aflatoxing-producing fungi. The treatment of the plant can be carried out at anytime, e.g. prior to, or after the onset of the infection. The control can be effectuated with the  B. subtilis  with which synthesizes the antifungal peptides or by the peptides themselves, one at a time or together. Several methods and means of control of aflatoxin producing fungi are provided. Any part of the plant, including the seeds can be treated. 
     The peptides can be used in a purified form or as crude extract, in aqueous or non-aqueous composition. 
     Isolation, sequencing and identification of the gene(s) expressing the antifungal peptides, or their functional equivalent, and the transformed plants resistant to aflatoxin-producing fungi, such crops like corn or peanut, susceptible to infection by aflatoxin-producing fungi, is being contemplated. 
     In particular, the invention provides a family of antifungal peptides, especially two which are of present interest which are antifungal against  A. flavus.    
     The present invention provides  B. subtilis , which shows high antifungal activity against  A. flavus  and also shows antibacterial activity against certain bacteria. The  B. subtilis  strain AU195 produces two small cyclic peptides exhibiting a range of antifungal activity against plant pathogenic fungi. Both peptides have a spectrum of activity against plant fungi and against bacteria heretofore not reported, which make these peptides, very valuable in the control of plant pathogens on plants susceptible to infection caused by pathogens, particularly to aflatoxin-producing fungi like  A. flavus  and  A. parasiticus.    
     The invention also related to a method for controlling the diseases of plants caused by such pathogens, and the plants treated with the microorganisms which generates these peptides or with compositions which comprises  B. subtilis,  extracts thereof or the peptides synthesized by the microorganisms. 
     The invention also related to a  B. subtilis  AU195 has been deposited at the ATCC under Accession number PTA-1767 on Apr. 27, 2000, at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, and was tested on May 4, 2000 and found to be viable. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG. 1 shows the in vitro inhibitory activity of the strain  B. subtilis  AU195. 
     FIG. 2 shows the production of antifungal peptides by  B. subtilis  AU195 and the inhibition of  A. flavus  growth. 
     FIG. 3 shows the inhibition of fungal growth by protein fractions. 
     FIG. 4 shows that the antifungal activity is associated with acidic peptides. 
     FIG. 5 shows the separation of the antifungal peptides a1 and a2 on superdex peptide Hiload 16/60 column. 
     FIG. 6 shows the FAB-MS spectrum of antifungal peptides a1 (a) and a2(b). 
     FIG. 7 shows the antifungal activity on three different fungi. 
     FIG. 8 shows the difference between the normal growth of  A. flavus  (A) and growth of  A. flavus  in the presence of an antifungal extract (B). 
    
    
     DETAILED DESCRIPTION OF THE FIGURES 
     FIG. 1 shows the in vitro inhibitory activity of the strain  B. subtilis  AU195. 
     FIG. 2 Effect of culture filtrate on growth of  Aspergillus flavus  as measured by absorbency at 560 nm daily during 6-day period. Control sample represents the fungal growth in PDB medium (500 spores/per well in 100 μl LB and 100 μl PDB). Other histograms represent the growth of the fungus in the presence of samples from culture filtrate obtained at day 1, day 2, day 3,day 4,day 5, day 6 and day 7 (100 μl of LB is replaced by 100 μl of culture filtrate). 
     FIG. 3 Inhibition of fungal growth by protein fraction precipitated with 20, 40, 60 and 80% ammonium sulfate. A mycelial plug of  Aspergillus flavus  was placed on PDA plate 3 days prior to application of samples to allow establishment of the fungus. The samples were placed at an equal distance (5 mm) from the edge of the mycelial growth. 50 μl of protein sample precipitated with 20-80% ammonium sulfate was placed on a Whatman filter disk. 
     FIG. 4 Inhibition of fungal growth by acidic peptides. After IEF electrophoresis, two independent gel segments (with a pH of 4.5 and 4.7, respectively) either containing the sample (top and bottom) or LB as control (left or right) were inoculated with  A. flavus.  This assay clearly demonstrated that the ampholytes are not antifungal and the activity was strictly associated with the a fraction of the gel representing a pH of 4.5. 
     FIG. 5 Separation of the antifungal peptides a1 and a2 on superdex peptide Hiload 16/60 column. 
     FIG. 6 shows the FAB-MS spectrum of antifungal peptides a1 (a) and a2(b). 
     FIG.  7 . shows the antifungal activity on three different fungi. 
     FIG.  8 . Pictures represent  A. flavus  growth on PDA plate view with Scanning Electron Microscope (SEM): A) normal hyphae; B) hyphae growing in presence of the antifungal fraction. Bar represent 10 μm 
     DETAILED DESCRIPTION OF THE INVENTION 
     The family of peptides, which have antifungal and antibacterial activity against a spectrum of fungi and bacteria include two small peptides designated herein as a1 and a2. These peptides were found to be associated and isolated from  B. subtilis  AU195. They were characterized as iturin and belong to the bacillomycin D family. Although as has been reported above, bacillomycin D peptides have been found to have antifungal activity, no peptide has been reported to date to have antifungal activity against certain specific fungi, to which plants are susceptible, specifically  Alternaria solani, Fusarium monoliforme, Pythium aphanidermatum,  and especially  A. flavus.    
     Other pathogenic fungi to which plants are known to be susceptible include  Puccinia ssp., Phytophthora ssp., Peronospora ssp., Rhizoctonia ssp., Botrytis ssp., Sclerotinia ssp.  and  Colletotrichum ssp , which can be controlled with the peptides of the invention. 
     Further, as has been reported, certain peptides which were isolated from bacteria, have been found to have antibacterial activity, which are human pathogens. In contrast, the peptides of the invention have been found to be effective against plant pathogenic bacteria such as,  Xanthomonas campestris  ( X. campestris ) is known to be pathogenic, for instance on cabbage plants and is responsible for black rot disease. Further, the peptides are effective against certain bacteria-like  Clavibacter michiganensis  and  B. cereus.    
     The composition of the invention comprises one or more of the principal two peptides discovered namely a1 and a2 in a mixture with conventional biologically acceptable inert or active carriers. The peptides can be used together or administered to the plant, or to the animal sequentially, or only one of the fungal peptides need to be used. 
     The amount in which the peptide is used in a minimum amount effective to control or inhibit the target fungi or bacteria to a maximum as may be needed for control. 
     Instead of the isolated peptide, the microorganism, the  B. subtilis  can be used by itself or in the composition, preferably with an appropriate ingredient that promotes the release of the peptide(s). It is not excluded that the spores of  B. subtilis  (e.g.  B. subtilis  AU 195) may be used. These would be allowed to grow on the plant and release the peptides. 
     The peptides a1 and a2 of the invention have been characterized as described hereafter. They have, respectively, a 1045 Da and 1059.5 ba molecular weight, determined by mass spectroscopy. It is believed that a1 has a β-amino fatty acid of 15 carbons and that a2 has a β-amino fatty acid of 16 carbons. The tandem mass spectrometry of a2 indicated a sequence very similar to a1 with an additional CH 2  at some point other than the Glu residue. It is not excluded that the length of these amino acids vary each by 1-3 carbons, i.e., be shorter or longer accordingly. The mass shift of a2 may be accountable by a homoserine for the serine residue or a fatty acid modifier one carbon longer than a1 or a single amino acid substitution of some type. 
     The target plant or mammal, which can be treated is any plant susceptible to the fungal or microbial infection to be controlled, like the major crops, whether monocotyledonous or dicotyledonous, like cereals, legumes, tubers, solanaceous plants, cucurbits, fibrous plants, corn, peanut, rice, wheat, etc. Of particular interest at this time in the treatment of peanut plants which are susceptible to aflatoxin-producing fungi. Peanut plants including the seeds can be treated in accordance with the invention. 
     Any mammal especially animals susceptible to  A. flavus  or and/or  A. parasitus  can also be treated with the compositions including the peptides of the invention. 
     The peptides of the inventions are generally obtained as a mixture of isomers in a mixture with other peptides that do not interfere with the activity of the peptides of the invention. The isomeric mixture can be separated into the respective isomers by know inventions. 
     Upon isolation and sequencing of the gene(s) which codes the peptides of the invention, the plants like corn, soybeans, peanut and other crops can be readily transformed (either by nucleus or chloroplast transformation) to produce plants resistant to the target infection, and likewise produce progeny (and maternally inherited) generations, that are likewise stably resistant to the target pathogen(s). 
     Any mammal, especially animals susceptible to  A. flavus  and/or  A. parasiticus  can be treated on accordance with the invention. 
     The peptides can be used for prophylactic control and/or therapeutic control. 
     Other embodiments of the invention will become apparent from the description herein. 
     The following Examples are not intended to limit the invention in any manner whatsoever, they are purely illustrative. 
     EXAMPLE 1 
     Description of the producing strain (see Table III). Table III gave the identification of the AU 195 as a  Bacillus subtilis  strain with gas chromatography of fatty acid methyl ester. AU195 produces a pigment, which gave an orange color to the culture filtrate. FIG. 1 shows the in vitro inhibitory activity of the strain  B. subtilis  AU195. 
     
       
         
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE III 
               
               
                   
               
             
             
               
                 RT 
                 Area 
                 Ar/Ht 
                 Respon 
                 ECL 
                 Name 
                 % 
                 Comment 1 
                 Comment 2 
               
               
                   
               
             
          
           
               
                  0.311 
                    450 
                 0.036 
                 . . . 
                  4.365 
                 . . . . . . . . . . 
                 . . . 
                 &lt; min rt 
                   
                   
                   
               
               
                  1.457 
                 360188400 
                 0.049 
                 . . . 
                  7.041 
                 SOLVENT PEAK . . . . 
                 . . . 
                 &lt; min rt 
               
               
                  5.923 
                    618 
                 0.039 
                 1.002 
                 13.617 
                 14:0 ISO . . . . . . 
                  0.90 
                 ECL deviates 
                 −0.001 
                 Reference 
                 −0.005 
               
               
                  7.295 
                   19890 
                 0.033 
                 0.971 
                 14.622 
                 15:0 ISO . . . . . . 
                 28.09 
                 ECL deviates 
                   0.001 
                 Reference 
                   0.000 
               
               
                  7.423 
                   27120 
                 0.034 
                 0.968 
                 14.712 
                 15:0 ANTEISO . . . . 
                 38.20 
                 ECL deviates 
                   0.001 
                 Reference 
                 −0.001 
               
               
                  8.818 
                    1476 
                 0.049 
                 0.946 
                 15.624 
                 16:0 ISO . . . . . . 
                  2.03 
                 ECL deviates 
                 −0.002 
                 Reference 
                 −0.003 
               
               
                  9.024 
                    1158 
                 0.042 
                 0.943 
                 15.755 
                 16:1 w11c . . . . . 
                  1.59 
                 ECL deviates 
                 −0.002 
               
               
                  9.409 
                    1938 
                 0.036 
                 0.938 
                 16.000 
                 16:0 . . . . . . . . 
                  2.65 
                 ECL deviates 
                   0.000 
                 Reference 
                 −0.001 
               
               
                 10.045 
                    2058 
                 0.039 
                 0.932 
                 16.384 
                 ISO 17:1 w10c . . . 
                  2.79 
                 ECL deviates 
                 −0.003 
               
             
          
           
               
                 10.195 
                    1014 
                 0.047 
                 0.930 
                 16.474 
                 Sum In Feature 5 . . 
                  1.37 
                 ECL deviates 
                 −0.002 
                 17:1 ISO I/ANTEI B 
               
             
          
           
               
                 10.453 
                    8904 
                 0.040 
                 0.928 
                 16.630 
                 17:0 ISO . . . . . . 
                 12.02 
                 ECL deviates 
                   0.001 
                 Reference 
                 −0.001 
               
               
                 10.605 
                    7692 
                 0.040 
                 0.927 
                 16.722 
                 17:0 ANTEISO . . . . 
                 10.37 
                 ECL deviates 
                 −0.000 
                 Reference 
                 −0.002 
               
               
                 19.443 
                    846 
                 0.186 
                 . . . 
                 21.927 
                 . . . . . . . . . . 
                 . . . 
                 &gt; max rt 
               
             
          
           
               
                 ******* 
                    1014 
                 . . . 
                 . . . 
                 . . . 
                 SUMMED FEATURE 5 . . 
                  1.37 
                 17:1 ISO I/ANTEI B 
                 17:1 ANTEISO B/i I 
               
               
                   
               
             
          
           
               
                   
                 Solvent Ar 
                 Total Area 
                 Named Area 
                 % Named 
                 Total Amnt 
                 Nbr Ref 
                 ECL Deviation 
                 Ref ECL Shift 
               
               
                   
                   
               
               
                   
                 360188400 
                 71868 
                 71868 
                 100.00 
                 68741 
                 7 
                 0.002 
                 0.002 
               
               
                   
                   
               
             
          
           
               
                   
                 TSBA [Rev 3.80] Bacillus . . . . . . . . . . . . . . . . . . . . . 
                 0.904 
               
             
          
           
               
                   
                   B. subtilis  . . . . . . . . . . . . . . . . . . 
                 0.904 
               
               
                   
                 
                   B. amyloliquefaciens . . . . . . . . . . . . . . 
                 
                 0.553 ( Bacillus subtilis  group) 
               
             
          
           
               
                   
                 CLIN [Rev 3.80] Bacillus . . . . . . . . . . . . . . . . . . . . . 
                 0.569 
               
             
          
           
               
                   
                   B. subtilis  . . . . . . . . . . . . . . . . . . 
                 0.569 
               
               
                   
                   
               
             
          
         
       
     
     EXAMPLE 2 
     Description of the method for the antifungal assays. Microtiter plate assay: Samples were tested for their antifungal activity against  Aspergillus flavus  in a microtiter plate (Falcon 3918, Becton-Dickinson and Company). Each well contained 100 μl Potato Dextrose Agar (PDA, Difco Laboratories) and 100 μl of sample and was inoculated with 500 spores of  Aspergillus flavus.  Fungal growth was monitored by reading optical density (O.D.) at 560 nm (Dynatech microplate reader). 
     Disc plate diffusion assay : Antifungal activity of the different purification steps were carried out under sterile conditions using the disc plate diffusion assay. Mycelium plugs from actively growing fungal cultures were placed in the center of the petri plate. After incubation at 27° C. to allow vegetative growth, samples were applied on sterile filter paper discs laid on the agar surface. 
     EXAMPLE 3 
     Production of the antifungal peptides. AU195 has been cultured in LB medium and culture filtrate was sampled daily. Samples were filter sterilized and incubated in presence of  Aspergillus flavus  spores in microtiter plates which enabled us to measure fungal growth spectrophotometrically as described in example 2. The antifungal peptides secreted into the culture filtrate by AU195 increased every day (FIG.  2 ). 
     EXAMPLE 4 
     Temperature stability and sensitivity to enzyme. 7-day culture filtrate was boiled for 20 minutes and then tested for antifungal activity. 50 μl of 7-day culture filtrate were incubated with 200 μg of lipase from wheat germ (sigma) in 50 mM tris HCl pH 7.5. Incubation with enzymes was carried out at 37° C. for 2 hours. The activity of the samples was then tested with a disc diffusion assay as described in example 2 and compared with that of a control. 
     There was no decrease in the antifungal activity when the 7-day culture filtrate is boiled, kept at room temperature for 7 days or one month or incubated with lipase. This indicated that the peptides are very stable. 
     EXAMPLE 5 
     Solubility. 7-day culture filtrate was freeze-dried and resupended in methanol, ethanol, butanol, ether, chloroform and acetone. Solvent was removed by evaporation, samples were dissolved in water and tested using the disc filter assay described in example 2. 
     The antifungal peptides were soluble in water, methanol, ethanol, butanol and insoluble in ether, chloroform and acetone. Solubility in polar solvent indicated that they are hydrophilic. 
     EXAMPLE 6 
     Precipitation with ammonium sulfate. The 7 th  day culture filtrate was sequentially precipitated with 20, 40, 60 and 80% ammonium sulfate. Using a disk filter assay as described in example 2, only fraction precipitated with 20% ammonium sulfate exhibited antifungal activity against Aspergillus flavus (FIG.  3 ). 
     EXAMPLE 7 
     PI of the antifungal peptides. To characterize the antifungal peptides, the fraction precipitated with 20% ammonium sulfate was further separated with isoelectro focusing (IEF) tube gel electrophoresis. For this purpose, 1 mg of protein sample was loaded on a tube gel containing ampholytes (pH 2-8) and after electrophoresis gels were cut in 1 cm pieces. Each piece (total of 15) was placed on an agar media that was already inoculated with the fungus. FIG. 4 represents the result of an antifungal assay against  Aspergillus flavus  after separation of the Bacillus proteins depending on their pI. 
     EXAMPLE 8 
     Purification of the antifungal peptides. Bacterial cells were removed by centrifugation after 7 days of culture and proteins were precipitated with ammonium sulfate. After centrifugation, the pellet was dialyzed against water, acidified to pH 3 with 12N HCl and centrifuged. After resuspending in water and adjusting pH to 7, pellet was loaded on a 5 ml anion exchange column (HiTrap Q pharmacia). The column was connected to an FPLC system (ConSep LC 100, PerSeptive Biosystems) and a linear gradient of 0 to 1.6M NaCl including 20 mM phosphate buffer pH 7 was applied. 5 ml fractions were collected, tested for antifungal activity using a filter disk assay as described in example 2 and dialyzed against water. All the fractions with antifungal activity were pooled together and loaded on a preparative superdex peptide hi load 16/60 column (Pharmacia). The antifungal fractions were separated twice on this column until the antifungal peaks were fully separated (FIG.  5 ). 
     EXAMPLE 9 
     Molecular weight of both antifungal peptides. The molecular weights of both peptides were determined by FAB mass-spectrophometry. The spectra for the antifungal peptide a1 indicated a mass [M+H] of 1045.5 and for the antifungal peptide a2 1059.5 (FIG.  6 ). 
     EXAMPLE 10 
     Amino Acid sequence of A1 and A2. Initial Edman sequencing was unsuccessful and tandem mass spectrometry of the molecular ion resulted in a highly complex spectrum which suggested a cyclic structure for both peptides. 
     A1 was digested to give a linear molecule and the sequence after digestion at the Glu—C amino acid was: NH2- STNYNPE-OH. A modification of the molecule at the Thr residue suggested the attachment of hydroxy fatty acid. A1 has the same amino acid sequence and mass than one of the bacillomycin D (21). 
     Tandem mass spectrometry of the cyclic form of A2 indicated a sequence very similar to A1 with an additional CH2. A2 has an amino acid sequence similar to bacillomycin D but its mass is higher m/z 1059.5 than the one published by Peypoux et al. (20) (m/z 1031 and 1045). 
     EXAMPLE 11 
     Antifungal activity of the crude fraction. The crude fraction included all the protein secreted in the culture filtrate of  B. subtilis  AU195 culture and precipitated with 20% ammonium sulfate. Both antifungal peptide a1 and a2 were present in the crude fraction in a mixture of 50/50. Antifungal activity of fraction obtained after ammonium sulfate precipitation was carried out under sterile conditions using the disc plate diffusion assay described in example 2. Mycelium plugs from actively growing cultures were placed in the center of a petri plate containing PDA for  Alternaria solani, Fusarium monoliforme  and  Pythium aphanidermatum.  After incubation at 27° C. to allow vegetative growth, samples were applied on sterile filter paper disc laid on the agar surface. The crude fraction is able to inhibit hyphal growth of  Alternaria solani, Fusarium monoliforme  and  Pythium aphanidermatum  (FIG.  7 ). 
     EXAMPLE 12 
     Antifungal activity of the purified peptides. After complete purification of the antifungal peptides as described in example 8, removing salt by purification on a Sepack cartridge (waters) and freeze drying, both peptides were quantified by weight. Table IV shows the result of the antifungal assay. The antifungal assays are described in example 2. 
     
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE IV 
               
               
                   
                   
               
               
                   
                 Disc plate diffusion assay 
                 Microtiter plate 
               
               
                   
                 Minimal amount to inhibit 
                 Minimal inhibitory 
               
               
                   
                 hyphal growth 
                 concentration (MIC) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Iturin A 
                 No inhibition with 50 μg 
                 250 μM 
               
               
                   
                 a1 
                 15 μg 
                 Not tested 
               
               
                   
                 a2 
                 3 μg 
                 130 μM 
               
               
                   
                   
               
             
          
         
       
     
     In a similar manner the purified peptides each one individually or in a mixture (40/60) control the growth of  A. parasiticus.    
     When peanut plants are treated with the compositions of Examples 11 and 12, the plants are expected to show improved resistance to  A. flavus.  Similarly improved resistance to  A. flavus  is expected by corn plants. 
     EXAMPLE 13 
     Antimicrobial activity of the crude fraction 3 or 5 ml of bacterial cell suspension (from 10 4  to 10 8  cells/ml) was widespread at the surface of LB plate and the excess was withdrawn. After 15 min dessication of plates under the hood, 4 discs were distributed containing different amount of the ammonium sulfate precipitate fraction. Plates were incubated for 48 hours at 27° C. and the inhibition zone was observed after 24 and 48 hours. Results are given in Table V for all the strains tested. 
     
       
         
               
               
             
           
               
                 TABLE V 
               
               
                   
               
               
                   
                 Antimicrobial activity of the ammonium 
               
               
                 Bacteria tested 
                 sulfate precipitated fraction 
               
               
                   
               
             
             
               
                 Escherichia coli 
                 − 
               
               
                 Clavibacter michiganensis 
                 ++ 
               
               
                 Pseudomonas fluorescens 
                 − 
               
               
                 Xanthomonas campestris 
                 + 
               
               
                 Erwinia carotovora 
                 − 
               
               
                 Bacillus subtilis AU195 
                 ++ 
               
               
                 Bacillus cereus 
                 ++ 
               
               
                   
               
               
                 −, no inhibition;  
               
               
                 +, poor inhibition;  
               
               
                 ++, good inhibition  
               
             
          
         
       
     
     All publications referenced herein are hereby incorporated by reference in their entirety. The invention is not limited to the embodiments described herein, but encompasses modifications with the scope of the following claims and equivalent thereof. 
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