MXPA99004568A - Biological control of plant fungal infections - Google Patents

Abstract

The present invention is directed to a unique strain of Bacillus subtilis for inhibiting growth of plant pathogenic fungi and bacteria and methods for treating or protecting plants from fungal and bacterial infections.

Description

BIOLOGICAL CONTROL OF FUNGIC INFECTIONS IN PLANTS FIELD OF THE INVENTION The present invention relates to a strain of B. subti l i s to inhibit the growth of pathogenic fungi in plants, and methods to protect a plant against bacterial and fungal infections.

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BACKGROUND OF THE INVENTION Microbial infections of plants, ie infections due to fungi, bacteria or viral agents, is a significant agricultural problem, often resulting in pronounced loss of quality and use of crops.

Plants are susceptible to attack by a variety of phytopathogenic fungi. A phytopathogenic fungus that particularly harms the plant Botrytis cirenea Pers, and plant diseases caused by Botrytis sp. are some of the most widespread and common diseases of crops grown in greenhouses, fields, vegetables, ornamentals and fruits throughout the world. A species of Botrytis, B. Cinérea, is the agent that causes several severe fruit diseases, including gray strawberry mildew (Fragaria ananassa Duchesne) and grapevine (Vitis vinifera L.) (Agrios, 1988). The diseases related to Botrytis cause losses not only in the field, but also in storage, transit and in wholesale and retail.

Other pathogenic fungi to plants include Fusarium oxysporum, which causes numerous plants to wilt, Sclerotinia sclerotiorum, which causes drying of sclerotium, and Rhizoctonia solani, which causes seed wetting and root disease. Additional genera of phytopathogenic fungi include Aspergillus, Penicillium, Ustilago and Tilletia.

The control of phytopathogenic fungi is of significant economic importance, since the growth of fungi in plants or parts of plants (for example, seeds, fruits, flowers, foliage, stems, tubers, roots, etc.) can inhibit production of foliage, fruits or seeds, as well as reducing the quality and quantity of the crop.Although many crops are treated with fungistatics or agricultural fungicides, fungi that damage agricultural crops typically result in losses to the agricultural industry of millions of crops. dollars annually.

Current forms of control against fungal diseases of plants, include creating crops to improve fungal resistance, cultural control, or the application of compounds or organisms that are toxic or another form of antagonism to pathogens or expression _of symptoms of the disease.

These approaches have several problems that limit their effectiveness. For example, genetic resistance is often lacking in the desired cash crops, while cultural methods, such as canopy management and reduction of plantation density, are labor intensive and of limited efficiency. The application of synthetic fungicides to control plant diseases by fungus could be expensive and have associated health risks.

The continuous economic toll taken by phytopathogenic fungi suggests a need to develop new more effective approaches to prevent fungal infection in plants. Additionally, these requirements should be met without significant adverse side effects to plants and the environment, and without severely restricting planting and growing conditions, or requiring costly chemical treatment of plant growth or fruit harvest.

BRIEF DESCRIPTION OF THE INVENTION The present invention is based on the discovery of a new strain of Ba ci l l us sub ti l i s, designated here as B. subti l i s strain ATCC No.55614, which is effective in inhibiting the growth of pathogenic bacteria and fungi in plants. The invention also encompasses a method "for protecting a plant against fungal or bacterial infections by applying to the plants or their environment B. subtili strain ATCC 55614, a supernatant obtained from a culture of the isolate, or a bioactive extract thereof.

In one aspect, the invention is directed to a biological culture of B. sub ti l i s strain ATCC 55614. Several incorporations include a bioactive extract of B. subti l i s strain ATCC 55614, a supernatant obtained from a culture of the isolate, and a composition containing one or more of the above. The composition could contain B. sub strain the strain ATCC 55614 in an impure state or in the form of a biologically pure culture, and is effective in inhibiting the growth of fungi and bacteria. In a particular embodiment, the composition is effective to inhibit the growth of Bo tryti s cinerea. In yet another embodiment, the composition is effective to inhibit the growth of Fusarium.

The present invention is also directed to a method for producing an extract of B. Subtypes that have antimicrobial activity. In a particular embodiment of the method, the extract is isolated by extracting the culture medium of bacterial isolate ATCC 55614 to produce a crude extract, separating the crude extract on a solid support to produce separate fractions, screening the separated fractions for antifungal or antibacterial activity. , and joining the active fractions. In yet another aspect, the invention provides a method for protecting a plant against fungal or bacterial infection by applying a composition containing Bacillus subtilis strain ATCC 55614, a supernatant or an extract thereof to a plant or its environment.

In one embodiment, the infectible surfaces of a plant susceptible to fungal or bacterial disease are covered with Bacillus subtilis strain ATCC 55614, a supernatant or an extract thereof. The invention thus provides a method for protecting plant against infection caused by various phytopathogenic fungi, e.g. ex. Diphtheria, Dreschslera, Fusarium, Geotrichum, Sclerotinia, Sclerotium, Erysiphe, Podosphaera, Uncinula, Puccinia, Plasmopara and Stemphylium.

In yet another embodiment, a method is provided for inhibiting the infection of Botrytis cin rea or Fusarium in a plant. Related embodiments include a method for inhibiting the growth of the vegetative hypha, and for inhibiting the formation of sclerotium by B. cirenea or Fusarium, by applying a composition containing Bacillus subtilis ATCC 55614 or an extract thereof, for a plant or its environment . A method is also provided to inhibit the conidium germination of B. cinerea or Fusarium.

The methods of the invention are useful to protect plants that have fruits, vegetables, floral plants, and their associated post-harvest crops, against fungal or bacterial infections.

These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES Figures 1A and IB are computer generated photographs of PDA plates containing B. cinerea alone (Figure 1A, negative control), and B. cinerea in combination with isolated B. subtilis ATCC 55614 (Figure IB).

Figs. 2A and 2B show photomicrographs generated in conidium germination computer on a PDA control plate (Fig. 2A) and on a PDA plate containing a stria of B. subtili s ATCC 55614 (Fig. 2B).

Figs. 3A and 3B show the percentage of total crop weight due to diseased fruit (y-axis), at the time of harvest (Fig. 3A), after storing at 40 ° C for 7 days under 95% RH ( Fig. 3B), in crops 1, 2 and 3 (x-axis) for fruit that received several treatments (indicated); Figs. 4A and 4B show the healthy fruit weight (y-axis), at the time of cultivation (Fig. 4A), and after storing at 40 ° C for seven days under 95% RH (Fig. 4B), at crops 1, 2 and 3 (x-axis) for fruit that received several treatments (indicated); Y Figs. 5A and 5B are computer generated photographs of PDA plates with Fusari um sp. , and a commercial biocontrol agent, MYCOSTOP (Streptomyces gri seoviridi s strain 61) and Fusari um sp. and B. subtili s strain ATCC 55614, respectively.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions "B. subtili strain ATCC 55614" includes mutants and variants thereof, mutants and variants particularly effective for inhibiting the growth of pathogenic fungi produced in soil or air of the Deuteromycota division (eg, Bo tryti s sp.) .

The microorganism, p. ex. B. subtype ATCC strain 55614 can be used in an impure state in combination with other materials that will not substantially interfere with the disease-suppressing characteristics of phytopathogenic fungi of B. sub tili s strain ATCC 55614, or in the form of a biologically pure culture.

By a "biologically pure culture" is meant a culture of the microorganism that does not include other materials (i) that are normally found in the soil in which the microorganisms grow, and / or (ii) from which the microorganisms are isolated.

The term "cultivar" refers to the propagation of organisms on or in the middle of various types. "Complete broth culture" refers to a liquid culture containing cells and medium. "Supernatant" refers to the liquid broth that remains when the cells growing in broth are removed by centrifugation, filtration, sedimentation or other means well known in the art.

As used herein, "biological control" is defined as the control of a pathogenic organism by the use of a second organism. The known mechanisms of biological control include enteric bacteria that control the decomposition of the root due to the competition of fungi for space on the surface of the root. The bacterial toxins, such as antibiotics, have been used to control pathogens. The toxin can be isolated and applied directly to the plant or the bacterial species could be administered to produce the toxin in itself.

A "bioactive" extract of B. Subtil i strain ATCC 55614 is one that possesses the antimicrobial properties of B. sub ti l i s strain ATCC 55614, p. ex. , is able to inhibit the growth of a microorganism. B. sub ti l i s strain ATCC 55614 inhibits the growth of bacteria and fungi.

A "microbial suppressant amount" of B. subtili s strain ATCC 55614 or an active component produced in this way is an amount sufficient to suppress the growth of a phytopathogenic microorganism by at least about 50% compared to microbial growth for an untreated plant. Preferably, the microbial suppressant amount will be sufficient to suppress from about 60-80% of the growth of fungi or bacteria that occur on an untreated plant, and will depend on various factors such as soil condition, climate, type of plant, conditions of planted and the like.

An "effective amount" is an amount sufficient for beneficial effects or desired results. An effective amount can be administered in one or more administrations. In terms of treatment and protection, an "effective amount" is enough to mitigate, improve, stabilize, reverse, slow down or delay the progress of the state of the fungal or bacterial disease. Plant, as defined herein, includes any and all portions of a plant, including the root system, the shoots, including the stem, nodes, internodos, petiole, leaves, flowers, fruits, and the like before and after harvest. Plant also means including any cellular derivative of a plant, including undifferentiated tissue (eg, calluses), as well as seeds, pollen, pro-glands and embryos.

By "flowering plant" any angiosperm is handled. Examples of angiosperms include almost all plants that have been domesticated for agriculture, such as wheat, corn, beans, rice, oats, potatoes and soybeans as well as ornamentals.

"Ornamental flowering plants" is managed to include ornamental flowering plants such as orchids, petunias, zinias, asters, begonias, geraniums, lilacs, African violet and rose.

"Plants that bear fruit" includes plants that produce fruits such as strawberry, raspberry, grape, tomato, pepper, cucumber, pumpkin, melon, sweet melon, watermelon, apple, peach, plum, nectarine, pear, peach, custard apple, banana, Avocado, currant, persimon, papaya, mango, guava and kiwi. A "vegetable plant" is a plant that produces vegetables; Examples of such plants include beans, beets, carrots, potatoes, spinach, celery, broccoli, cauliflower, cabbage and lettuce.

II. Isolation and identification of B. subtilis strain ATCC 55614 The present invention is based on the discovery of a single strain of B. subtilis, a bacterium that forms "spores, aerobic, flagellate, which exhibits potent antifungal and antibacterial properties against a wide range of filamentous and non-filamentous bacteria and fungi. The isolation and identification of B. subtilis strain ATCC 55614 will now be described.

A. Source of B. subtilis strain ATCC 55614 and Preliminary Screening for Antifungal Activity The microorganism of the present invention,. a strain of Bacillus subtilis, can be obtained from soil or plant hosts. The preferred sources of the bacteria are rhizosphere and root tissue. of a raspberry plant. A particularly preferred vegetable host is the cultivation of raspberries, cv Meeker. By way of example, the collection and isolation of bacterial samples by antifungal screening, which led to the discovery of the only Baclillus strain described here, is presented in example 1.

Once a plant host and / or soil sample is obtained, the bacteria associated with the sample are isolated and cultured as follows. The sample is typically washed with sterile water, soaked in phosphate buffered saline (PBS), or sterilized surface. In cases where the bacterium is obtained from the plant tissue, the tissue is lowered and typically aligned in agar medium, typically with a wire.

A number of different common agar media can be used to grow the bacteria, such as nutrient glucose agar (NDA; Difco, Detroit, MI), yeast-dextrose-calcium carbonate extract, yeast extract broth nutrient agar (NBY) ), and B agar with King medium (KB), potato dextrose agar (PDA), receptors for this are provided in Schaad (Schaad, 1988, page 3). Colonies typically appear in the middle in about 1 - 5 days.

These colonies are then tested for antifungal and antibacterial activity, to identify colonies that produce compounds that have antimicrobial activity. Any of a number of preliminary screening tests can be employed to determine antifungal activity for bacterial colonies. Such a test, referred to herein as a stria test, is conducted by first striating simple colonies of bacterial isolates on suitable agar medium, such as PDA. The sample is incubated for approximately 2-5 days, followed by the addition of a mass of pathogenic fungi to the previously incubated culture, at a specified distance from the bacterial striae. The resulting culture is then examined for areas in which the growth of pathogens is inhibited. A representative fungal pathogen against which isolates can be screened is Bo tryti s, p. ex. , Bo tryti s ci renea. Additional examples of fungal pathogens against which antifungal activity may be preliminarily evaluated, such as by spot test, include Fusari um, Dipl odia, Drechsl era, Fusari um, Geo tri ch um, Scl ero tina, Scl ero ti um, Erysiph e, Podosphaera, Uncinula, Puccinia, Plasmopara and Stemphyl i um.

By way of example, the screening of bacterial isolates obtained as described above resulted in the initial identification of several isolates capable of inhibiting the mycelial growth of B. cinerea (Example 2). The tests were carried out in two different media, papa dextrose agar (PDA) and 25% tryptic soy agar (TSA). About 1211 isolated bacteria tested, 12 were identified as inhibitors of mycelial growth of B. cin rea by at least 50% compared to the growth of B. cinerea under control conditions.

In addition, the screening experiments to evaluate the antagonistic effect of each of the 12 isolates on another fungal pathogen, Fusari um sp. , only 1 of the isolates identified as inhibitor of B growth. The area severely limited the growth of Fusari um sp.

Based on its ability to severely inhibit the growth of B. In the middle between PDA and 25% TSA, and its ability to effectively inhibit the growth of Fusarium, this isolate, called strain ATCC 55614, was chosen for further evaluation. The results of relevant in vi tro screening tests are described in Examples 2 and 3. To summarize the results presented here, strain ATCC 55614 was effective in inhibiting the growth of the fungal pathogen Bo tryti s cinerea by approximately 70% in the media. PDA and TSA compared to untreated controls. Another screening test that can be used to identify effective isolates to inhibit the growth of several pathogenic fungi is a spot test or overlap test (Gross and Devay, 1977; Gross, et al., 1977). Conducting an overlay test, cells from a single colony are stained on agar-containing plate (eg, PDA) and incubated as described above). The colonies are then typically removed with a sterile sponge and the plate is exposed to chloroform vapors for an extended period of time (eg 20 minutes) followed by dissipation of the chloroform vapors to kill the remaining bacterial cells. To identify "an isolate that possesses antifungal activity, the plate is sprayed with a spore suspension of fungal pathogen, and then examined for areas in which the growth of the fungal pathogen is inhibited." Representative fungi against which antifungal activity can preliminary assessment, such as by spot test, include but are not limited to those described above, eg 'Botryti s, Dipl hate, Dreschslera, Fusari um, Geotri chum, Scl erotinia, Scl ero ti um and Stemphyl i um. A bacterial isolate is considered to be antagonistic against the growth of a given microbial pathogen if the growth of the target phytopathogenic microbe is mitigated, improved, stabilized, reversed, slowed down or delayed, and preferably, if the progression of the fungal disease state or bacterial is inhibited by at least about 50 percent compared to microbial growth in the absence of the bacterium.

After identifying a culture that is active in spot tests (eg, ATCC 55614 isolated), then the bacterial strain is further characterized. Based on a number of diagnostic tests described in Example 4, the bacterial isolate ATCC 55614 was characterized as Ba ci l us subtili s. Diagnostic tests to determine the identity of a bacterial isolate are described in full below.

B. Characteristics of B. sub ti li s strain ATCC 55614 The bacterium that belongs to species B. subtilis has a number of characteristics, which have been described in detail in Schroth, et al. (1983) and in Schaad (1988) and are representative of strain B. subti l i s strain ATCC 55614.

The characteristics of B. Subtili s are briefly described below. 1. LOPAT test. LOPAT tests (yeast formation, Oxidase tests, potato decomposition capacity, Arginine dehydrolase production, and hypersensitivity to tobacco) (Schaad, 1988) are used to determine a number of identification characteristics of a bacterial isolate. The bacteria that belong to species B. subtili s will present, globally, LOPAT features as described in Schaad. 2. Additional Diagnostic Tests. Once an isolate has been preliminarily identified as B. sub ti lis (eg, by means of LOPAT tests), a positive identification is made by submitting the isolate to additional diagnostic tests according to conventional diagnostic test protocols common in the art, as described in FIG. Schaad (1988).

Typically, the secondary test is carried out afterwards, the results of which are based on an ability of the isolate to utilize a particular substrate or to form a particular product. By way of example, the identity of the ATCC isolate 55614 was confirmed by GC-FAME and Biolog MicroPlateÓ analysis (Biolog-Hayward, CA), which provides a standardized micro-test method that uses 95 different tests to -identify a broad range of enteric bacteria , non-fermenting, Gram-negative. The microplate test method examines the ability of the bacteria to use or oxidize a preselected panel of different carbon atoms, as described in GN MICROPLATE INSTRUCTIONS FOR USE, BIOLOG, Hayward, CA September 1993.

Based on these test results, strain of bacterial isolate ATCC 55614 was identified as B. sub ti l i s, as described in Example 4. B. sub ti lis ATCC 55614 has been deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852, and assigned the following designation, ATCC 55614. The deposit was accepted by the International Depositary Authority on 21 September 1994 3. Impact on Manifestations of Fungal-Induced Disease. The experiments carried out in support of the invention indicate that B. subtili s strain ATCC 55614 is effective in inhibiting not only growth of vegetative hypha, but also in inhibiting the formation of sclerotium, e.g. ex. , by B. cyrene (Examples 3 and 5, respectively).

Referring to the results in-vi of examples presented in Table 2 of Example 5, column 2 (Rank #), the absolute number of sclerotium of a sample of B. sub tili s strain ATCC 55614 treated was greatly reduced from an untreated control sample (non-bacterial treatment). Based on the average values of column 3, an approximate 75-80% reduction in sclerotium growth was observed in samples treated with B. subtil i strain ATCC 55614 when compared to control samples lacking the bacterial antagonist.

To determine whether the reduction was simply due to a reduction in vegetative mycelial growth, the results were also calculated as a ratio of mycelial growth to sclerotium number. If the reduction in sclerotium for the treated sample was simply a result of a reduced number of mycelium, the relationships for the control sample and treated with B. subtilis should be expected to be similar. Based on the average values presented in column 5 of Table 2, the ratio for the treated sample is greater than for the corresponding control. This result indicates a decrease in sclerotium for a given number of mycelium (smaller denominator), and thus a great value for the complete relation in comparison to the control. These results indicate that the isolated Bacillus of the invention is effective in inhibiting not only the growth of the vegetative hypha, but also in inhibiting the formation of sclerotium, e.g. ex. , by B. cinerea Additionally, the isolate of B. subti l i s ATCC 55614 is effective in inhibiting the germination of conidium and of a fungal pathogen, for example B. cinerea, as supported by the illustrative results presented in "Example 6. Conidio, which is produced by mycelium, is the main source of inoculum in the field." The conidio (or spore) lands on or near a plant, germinates to produce mycelia, which then produce conidioespores with conidium, which leads to the growth of the fungi and to the development of the disease.As described in detail in Example 6, the conidia of Biosphere were isolated and used for To prepare a conidial suspension, the suspension was added to agar plates with and without B. subtius ATCC 55614, followed by incubation.The degree of germination was then evaluated by means of a light microscope (Figs 2A and 2B). they are summarized in Table 3. Compared to the photomicrographs presented in Figs 2A and 2B, it can be seen that germination was inhibited by approximately 85 percent in the presence of B. subti lis ATCC 55614, in comparison to a control sample.

III. Isolation of a Bioactive Extract from B. subti l i s strain ATCC 55614 A. Separation of the Bioactive Component As discussed above, the composition of the invention could contain B. subti l i s strain ATCC 55614, a culture supernatant or an extract. In compositions containing an extract of B. subtili s strain ATCC 55614 or a bioactive extract produced by the isolate, the isolate is typically at least partially purified from cellular components, to provide an extract having antimicrobial activity as described in section II above.

A bioactive extract produced by B. subti li s strain ATCC 55614 is obtained in general as follows.

Typically, B. subtil is strain ATCC 55614 is grown in potato dextrose broth, or Antibiotic Medium 3 (Difco Laboratories (Detroit, MI) as stationary cultures over a period of several days.The culture medium is then extracted with a suitable solvent, eg ethyl acetate, chloroform, acetone, to remove various contaminating materials and obtain a bioactive extract.The resulting bioactive extract can be further separated by means of any of a number of separation techniques, such as adsorption chromatography. Suitable solids for separations based on chromatography include supports for gel filtration such as DEAE, C-18, for reverse phase applications, and XAD-type nonionic supports.The fractions recovered, are tested for antifungal activity, such as by tests of stain as previously described Fractions that exhibit bioactivity are then collected, and could be concentrated in dry form.

The resulting semi-purified preparation containing a bioactive component could then be used directly for the treatment of fungal or bacterial infection, such as in topical applications (eg to inhibit the growth of fungi and bacteria in plants).

IV. Composition sue Contain B. subtili s ATCC strain 55614 A. Antifungal Formulations The compositions containing B. subtili s strain ATCC 55614 could contain the isolate (i) in an impure state (eg, culture broth) in combination with other materials that will not adversely impact characteristics that suppress the phytopathogenic disease of the isolate, nor adversely affect health of the middle plant or the surrounding medium, (ii) as a biologically pure culture, (iii). a supernatant, or (iv) as an extract. The composition could include mutants and variants of bioactive component of B. subtype ATCC strain 55614 or a mixture thereof, particularly mutants and variants exhibiting features that suppress phytopathogenic disease that are substantially the same or improved over B. subtle strain ATCC 55614. To prepare such a composition, the subtype strain ATCC 55614 is grown using conventional techniques as previously described. The cultivation of B. sub ti l i s can be carried out in liquid nutrient medium or in solid at a temperature of about 220-23'OC. If it is desired to limit the quantities of microorganism, surface cultures and bottles could be used.

The microorganism is typically grown in a nutrient medium containing a carbon source, such as an assimilable carbohydrate, and a nitrogen source, e.g. ex. , an assimilable nitrogen compound or protein material. Representative carbon sources include glucose, brown sugar, sucrose, starch, lactose, and the like. Representative nitrogen sources include yeast, soybean meal, corn flour, milk solids, and the like. Trace metals (eg, zinc, magnesium, cobalt, iron) could optionally be added to the fermentation medium.

If a large quantity of microorganisms is required, a vegetative inoculum is prepared in a culture of nutrient broth, e.g. ex. , inoculating the broth culture with an aliquot of soil, root tissue, or slope culture, and aseptically transferred to a large container or tank. The optimal amounts of Bacillus endospores subtypes of strain ATCC 55614 employed in the plant treatment composition herein for a particular application can be readily determined by those skilled in the art. In general, the portion of the active ingredient of a composition according to the invention may contain from about 0.001% to about 50% by weight, and preferably from about 0.01% to about 30% by weight, of Bacillus subtilis s endospores. .

The composition of the present invention could contain one or more biologically inert components, e.g. ex. , carry materials such as talc, gypsum, kaolin, attapulgite, wood pulp and / or binders such as ethylene glycol, mineral oil, polypropylene glycol, polyvinylacetate, plant growth hormones and nutrients.

The antimicrobial composition can be formulated in a variety of formulations including powder, aqueous, fluidizable, dry fluidizable, or granular applications. The composition could also be microencapsulated. Powder formulations are generally prepared by mixing the dry components together, including any vehicle and / or other additives, until a homogeneous mixture is formed. A binder, if employed, could be added later and the whole mass mixed again until it becomes essentially uniform in composition.

A liquid formation could, for example, include organic solvents- such as xylene, methanol, ethylene glycol and mineral oil. Additional components could include active surface agents, e.g. ex. , calcium dodecylbenzenesulfonate, polyglycol ether, ethoxylated alkyl phenol or alkyl aryl sulfonates. Alternatively, the liquid formulation could be an aqueous base suspension. Such formulations will typically contain 106-109 colony forming units (CFU) of B. sub ti l i s strain ATCC 55614 per mL of aqueous vehicle. Also for that. contain water, an aqueous vehicle could optionally contain a wax such as paraffin wax. For granular applications, the vehicle can be an inorganic or organic material. Representative examples include attapulgite, montmorillonite, bentonite, wood pulp, starch, cellulose, bran, etc. The formulation could also include a binder such as mineral oil, lignosulfonate, polyvinyl alcohol or sucrose, to maintain granular integrity.

In addition, the composition of the invention can be used together with one or more additional fungicidal or pesticidal materials Examples of insecticides for inclusion in the composition are, for example, organochlorinated components such as lindane (1,2,3,4,5, 6-hexachlorocyclohexane (gamma isomer)), organophosphoric esters such as diazinon (0, 0-diethyl 0-2-isopropyl-6-met ilpyrimidin-4-yl phosphorothiate), isazophos (0-5-chloro-1-phosphorothioate) isopropyl-lH-1,2,4-triazol-3-yl-0, O-diethyl), thiofanox (1- (2,2-dimethyl-1-methylthiomethylpropylidenaminoxy) - N -methylformamide), and the like; carbamates such such as carbofuran (2, 3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbonate), mercaptodimether (3,5-dimethyl-4- (methylthio) phenol methyl carbamate), bendiocarb (2, 2-methylcarbamate) -dimet il-1, 3-benzodixol 4-yl); repellents such as anthraquinone, thioanthraquinone, benzatrone, zira (zinc bis (dimethyldithiocarbamate)) and diphenylguanidine.

In general, the Bacillus subtilis ATCC 55614 endospore component of a composition of the invention will typically retain at least about 50%, and preferably at least about 70% of its original viability after storage of the composition during a period of up to 36 months.

B. Method to Avoid the Fungal or Bacterial Growth The method of the present invention comprises applying to plants an effective amount of the biocontrol composition described herein. The composition can be applied to any portion of a plant including its foliage, fruits, root system, or it could be used as a seed complement. When used as a seed supplement, the composition is generally applied to the seed in a ratio of approximately 125 to approximately 2000 grams, and preferably in a proportion of approximately 250 to approximately "approximately 750 grams per 100 kilograms of seed.

In an approach to prevent fungal or bacterial growth in plants, B. subti li s strain ATCC 55614 is used to cover infectious surfaces of a plant susceptible to disease promoted by fungi or bacteria. For such topical applications, the isolate will typically be formulated as a liquid (for spray applications), aerosol, or powder (to powder infectious plant surfaces) as described above. Alternatively, the strain could be used for systemic treatment of fungal plant diseases, for intake by the root system. In such cases, the isolate is typically formulated as granules, usually for convenience in the application. Granule formulations are typically activated by the application of water, and release of the active component typically occurs over an extended period of time, such as 2-12 weeks. As the following examples demonstrate, the plant protection compositions according to the invention exhibit potent protection against phytopathogenic fungi, particularly Botryti s, Phytoph thora, Ri zoctonia, Al ternarla, Monilinia, and Fusa ri um, and also against Great-negative bacteria, Erwinia.

According to another aspect of the invention, there is provided a method for forming a transgenic plant resistant to attack by fungi or bacteria. The transgenic plant contains a chimeric gene corresponding to the gene attributable to the antagonistic phenotype of B. subtilis strain ATCC 55614. ' The chimeric gene is operably linked to a promoter compatible with the plant effective to drive the expression of the chimeric gene, and encodes an effective product to confer resistance to a fungal or bacterial pathogen (eg, Botrytis, Fusarium) on plant cells Transformed Briefly, by carrying out the method, a genetic locus attributable to the antagonistic phenotype of B. subtilis strain ATCC 55614 is first identified, e.g. ex. , by mutagenesis of B. subtilis. Before mutagenesis, the ability of B. subtilis strain ATCC 55614 wild-type to antagonize the growth of B. cinerea is confirmed. The mutagenesis of B. subtilis is then carried out according to standard protocols. After mutagenesis, the mutants are tested for their ability to inhibit vegetative mycelial growth essentially as described above and in Example 2. Putative mutants deficient in B. cinerea antagonism are then used to identify a genetic locus attributable to the phenotype. antagonistic of B. subti li s ATCC 55614.

In the identification, cloning and sequencing of the gene, the transgene can then be used to form a transgenic plant in which expression of the transgene is effective to confer resistance to a fungal pathogen to the plant (eg, Botrytis, Fusarium). Additionally, the transgene could be introduced into a prokaryotic or eukaryotic organism to form a recombinant organism capable of producing the antifungal compound described above secreted by B. sub ti li s strain ATCC 55614.

V. Utility.

The experiments conducted in support of the invention show that B. subti l i s strain ATCC 55614 - is effective in inhibiting the growth of pathogenic fungi of plants including but not limited to Botryti s, Phyt oph thora, Pseudomonas s, Erwinia, Al ternari a, Tri choderma, Monilinia, Puccinia, Rhi zoctonia, Phythi um and Plasmopara and Fusarium, and is also effective in preventing the development of plant diseases associated with these pathogens.

The bioactive strain can be used to treat plant diseases such as seed flood and root rot disease, vascular wilt diseases, or any disease state caused by attack of one of the pathogens of fungal or bacterial plants described above. (eg, Table 13).

The composition of the invention is used to avoid Bo tryti-related diseases such as gray strawberry and raspberry mold, decomposition of grape clusters, gray mold rot of vegetables such as beans, beets, carrots, and cucumbers, the tip of the lettuce, pepper, tomato and squash, rotten disease of dry eye in apple, and gray mold or burn of numerous ornamental plants such as begonia, geranium, lilac, African violet and rose; The composition can also be used to treat vascular wilt diseases induced by Fusa ri um, such as wilt diseases of tomato, pears, banana and cotton.

The composition and method of the invention could be employed to protect commercial raspberry cultures such as Canby, Chilcotin, Amity, and Willamette against gray mold. These crops are particularly susceptible to damage by gray mold caused by Botryti s. Similarly, the composition and method described herein can be used to protect commercial varieties of strawberries susceptible to attack by gray mold, e.g. ex. Chandler, Pajaro and Selva.

By way of example, greenhouse tests conducted in support of the invention (Example 7) revealed the effectiveness of B. subtil i strain ATCC 55614 to reduce losses in greenhouse strawberry crops (Pajaro variety) due to gray mold caused by B. ci n area The details of the study are briefly summarized below.

In conducting the tests described in Example 7, strawberry plants inoculated with B. nei were subjected to several treatment regimens. The treatment included 5 foliar applications of (i) Rovral 50 VP, a commercially available fungicide used to control mold of Bo tryti fruit in strawberries, (ii) potato dextrose broth, (iii) B. sub ti l i s strain ATCC 55614, and (iv) untreated control series. Plants, including flowers, were treated before inoculation.

The results were evaluated based on a number of factors, including number of healthy fruits, weight of healthy fruit recovered, weight of diseased fruit, and percent of the total weight of the crop attributable to the diseased fruit. Examining the results provided in Tables 4, 5, 6, and 7, it can be seen that_the foliar treatment with a composition containing B. subti l i s strain ATCC 55614 was effective in controlling fruit losses due to gray mold, as indicated by the amount of diseased fruit expressed as a percentage of total crop weight, for treated and untreated fruit (control). Additionally, the treatment with B. subti l i s strain ATCC 55614 does not adversely impact the yield (eg weight) of healthy strawberry fruits. " The antifungal composition described herein can also be applied to grape varieties, such as Chite Riesling and Pinot Noir, Chardonnay, Zinfandel and Chenin blanc to avoid infection of gray mold. All these varieties are susceptible to attack by B. c ren (Pscheidt, 1990, 1991; 1993 PACIFIC NORTHWEST PLANT DISEASE CONTROL HANDBOOK; English, et al., 1989; Gluber, et al., 1987).

The experiments carried out in support of the invention also indicate that the bacterium isolate of the invention is effective to inhibit (i) growth of vegetative hyphae, and (ii) sclerotium formation, e.g. ex. , by B. cinerea (Examples 3 and 5, respectively).

A component of the disease cycle of B. Cinérea in long-lived perennial crops (eg, grapes and raspberries) is the production of sclerotium in winter on dead waste. The sclerotium germinates in the spring and initiates the infection of the new crops of the season (Weller, 1988). Thus, the application of a composition that contains, B. subtype strain ATCC 55614, a bioactive extract or meta bolite thereof, could be used to reduce the source of inoculum in winter on dead, necrotic or senescent tissue.

Other important components of the B disease cycle. Cin rea is the production of conidium. Conidia serves as a primary source of inoculum under field conditions. The experiments carried out to support the invention indicate that the isolate of B. subtilis ATCC 55614 is also effective to inhibit the conidium germination of B cinérea (Example 6). By inhibiting conidium germination, the compositions and methods described herein can be used as a preventive measure to reduce losses related to Botrytis.

Due to the effectiveness to inhibit the conidium gemination, the antifungal composition could also serve as a control for triggered genera of phytopathogenic fungi that are members of the classes Ascomycota and Deuteromycota (imperfect fungi). Representative genera include Verticillium sp. , Fusarium sp. , Macrophomina sp. , Thielaviopsis sp., And genera of fungi that are causative agents of the soft cottony fungus.

Additionally, an antifungal composition comprising an extract produced by B. subtilis strain ATCC 55614 could be used to treat human fungal diseases in which the propagule of disseminated disease is a conidium, for example, Aspergillus sp. , Histoplasma sp. , and Tinea sp.

The following examples illustrate, but in no way attempt to limit the present invention.

MATERIALS AND METHODS Unless otherwise indicated, the chemicals and reagents, p. ex. , potato dextrose agar (PDA), potato dextrose broth (PDB), tryptic soy agar (TSA) and malt extract agar (MEA), were obtained from Sigma Chemical Company, St. Louis, MO.

A. Fungal cultures Pure cultures of Botryti s cinerea (B. cinerea) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, accession number 11542) and were maintained at 25 ° C in PDA.

EXAMPLE 1 Collection / Isolation of a New Bacterial Strain Bacteria were isolated from the rhizosphere and root tissue of raspberry plants (cv. Meeker) located at Washington State University - ARS (Vancouver, "WA).

The soil and root materials were placed in a test tube. Five volumes of phosphate buffered saline (PSB, pH 7.3; Wollum, 1982) were added and the tube vortexed for 1 minute. A series of dilutions (10_1 to 10"8) were made using PSB, one hundred and eleven of each dilution were plated into (petri-containing PDA) boxes (ATCC Media Handbook, 1984) .The plates were incubated at 200C in a growth chamber, which uses a duration of 16 hours a day.

EXAMPLE 2 Bacterial Isolates Screening for Antifungal Activity Simple colonies of bacterial isolates were striated in a straight line approximately 12 mm from the edge of a 100 mm '15mm petri dish (plate) containing PDA. The striated plate was incubated for 2 days in a growth chamber as in Example 1 above. After the 2 days of the incubation period, a mass of 5 mm of hypha of B. Cinérea growing in PDA se. added to each plate about 55 mm from the edge of the bacterial striae closest to the center of the plate. The control plates lacking bacterial striae were similarly inoculated to evaluate the growth of B. neerea in the absence of bacteria. All plates were incubated as in Example 1 above.

Each bacterial isolate was initially tested twice for the ability to inhibit the growth of B. cinerea The putative antagonistic isolates were identified and challenged on PDA plates, as well as on plates containing 25% tryptic soy agar (TSA). The bacterial isolates identified in the initial screening as having fungal activity were subsequently challenged using plates with 10 replicates of each PDA and 25% of TSA, to determine the relative ability of each isolate to inhibit the growth of B. Cinerea.

De-1211 bacterial isolates tested for antagonism against B growth. cinérea, twelve were identified as inhibitors of mycelial growth of B. Cinérea by at least 50% compared to the growth of B. nee under control conditions.

The 12 isolates - which were shown to inhibit the growth of B. cinerea were tested at least partially for their ability to inhibit the growth of Fusarium sp. The tests were carried out essentially as described above for antagonism against B. cinerea, using PDA plates in which a 5 mm hypha mass of Fusarium sp.

Of the 12 isolates, only one (the last one designated as B. subtilis strain ATCC 55614) severely limited the growth of Fusarium. Based on the results of the in vitro test, strain ATCC 55614 was essentially as effective as a known commercial biocontrol agent, "MYCOSTOP" (S treptomyces griseoviridis strain 61) in inhibiting the growth of Fusarium sp.

The results are presented in Figs. 5A and 5B EXAMPLE 3 Effects of Isolates of Hifa Necrosis Of the 12 isolates identified above, 1 was selected for further analysis based on its ability to severely inhibit the growth of B. Cinérea on PDA and 25% TSA plates. This isolate was designated isolated ATCC 55614.

The effects of the isolate on the mycelial growth of B. cin erea was tested as before, and the results are summarized in Table 1, below. The antagonism towards Bo tryti s was evaluated by determining the degree of growth of the fungus, it was reported as the distance of the mass of agar that contained the inoculum. As can be determined in Table 1, larger numbers indicate a higher degree of fungal growth, and therefore a lower degree of antifungal activity.

Table 1 Antagonism in vi tro Growth of Bo trvti s ci n erea1 1. to. Growth tested 7 days after inoculation with a mycelial mass of 5 mm. b. Growth conditions = 200C, period of 16 hours a day. c. Mean and DS calculated for n = 10. 2. The means are significantly different from the control (alpha = 0.01), * Dunnett's test.

The results presented in Table 1 indicate that the ATCC isolate 55614 inhibited the growth of B. cinerea in 71.3% and 69.9% in PDA and TSA plates, respectively.

Figures 1A and IB show computer generated photographs of PDA plates in which they are placed on a B plate. cine and Isolated ATCC 55614 (Fig. IB), and a negative control plate containing only B. cinerea (Fig. 1A). The images were obtained 14 days after the application of a 5 mm hyphal mass of B. cinerea The maintenance of the health of the hypha of B. Cinérea was severely affected by the bacterial isolate. The necrosis of the hyphae first became visible after approximately 5 days and continued in a linear fashion as the hypha closest to the bacterium that became necrotic, followed by necrosis of the hyphal more distal to the bacterial stria. Very little mycelial growth (approximately 3 mm) that had the characteristics of cottony white B mycelium. Healthy area (Fig. 1A) was observed after 14 days on the plate containing the ATCC isolate 55614.

EXAMPLE 4 Characterization of the Isolated and Identification of Species The isolate identified earlier as having activity against B. cinerea was further characterized using conventional methods to identify pathogenic plant bacteria (Schaad, 1988).

Specifically, isolate identification was determined using GC-FAME analysis and the Biolog Microtiter System. The Biolog Microtiter system is based on a capacity of the isolate to use or oxidize a preselected panel of 96 different carbon sources using the GN MICROPLATEÓ test panel (Biolog, Hayward, CA), according to the protocol recommended by the manufacturer (GN Microplated , Instructions for Use, Biolog, Inc., Hayward, CA, 1993). The wells in which no reaction occurred remained colorless; positive results were indicated by the appearance of a purple color. The results of the test were processed using the MICROLOG 30 computer program available through Biolog, Inc. (Hayward, CA), which automatically crosses the purple well pattern reference to an extensive species library.

Based on these tests, the ATCC isolate 55614 was identified as a strain that produces antimicotic from Ba ci l l us sub ti l i s (B. sub ti l i s).

The bacterial isolate of B. subti li s ATCC 55614 has been deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852, and assigned a designation number of ATCC 55614. The deposit was received by the ATCC on September 21 of 1994.

EXAMPLE 5 Sclerotium Test The effect of B was also examined. sub ti l i s ATCC 55614 in the production of sclerotium. The PDA plates (with and without bacterial isolates) were inoculated with a 5 mm mycelium mass containing B. cin rea as described above. The sclerotium was counted 21 days after the inoculation of the PDA plates.

Due to the observation of sclerotium production significantly decreased by B. cine in TSA medium, the experiment was not carried out on TSA plates.

Sclerotia count: Due to the possibility that any reduction in the number of sclerotia counted in the test was a consequence of the reduction in vegetative mycelium growth, the ratio of mycelial growth to sclerotium number was calculated for each condition .

The results of these studies are summarized in Table 2, below. The range (#) indicates the total number of sclerotium present on a plate, while the column with the rank heading represents the growth ratio of mycelium to sclerotium number.

Table 2 Antagonistic Effects on Sclerotium Growth1 1. to. Sclerotium counted 21 days after inoculation of the PDA plate with a mycelial mass of 5 mm. b. Growth conditions = 20 ° C, 16-day period. c. Mean and DS calculated for n = 5. 2. The means are significantly different from the control (alpha = 0.01), Dunnett's test The results show that the bacterial isolate of B. subiilis ATCC 55614 reduced the number of sclerotium compared to the negative control. Furthermore, the inhibition of sclerotium production by B. subtilis can not be a consequence of the reduced mycelial growth, since the growth rate at the sclerotium number is significantly different between the negative control and the B treatment. subtilis (comparing, for example, the values in the last column).

Thus, the in vitro analyzes described here indicate that B. subtilis ATCC 55614 has the ability to inhibit not only the vegetative growth of the hypha, but also to inhibit the formation of sclerotium by B. cinerea EXAMPLE 6 Conidio Test The impact of B. subtilis ATCC 55614 on another aspect of the B disease cycle. cinerea, p. For example, conidio production was also conducted. Conidia serves as the main source of inoculum for disease under field conditions.

The conidium was produced in growing culture B. cinerea on malt extract agar (MEA) for 5 days at 25 ° C with a duration of 12 h day, followed by exposure of the plates to ultraviolet (UV) light for 3 days (Leifert, et al., 1993). The plates were then flooded with 10 ml of double deionized H20 and the conidium was removed from the conidiophores by gently scraping the plates with a rubber guard The liquid was pipetted from the plates and filtered 4 times through a double layer of The resulting suspension was vortexed for 1 minute to separate the conidium into individual propagules, the density of conidium per ml of H, 0 double deionized was determined using a hemocytometer, and the suspension was diluted to reach a reserve concentration of 0.10% conidium / ml. μl of the conidium suspension (approximately 1000 conidia) were placed on PDA plates and incubated for 12 hr in the dark at 250 ° C. Another 10 μl of the suspension was placed on PDA plates in which a stria of B had grown. subti li s ATCC 55614 for 2 days. The conidial suspension was placed 1 cm apart from the bacterial stria and similarly incubated.

Germination was evaluated after 12 hr incubation in the dark at 28 ° C. The plates were observed under a light microscope (100X), and 100 conidia were evaluated in each germination plate. Examples of photomicrographs of conidia germination obtained in this experiment are presented in Figures 2A and 2B. The image of Figure 2A illustrates the complete germination of conidia in PDA control plates. The image of Figure 2B shows an example of inhibition of the germination of conidia that were placed 1 cm apart from the stria of B. subtili s.

The results of these experiments are summarized in Table 3, below.

"TABLE 3 Conidia Germination Tests1 1. Approximately 1000 conidia were placed 1 cm away from a 2-day age stretch of each biocontrol agent in Potato Dextrose Agar. The plates were incubated overnight at 25 ° C. 100 conidia per plate were randomly tested for germination. The experiment was repeated 5 times.

The results in Table 3 demonstrate the effectiveness of B. sub ti l i s ATCC to inhibit the germination of B conidia. cinerea EXAMPLE 7 Greenhouse Testing The greenhouse tests were conducted to determine the effectiveness of Bacillus stilts ATCC 55614 in controlling losses in strawberries grown in the greenhouse (cv. Pajaro) due to the gray mold caused by Botryti s cinerea.

The effectiveness of B. subtili s ATCC 55614 was compared to: (i) a foliar application of PDB alone (negative control), and (ii) Rovral 50 WP ("IPRODIONE", Rhone-Polenc, Paris, France), a commercially available and widely used fungicide for control of mold Bo tryti s of fruit in strawberries.

A. Strawberry Growth Strawberry plants (cv. Pajaro) were obtained as rooted crowns from BHN Research (Watsonville, CA). Plants planted in 6"plastic pots (one plant / pot) were filled with 100% BlackGold organic soil (BlackGold, Inc., Hubard, OR) .The plants were watered, fertilized regularly with Peters 20 fertilizer: 20:20 and they grew up under a cycle of 18 light hours / 6 dark hours.

B Bacterial cultures The simple colonies of B. subtypes ATCC 55614_ were used to inoculate two 2 L flasks containing PDB (1 liter / flask). The flasks were placed on a rotary shaker and grown for 48 hours at 28 ° C, 225 rpm. The cell density was measured by absorbance at 600 nm using a spectrophotometer. Based on this reading, the cultures were adjusted with PDB to a final cell density of approximately 5 x 10 ~ 8 cells / ml.

C. Tr_attacks Treatments included foliar applications of (i) Rovral 50 VP at 1.5 lb / 100 gallons of water, (ii) no treatment (control), (iii) PDB alone, or (iv) Ba ci ll s ubtl l s s strain ATCC 55614 (5 x 10-8) CFU / ml in PDB).

The treatments were administered by aspiring the plants with a manual scavenger containing one of the above compositions. The plants were harvested until drained (approximately 75 ral / plant), treatments (i) to (iv) were applied 5 times during the test, the first application was 10% of the flowering of the flower and the following four in Two weeks intervals after this The fifth treatment was applied one day before the first harvest.

D. Artificial Inoculation in Greenhouse of Strawberry Plants with B. cin rea The plants were inoculated with B. cinerea aspiring to drain with a suspension of 4 X 10 ~ 3 conidia / ml (approximately 75 ml / plant) in three sessions of aspreado. The first aspreado was administered on the day that the first inflorescence was observed, and the next two were administered after this at two week intervals.

Harvest All ripe berries were harvested from all plants, once a week, "for three consecutive weeks, the first harvest was the day after the last treatment.

Sample size The treatments were conducted in "blocks" of 5 plants each and each treatment was replicated 4 times (A, B, C and D), resulting in 20 plants per treatment.

G. Evaluated parameters The following parameters were evaluated after each harvest: (i) number of healthy fruits, (ii) weight of the healthy fruit (g), (iii) weight of the diseased fruit (g), and (iv) percent of the total weight of the fruit the harvest attributable to diseased fruit.

After evaluating the above parameters; the healthy fruits were placed in plastic trays with individual compartments (so that the fruits were not in contact with each other) and the trays were covered with plastic wrap and stored at 4 ° C for 7 days. This treatment of 4 ° C, 95% relative humidity (RH) was to simulate storage conditions in the wholesale / retail distribution chain.

After 7 days of storage at 4 ° C, 95% RH the healthy fruits were evaluated as before by (i) weight of the healthy fruit (g), (ii) weight of the diseased fruit (g), and (iii) percent of the total weight of the crop attributable to the diseased fruit.

The following equation was used to determine the percent of the total weight of the crop attributable to the diseased fruit: (g) sick fruit (T = 0) + (g) ~ diseased fruit (T = l) (g) healthy fruit (T = l) +? 7r = 0 (g) diseased fruit H. Results Examples of results of the greenhouse tests are summarized in Tables 4, 5, 6 and 7, and Figures 3A, 3B, 4A and 4B.

Table 4 shows the percentage of total harvest weight due to diseased fruit at the time of harvest (T = 0), in crops 1, 2 and 3, for control fruit and fruit treated with Rovral or B. subtilis ATCC 55614. These results are also presented graphically in Figure 3A. The y-axis in Fig. 3A shows the percent of crop weight due to diseased fruit in each of the three crops (1, 2, and 3) indicated on the x-axis.

Table 4 Sick Fruit as Percentage of Harvest Weight at the Harvest Moment In the first harvest, the average weight (and per scientist - of the total) of sick fruit treated with Rovral and B. subtilis ATCC 55164 was significantly lower than that of the control treatment. In the second harvest, the results are segregated into two subgroups. The average weight (and percent of the total) of the diseased fruit of the samples treated with Rovral or Bacillus was significantly lower than the weight and percentage of the diseased fruit of the samples that had not received treatment (control), or treated samples only with PDB. Non-significant differences were observed in the two subgroups. In the third harvest, the average percentage of diseased fruit was significantly lower in samples treated with Rovral than in samples that received any of the other three treatments.

Table 5, below, shows the percentage of total weight of the crop due to diseased fruit, after storing at 4 ° C for 7 days under 95% RH (T = 7), in harvests 1, 2 and 3 for control fruit and fruit treated with Rovral or B. subti li s ATCC 55614. These results are also presented graphically in Figure 3B. The y-axis in Fig. 3B shows the weight percent of the crop due to sick fruit in each of the three crops (1, 2, and 3) indicated on the x-axis.

Table 5 Sick Fruit as Percentage of Harvest Weight After 7 Days of Storage (4 ° C, 95% RH) In the first harvest, the average weight (and percent of the total) of sick fruit treated with Rovral or Bacillus was significantly lower than the corresponding measurements of the samples receiving control treatments. In the second harvest, the average weight (and percent of the total) of diseased fruit subjected to PDB and control treatment was significantly higher than the corresponding measurements of the samples that received Rovral or Bail lus treatments. As it was the case of the samples examined immediately after the harvest, the average percentage of sick fruit of the third harvest was significantly lower in samples treated with Rovral than in samples that received any of the other three treatments. Significant non-consistent differences were observed between treatments in fresh and stored analyzed fruits.

Table 6, below, shows the weight of healthy fruit, at the time of harvest (T = 0), in crops 1, 2 and 3 for the control fruit and fruit treated with Rovral, or B. subti li s ATCC 55614. These results are also presented graphically in Figure 4A. The y-axis in Fig. 4A shows the weight of the healthy fruit (in grams) in each of the three crops (1, 2 and 3) indicated in the ej e-x.

Table 6 Weight of Healthy Fruit in Harvest Time In the first harvest, non-significant differences were observed in the average weight of healthy fruit that each of the four treatments received. In the second harvest, the results were segregated into two subgroups. The average weight of healthy fruit of the samples treated with Bacillus was significantly higher than the average weight of healthy fruit of the control, PDB or Rovral treated samples, with no significant differences between treatments in each subgroup. the third harvest, there were no other significant differences.

Table 7, below, shows the weight of the healthy fruit, after storage at 4 ° C for 7 days under 95% RH (T = 7), in crops 1, 2 and 3 for control fruit and treated fruit - with Rovral or B. subtilis ATCC 55614. These results are also presented graphically in Figure 4B. The y-axis in Fig. 4B shows the weight of the healthy fruit (in grams) in each of the three crops (1, 2 and 3) indicated on the x-axis.

Table 7 Weight of the Healthy Fruit After 7 Days of Storage (4 ° C, 95% RH) In the first harvest, non-significant differences in the weight of the healthy fruit were observed between the different treatments. In the second harvest, the control and PDB treatments produced significantly lower healthy fruit weight than the Bacillus treatment, with the Rovral treatment that produced significantly lower fruit weight than the Bacillus treatment. In the third harvest, non-significant differences were observed between the 4 treatments.

Taken together, the above results indicate that Bacillus ATCC 55614 treatments were as effective as Rovral in controlling losses due to gray mold (% of total crop that is of diseased fruit) for the first 2 or 3 crops.

With respect to healthy fruit, Bacillus treatments did not result in significantly lower yields than control or PDB treatments, and Bacillus treatments were not significantly less effective than Rovral treatments.

EXAMPLE 8 Fungicide Activity of ATCC Culture 55614 To determine whether ATCC 55614 was effective against the fungi Phytoph thora infes tans, Pythi um ul timu, Botryti s cinerea, Rhi zoctonia solani, and Al ternaría solani, the following experiments were performed. The petri dishes were filled with agar medium (PDA-potato dextrose agar, Difco). Cultures of the above fungi were grown for three days in YPG-1 liquid medium (0.4% yeast extract, 0.1% KH2P04, 0.05% MgSO4 »7H20, 1.5% glucose). Aliquots of 0.1-0.2 mL of spore suspension (concentration approximately 2 x 106 spores / mL) of pathogen were assed on the agar. ATCC 55614 was grown in potato dextrose broth (PDB) (Difco) for three days. To test the cultures in complete broth, the strain was grown at approximately 1 x 106 to 6 x 106 CFU / mL and the aliquots were taken from these cultures. The supernatant was obtained by centrifugation of the culture density at 5,200 rpm for 20 minutes.

Two 7 mm holes were made in each petri dish. A volume of 100 μL of the test sample (supernatant or full broth) was added to each 7 mm hole. Each test was performed in duplicate. A test sample without microorganisms was added to a control plate. The zone of inhibition, measured in millimeters around each hole, was measured after 3 to 10 days. The results for Phytoph thora, Botryti s, Rhi zoctonia, Al ternaria and Pythi um are shown in Table 8. The control plates do not show zone of inhibition.

Table 8 The same previous experiment was carried out using a different medium (dextrose, bactopeptone, yeast extract, malt extract) and including the gram negative bacterium Pseudomonas s syringae, an important plant pathogen. The results are shown in Table 9.

Table 9 The same experiment was repeated to test ATCC 55614 against the fungus Tri choderma harzi anum, a major fungal pathogen. Tri ch oderma harzian um, strain T-14, was obtained from Campbell. (Davis, CA). A perforated plug of number four was used to make four wells on PDA plates. ATCC'55614 was applied to one of the four wells. Two disks of mycelial mass of Tri ch oderma of the size of the perforated plug of number 'four were added to each plate between two wells on each side of the plate. The results were recorded 24 hours later. The size of the clarified zone between the bacteria and the mycelium was recorded. ATCC 55614 produced a 4 mm zone.

To determine the effectiveness of ATCC 55614 against the gram negative bacteria, Erwinia herbi cola, and the fungus, Monilinia fructi cola, the following experiment was conducted. Bacterial strains were cultured as described above. The cultures of Monilini a fructi col a were grown on V-8 agar (20 g of agar, 4 g of CaCO3, 200 mL of V-8 juice) in the dark at room temperature. The spores were harvested by placing sterile distilled water on the surface of the culture plates and scraping the surface with a sterile needle to dislodge the spores. The spore concentration was adjusted to 3.3 X 106 spores / L and 400 μL was added to 4 mL of soft potato dextrose agar. This mixture was poured on the surface of the potato dextrose agar culture plates and the soft agar allowed to solidify. Erwinia herbi col a was fermented overnight in TSA of medium resistance and 1 X 10? cells were harvested on a TSA agar plate. Using a sterile perforated no. 4, 5 wells were made in each plate and 100 μL of a three day old culture of ATCC 55614 was added to each well. The plates were incubated at room temperature in the dark and areas without growth around each well were measured. The results are summarized in Table 10.

Table 10: Bacterial Inhibition of M. fru c ti col a and E. herbi cabbage EXAMPLE 9 Fungicidal Activity of ATCC 55614 Using Complete Plants The ability of ATCC 55614 to control late aphid infection (P. infes tans) was tested on whole tomato plants. Tomato plants (Ace and Patio varieties) were purchased at the Ace store and transplanted into 6 packages that had three plants per package. ATCC 55614 was grown in Trypticase Soya Broth (TSB) (Difco) for 72 hours and a concentration of 5 X 106 CFU / mL was reached. One plant of each tomato variety was harvested until drained with a full culture broth or supernatant from ATCC 55614 and then air dried at approximately 21 ° C. Two control plants were untreated. All the plants were then harvested until drained with a culture of P. i nfes tans at 1.55 X 105 CFU / mL. The plants were air-dried at 21 ° C, darkened slightly with deionized water, enclosed in a clear plastic bag and incubated at approximately 16 ° C. The number of late aphid infestations was recorded fifteen days after treatment. The results are shown in Table 11.

Table 11 Infection of late aphid and bacterial spot fifteen days after treatment with ATCC 55614 These results show that the strain producing antibiotic in the present invention is effective against late aphids and bacterial blotches.

EXAMPLE 10 Fungal Activity of Supernatant of ATCC 55614 against B. cinerea To test the effectiveness of the supernatant of the bacterial strain ATCC 55614 of the present invention against B. cinerea, the fresh strawberries harvested on the day of testing were used. For test # 1, the frozen supernatant of ATCC 55614 was used. ATCC 55614 was grown in potato dextrose broth as previously described. The supernatants were frozen for 1 to 1.5 months before the test. In test # 2, ATCC 55614 was grown in medium-strength TSB or potato dextrose broth (PDB) and the broth or supernatant was tested without freezing. The cultures of full broth and supernatants were harvested in the strawberries until they were drained, then allowed to air dry.

The spores of B. cinerea were grown on potato dextrose agar in a petri dish and scraped in deionized water to form a liquid inoculum. The inoculum of B. cinerea, which measured approximately 5.8 x 10 5 cells per mL, was sprayed on the berries until it drained, and the berries were allowed to air dry. In test # 1, the berries were placed inside a cardboard container with plastic wrap at 25 ° C. In test # 2, all the berries were placed uncovered in an incubator at approximately 16 ° C. The results are shown in Table 12.

Table 12 The frozen supernatant of ATCC 55614. was completely effective in inhibiting B infection. cin rea in living strawberry plants. In addition, the full broth culture of ATCC 55614 was completely effective in preventing B infection. cinerea, ignoring the medium used. The supernatant of ATCC 55614 grown in TSB was partially effective but, when it grew in PDB, it was 100% effective against B. cinerea EXAMPLE 11 ATCC Activity 55614 Against Fungal Pathogens To test ATCC 55614 against a number of fungal pathogens, the strain was grown in potato dextrose broth. The cells were cultured at 5 x 10 6 cells / mL. Replicates of three test plants and three control plants were used per pathogen. The test plants were each sprayed with a full broth culture of ATCC 55614 until drained with a hand-held burner. When the foliage had dried, each test plant was burned a second time. After the second culture application of the bacterial strain had dried, the test plants and the control plants were inoculated with the appropriate fungal pathogen. The plants were incubated under conditions conducive to the development of the disease. In addition, positive controls were used by testing known pesticides against appropriate fungal pathogens in the same way that the culture of the bacterial strain was tested. Each plant was evaluated by estimating the percent of the control disease using a scale of 0% control to 100% control. (0 = disease level of the untreated control, 100 = plants without visible lesions). The fungal pathogens, resulting diseases, host plant and control pesticides are presented in Table 13. The results are shown in Table 14.

Table 13 Table 14 ? Pi = P. infestans; As = A. solani, Pv = P. viticultural, Un = U. necator, Bc = B. cinerea, Sn = S. nodorum, Pr = P. recondite f. sp. tritici (%) z Disease rate = percent of diseased tissue in untreated inoculated plants.

ATCC-55614 -provided complete "control of B. cinerea, ATCC 55614 was also highly active against the mild cottony fungus of grape and leaf blight, and had slight activity against Phytoph thora in this test.

EXAMPLE 12 ATCC Activity 55614 against brown rot, Moni l i ni fructi cola A 250 mL culture of ATCC 55614 was grown for 3.5 days in PDB as previously described. The peaches were purchased at a local candy store (Safeway) and the surface was sterilized with a 10% Clorox solution, rinsed with deionized water and air dried. The complete fermentation broths of ATCC 55614 (8.7 x 106 CFU / mL) were scaled with a hand held in two peaches until drained (approximately 50 mL by two peaches). The peaches were allowed to air dry. Moni lia spores were scraped from a petri dish and suspended in deionized water for a concentration of 1.09 x 105 spores / mL. The peaches were then sprayed with the spore suspension until it was drained and allowed to air dry. Two peaches were untreated and two peaches were only eaten with Moni l i ni a. The peaches were placed in a polypropylene container in an incubator in the dark at 18 ° C for four to six days. The amount of brown rot in each peach was measured, as shown in Table 15.

Table 15 Inhibition by ATCC 55614 of Monilinia in Peaches At four and six days, ATCC 55614 suppressed the brown rot of Monilinia compared to the untreated controls and peaches with only Monilinia. After six days, the peaches-control were all excessively infected with brown rot, while only one of the peaches of ATCC 55614 showed infection. In addition, infected ATCC 55614 peach had a lesion that was much smaller than those in the untreated or controls with only Moni linia.

Comparison of ATCC 55614 and Inhibition of Chemical Fungicide of Monilinia fructi cola Using the same protocol as described above to grow ATCC 55614 cultures, and spores of Moni l ini a, the effect of ATCC 55614 was compared to the commercially available fungicide Benlate® (benomyl). The results are shown in Table 16.

Table 16 ATCC 55614 and Benlate® Inhibition of Monilinia in Peaches These results show that ATCC 55614 is as effective in controlling Monilinia infection as the commercial fungicide, Benlate®.

While the invention has been described with reference to specific methods and modalities, it will be apparent that various modifications and changes could be made without departing from the invention.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

Having described the invention as above, the content of the following is claimed as property.

Claims (18)

1. "A biologically active culture of Bacillus subtilis strain ATCC Accession No. 55614.

2. A composition, characterized in that it contains Bacillus subtilis strain 55614, a supernatant or a bioactive extract thereof.

3. A method for producing a composition of B. subtilis having antimicrobial activity, characterized in that it comprises: (a) cultivating, in a culture medium effective to support growth of fungal cells, a culture of Bacillus subtilis strain ATCC 55614 (ATCC No. 55614).

4. The method of claim 3, characterized in that it further comprises isolating from the culture medium a bioactive extract produced by the Bacillus strain, subtili s.

5. The method of claim 4, characterized in that it comprises: (i) extracting a bioactive component present in the cell culture medium to produce a crude extract, (ii) separating the crude extract on a solid support to produce separate fractions, (iii) screen the separated fractions for antimicrobial activity, and (iv) collect the active fractions identified in (iii).

6. A method for protecting a plant against fungal or bacterial infection, characterized in that it comprises: applying to a plant or its medium an effective amount of a composition containing Bacillus subtilis strain ATCC 55614 (ATCC No. 55614), a supernatant or extract thereof.

7. The method of claim 6, characterized in that the infection is caused by an organism selected from the group consisting of Botrytis, Fusarium, Phytophthora, Pseudomonas s, Erwinia, Alternate it, Trichoderma, Monilinia, Puccinia, Rhizoctonia, Phytium and Plasmopara.

8. The method of claim 6 or claim 7, characterized in that the supernatant is cooled or frozen before application to the plant.

9. The method of any of claims 6 to 8, characterized in that the supernatant is diluted.

10. The method of any of claims 6 to 9, characterized in that the composition is applied as a wettable powder, granules, aqueous fluid, dry fluid or is microencapsulated in a suitable substance.

11. The method of claim 6, characterized in that the plant is a plant that has fruits.

12. The method of claim 6, characterized in that the plant is a plant of legumes.

13. The method of claim 6, characterized in that the plant is an ornamental flowering plant.

14. A method of claim 11, characterized in that the plant that has fruits is raspberry or strawberry.

15. A method for inhibiting Botryti s cinerea infection in a plant, characterized in that it comprises applying a composition of claim 2 to the plant, in an amount effective to inhibit the growth of Botrytis cin rea.

16. A method for inhibiting the infection of Fusa ri um in a plant, characterized in that it comprises: applying a composition of claim 2 to the plant, in an amount effective to inhibit the growth of Fusarium.

17. A plant treated with the composition of claim 2.

18. The method of claim 6", characterized in that the composition is applied in combination with at least one chemical pesticide.