JP2001503642A - Biological control of fungal infection of plants - Google Patents

Abstract

  (57) [Summary] The present invention relates to unique strains of Bacillus subtilis for inhibiting the growth of phytopathogenic fungi and bacteria, and methods of treating or protecting plants from fungal and bacterial infections.

Description

DETAILED DESCRIPTION OF THE INVENTION                   Biological control of fungal infection of plants Field of the invention   The present invention relates to B. subtilis strains for inhibiting the growth of phytopathogenic fungi, as well as plants. Methods for protecting objects from fungal and bacterial infections. Background of the Invention   Microbial infection of a plant means infection by fungi, bacteria, or viral factors, Serious agricultural problem, causing serious losses in crop quality and availability There are often.   Plants are susceptible to attack by various phytopathogenic fungi. Damaging plants One particular phytopathogenic fungus is Botrytis cinerea Pers, Botrytis sp. . Plant diseases caused by greenhouse crops, farm crops, vegetables, ornamental plants, And the most widespread and common disease on fruits. Botrytis, B.cinere One species of a is a causative agent of some severe fruit diseases, and strawberry (Fragaria 'Ananassa Duchesne) and grape vines (Vitis vinifera L.) ey mold) (Agrios, 1988). Botrytis-related diseases only occur on farms Without storage, in transit, and during targeted wholesale and retail markets Also cause damage.   Other harmful fungal plant pathogens are Fusarlumoxysporu, which makes many plants wilting m, Sclerotinia sclero causing scelerotinia wilt tiorum, and Rhizoctonia sola, which can cause seedling wilt and root rot Including ni. Further genera of phytopathogenic fungi are Aspergillus, Penicillium, Ustila go, and Tilletia.   Plant or plant part (eg, seeds, fruits, flowers, leaves, stems, tubers, and roots, etc.) )) Growth of the fungus on the top inhibits the production of leaves, fruits, or seeds, and is harvested Control of phytopathogenic fungi is significantly less economical as crop quality and yield can be reduced Is important. Most crops use agricultural fungicides or fungicides. Although treated, fungal damage to agricultural crops is typically millions of dollars annually. Bringing lost income to the agricultural industry.   Current control measures for plant fungal diseases are varieties for improved fungal resistance Breeding, cultivation control, or toxicity to pathogens or the manifestation of disease Or the application of an otherwise antagonistic compound or organism.   These approaches have various problems that limit their effectiveness. For example, Genetic resistance is often not sufficient in the desired commercial variety, while Cultivation methods such as canopy management and reduced planting density are labor intensive. It is labor intensive and has limited effectiveness. Controls fungal disease in plants The application of synthetic fungicides for medicine can be expensive and has been associated with health risks. Was.   The constant economic damage paid by phytopathogenic fungi is Suggests the need to develop new and more effective approaches to prevent infection. Sa Furthermore, these requirements are met without significant harmful side effects on plants and the environment. Strictly restrict cultivation and growth conditions, or Without any need for expensive chemical treatments. Should be.                                Summary of the Invention   The present invention relates to a new strain of Bacil, referred to herein as B. subtilis strain ATCC No. 55614. Based on the discovery of lus subtilis. This strain inhibits the growth of plant pathogenic bacteria and fungi. Effective against harm. The present invention also provides a culture of B. subtilis strain ATCC 55614, an isolate thereof. The supernatant obtained from or the biologically active extract thereof to the plant or its environment. Thereby protecting the plant from fungal or bacterial infections.   In one aspect, the invention relates to a biological culture of B. subtilis strain ATCC 55614. Related. Various embodiments include bioactive extracts, isolates of B. subtilis strain ATCC 55614 And a composition comprising one or more of the foregoing. This group The adulteration of B. subtilis strain ATCC 55614 in impure or biologically pure form And is effective for inhibiting fungal and bacterial growth. One feature In certain embodiments, the composition is for inhibiting the growth of Botrytis cinerea. It is effective for In yet another embodiment, the composition is for the growth of Fusarium. It is effective to inhibit.   The present invention also provides a method for producing a B. subtilis extract having antimicrobial activity. About. In one particular embodiment of the method, the extract is isolated from a bacterium. Extracting the cell culture medium of the product ATCC 55614 to produce a crude extract, the crude extract Separating the separated fractions on a solid support to produce separated fractions. Screening for bacterial or antibacterial activity, and pooling active fractions To be isolated. In yet another aspect, the invention relates to Bacillus s ubtilis strain ATCC 55614, or a composition comprising a supernatant or extract thereof, in a plant or plant. Protects plants from fungal or bacterial infection by applying to their environment Provide the law.   In one embodiment, the infectious of a plant susceptible to a fungal or bacterial disease Surface is coated with Bacillus subtilos strain ATCC 55614, its supernatant or extract . Therefore, the present invention relates to Diplodia, Drechslera, Fusarium, Geotrichum, Scle rotinia, Sclerotium, Erysiphe, Podosphaera, Uncinula, Puccinia, Plasmopara And infections caused by various phytopathogenic fungi such as Stemphylium Methods for protecting plants are provided.   In yet another embodiment, a Botrytis cinerea or Fusarium infection in a plant Are provided. A related embodiment is a Bacillus subtilis strain ATCC By applying a composition comprising 55614 or an extract thereof to a plant or its environment Thus, methods for inhibiting hyphal growth and B. cinerea or Fusarium And methods for inhibiting the formation of sclerotia. Also, B.cin A method is provided for inhibiting the germination of conidia of erea or Fusarium.   The method of the present invention relates to fruit plants, vegetable plants, flowering plants, and related plants. It is useful for protecting harvested crops from fungal or bacterial infection.   These and other objects and features of the present invention are described below in conjunction with the accompanying drawings. It will be more fully understood when reading the detailed description of the invention.                             BRIEF DESCRIPTION OF THE FIGURES   FIGS. 1A and 1B show PDA plates containing only B. cinerea (FIG. 1A; negative). Control), and P containing B. cinerea in combination with the B. subtilis isolate ATCC 55614. It is a computer-generated photograph of a DA plate (FIG. 1B).   2A and 2B show conidia germination on control PDA plates (FIG. 2A) Germination on PDA plates containing streaks of B. subtilis ATTC 55614 ( FIG. 2B shows a computer-generated micrograph of FIG. 2B).   FIGS. 3A and 3B show the harvest of the variously treated fruits (shown) 1, 2, and 3. Diseased fruit at harvest (FIG. 3A) and 95% RH at 3 and 3 (x-axis) Below, the diseased fruit after storage at 40 ° C. for 7 days (FIG. 3B) The percentage of total harvest weight (y-axis) is shown.   FIGS. 4A and 4B show the harvested fruits 1,2, and 4 with various treatments (shown). And 3 (x-axis) healthy fruits at harvest (FIG. 4A) and under 95% RH 4 shows the weight (y-axis) of healthy fruits (FIG. 4B) after storage at 40 ° C. for 7 days.   Figures 5A and 5B show Fusarium sp. And commercial biological controls, respectively. PDA play with MYCOSTOP (Streptomyces griseoviridis61 strain) And PDA plated with Fusarium sp. And B. subtilis strain ATCC 55614 It is a computer-generated photograph of the plate.                             Detailed description of the invention I.Definition   "B. subtilis strain ATCC 55614" is a variant and variant thereof, especially soil or air. Pathogenic fungi from the division Dcuteromycota (eg, For example, mutants and variants effective to inhibit the growth of Botrytis sp.).   The microorganism, B. subtilis strain ATCC 55614, is a plant of B. subtilis strain ATCC 55614. Pure in combination with other materials that do not substantially interfere with the inhibitory properties of the pathogenic fungal disease Or in the form of a biologically pure culture.   "Biologically pure culture" is usually found in soil where microorganisms grow. And / or (ii) other materials from which the microorganisms are isolated. Means no culture of microorganisms.   The term "culture" refers to the propagation of an organism on or in various types of media. "Whole broth culture" means a liquid culture that contains both cells and media. " `` Supernatant '' refers to cells grown in the broth that have been centrifuged, filtered, sedimented, or otherwise purified. It refers to the liquid broth that remains when removed by means known in the art.   As used herein, a "biological control" refers to a second biological organism. Is defined as the control of pathogenic organisms by the use of Biological control A known mechanism of root rot by competing for fungi to gain space on the root surface Contains intestinal bacteria that control it. Bacterial toxins such as antibiotics control pathogens Has been used to troll. This toxin can be isolated and directly Or the bacterial species can be administered, thus producing the toxin in situ. To achieve.   The "biologically active" extract of B. subtilis strain ATCC 55614 is An extract having antimicrobial properties of i.e., capable of inhibiting the growth of microorganisms. B. subtilis strain ATCC 55614 inhibits both bacterial and fungal growth.   B. subtilis strain ATCC 55614 or the active compound produced thereby An `` inhibition amount '' is at least about 50% as compared to microbial growth on untreated plants. An amount sufficient to suppress the growth of plant pathogenic microorganisms. Preferably, this microbial control Dosage should reduce about 60% to 80% of fungal or bacterial growth on untreated plants. Sufficient and subject to various factors such as soil conditions, climate, plant types, cultivation conditions, etc. Dependent.   An “effective amount” is an amount sufficient to effect beneficial or desired results. Effective dose is It is administered in one or more administrations. In terms of treatment and protection, an “effective amount” is Or alleviate, ameliorate, stabilize, reverse, slow down, or progress the state of the bacterial disease It is enough to delay.   As defined herein, a plant may be a single plant, either before or after harvest. Contains any and all parts of the plant, i.e., including the root system, shoots, Includes stems, nodes, internodes, stalks, leaves, flowers, fruits, etc. Plants are also derived from one plant It is meant to include any cells, ie, plant seed, pollen, progag (prog agule), and embryos, as well as undifferentiated tissues (such as callus).   "Flowering plant" means any angiosperm. Examples of angiosperms include ornamental plants, And wheat, corn, beans, rice, oats, potatoes, and soybeans. Which includes almost all plants that have been planted for agriculture.   "Ornamental flowering plants" include orchids, petunias, zinnias, asters, begonias, Includes flowering ornamental plants such as ranium, lilies, African violets, and roses Means   "Fruit plants" include strawberries, raspberries, grapes, tomatoes, pepper, and cucumber. Uri, pumpkin, melon, cantaloupe, watermelon, apple, peach, plum, neck Tallinn, pear, antelope, cherimoya, banana, avocado, currant, oyster Produces fruits such as papaya, mango, guava, and kiwifruit Plants.   A "vegetable plant" is a plant that produces vegetables; examples of such plants are beans, beans, Carrots, carrots, potatoes, spinach, celery, broccoli, cauliflower , Cabbage, and lettuce. II.B.subtilis Isolation and identification of strain ATCC 55614   The present invention provides a unique strain of B. subtilis, a broad spectrum of bacteria and filamentous and A good spore-forming agent with strong antifungal and antibacterial properties against non-filamentous fungi Based on the discovery of a temper flagellated bacterium. Isolation and identification of B. subtilis strain ATCC 55614 Is described here.   A.B.subtilis Preliminary screening of source and antifungal activity of strain ATCC 55614 G   The microorganism of the present invention, i.e., the Bacillus subtilis strain, can be obtained from soil or a plant host. Can be obtained. Preferred sources of this bacterium are rhizosphere soil from raspberry plants or Root tissue. One particular preferred plant host is a cultivar of strawberry. It is a cultivar variety me car (cv Meeker). By way of example, the Bacillus described herein Bacterial samples for antifungal screening, leading to the discovery of unique strains of And isolation is provided in Example 1.   Once a plant host and / or soil sample has been obtained, it is associated with the sample (as The sociated bacteria are isolated and cultured as follows. Typically, the sun Wash the pull with sterile water and soak in phosphate buffered saline (PBS) solution, or Surface sterilize. When bacteria are obtained from plant tissue, this tissue is typically Streak and typically streak on an agar medium using a wire loop.   Many different common agar media can be used to culture this bacterium. This agar Examples of culture media include nutrient glucose agar (NDA; Difco, Detroit, MI), yeast extract Kistulose calcium carbonate, nutritive broth yeast extract agar (NBY), and quin Medium agar (KB), potato dextrose agar (PDA) The method is provided in Schaad (Schaad, 1988, p. 3). Typically, the colony -Appears on the medium in about 1-5 days.   These colonies are then assayed for antifungal and antibacterial activity and A colony producing a compound having a biological activity is identified. Many preliminary screenini Any of the screening assays can be used to determine antifungal activity on bacterial colonies. Can be. One such upgrade, referred to herein as the streak test. Say first stripes a single colony of bacterial isolate on a suitable agar medium, such as PDA. Do by doing. Incubate the sample for about 2-5 days, then The fungal pathogen of the turf (plug) is added to the previously incubated culture, Add at a fixed distance. The resulting culture is then transferred to the area where pathogen growth was inhibited. Inspect for One representative fungal pathogen from which isolates can be screened is And Botrytis such as Botrytis cinerea. Antifungal activity by spot test etc. Further examples of fungal pathogens whose sex can be preliminarily assessed are Fusarium, Diplodia, Drec hslera, Fusarium, Geotrichum, Sclerotinia, Sclerotium, Erysiphe, Podosph Including aera, Uncinula, Puccinia, Plasmopara, and Stemphylium.   As an example, the results of screening bacterial isolates obtained as described above, B. cine Several isolates capable of inhibiting hyphal growth of ra were initially identified (Example 2). Exams Potato dextrose agar (PDA) and 25% trypsin digested soy agar (TSA) On two different media. Of the 1211 bacterial isolates tested, B. cinerea bacteria at least 50% compared to B. cinerea growth under roll conditions Twelve were identified as inhibiting thread growth.   Assess each antagonistic effect of 12 isolates against another fungal pathogen, Fusarium sp. In a further screening experiment, which inhibits the growth of B. cinerea Only one of the identified isolates severely restricted the growth of Fusarium sp.   The ability to severely inhibit the growth of B. cinerea on both PDA and 25% TSA medium. ATCC 55614 strain based on its ability to effectively inhibit the growth of Fusarium and Fusarium This isolate was selected for further evaluation. Related in vitro screens The results of the ning assay are described in Examples 2 and 3. Results presented there In summary, the ATCC 55614 strain was compared to untreated controls in PDA and About 70% inhibits the growth of the fungal pathogen Botrytis cinerea on both TSA and TSA media Was effective.   Used to identify isolates that are effective at inhibiting the growth of various pathogenic fungi Another screening assay that can be used is spot testing or overlay ) Test (Gross and Devay, 1977; Gross et al., 1977). When performing an overlay test Transfer cells from a single colony to an agar-containing plate (eg, PDA) as described above Spot and incubate. Next, the colonies are typically Remove with a bub and plate the plate for a long time to kill the remaining bacterial cells. Exposure to lume vapor (eg, 20 minutes), followed by emission of chloroform vapor. Anti Plate a spore suspension of fungal pathogen to identify isolates with fungal activity Overspray and then inspect areas where fungal pathogen growth is inhibited. Spot Typical fungi for which antifungal activity can be preliminarily evaluated, such as in ytis, Diplodia, Drechslera, Fusarium, Geotrichum, Sclerotinia, Sclerotiu m, and Stemphylium. Bacterial isolate targeted Slows, improves, stabilizes, reverses, slows, or slows the growth of phytopathogenic microorganisms And preferably compared to the growth of the microorganism in the absence of bacteria. If at least about 50 percent of the fungal or bacterial disease state progression is It is thought to antagonize the growth of certain microbial pathogens.   Identify active cultures (eg, isolate ATCC 55614) in spot tests After doing so, the bacterial strain is then further characterized. Many diagnostic tests described in Example 4 The bacterial isolate ATCC 55614 was characterized as Bacillus subtilis. Bacteria Diagnostic tests for determining the identity of an isolate are described generally below.   B.B.subtilis Characteristics of ATCC 55614 stock   Bacteria belonging to the species B. subtilis have functions of many features and are described by Schroth et al. (1983). And Schaad (1988) and are described in detail in B. subtilis strain ATCC 55614. To represent.   The functioning of the features of B. subtilis is briefly described below.   1.LOPAT test. LOPAT test (Levan formation, oxidase test, potato rotting ability , Arginine dehydrolase production, and tobacco sensitivity) (Schaad, 1988) Useful for determining many identifying characteristics of bacterial isolates. Belongs to the species B. subtilis Bacteria generally exhibit LOPAT characteristics as described in Schaad.   2.Further diagnostic tests. Once the isolate has been preliminarily identified as B. subtilis (Ie, by the LOPAT test), as described in Schaad (1988). Isolate further diagnostic tests with conventional diagnostic test protocols common in the art By doing so, reliable identification is performed.   Typically, a secondary test is then performed, where the results are isolates utilizing a particular substrate. Or form a specific product. By way of example, the isolate ATCC 5 (This includes 95 different assays that identify a wide range of intestinal, non-fermentative, and gram-negative bacteria. (Provides a standardized microtest method using a test). My The Cloplate test method uses a pre-selected panel of different carbon sources in bacteria. Test the ability to utilize or oxidize (GN MICROPLATE INSTRUCTIONS FOR  USE, BIOLOG, Hayward, CA, as described in September 1993).   Based on these test results, as described in Example 4, the bacterial isolate ATCC 5 5614 was identified as B. subtilis. B. subtilis ATCC 55614 is American Type Cul Nature Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 And has been assigned the following name, ATCC 55614: Contracted on September 21, 1994 Received by the International Depositary Authority on the day.   3.Effects on fungal-induced disease symptoms.Experiments performed in support of the present invention show that B.su btilis strain ATCC 55614 not only inhibits the growth of vegetative mycelia, but also (See Examples 3 and 4, respectively) 5).   Exemplary in vitro results shown in column 2 (range number) of Table 2 of Example 5 As a reference, the absolute number of sclerotia of the sample treated with B. subtilis strain ATCC556l4 was untreated. From the absolute number of control samples (no bacterial treatment). Third The average value in the column indicates that the ratio is lower than that of the control sample lacking the bacterial antagonist. When compared, about 75-80% reduction in sclerotia growth was observed in B. subtilis strain ATCC 55614. Observed in the treated samples.   To determine if this reduction was simply due to reduced vegetative mycelium growth In addition, data were calculated as the ratio of mycelial growth to the number of sclerotia. Processing sump If the decrease in bacterial sclerotia is simply the result of a decrease in the number of mycelium, control and B . The ratio with the subtilis strain treated sample is expected to be similar. Column 5 of Table 2 According to the average values shown in the above, the ratio of the treated samples is greater than that of the corresponding control. Is also big. This result indicates that for a given number of mycelia (smaller denominator), the sclerotium Less, and therefore a larger overall ratio compared to the control Value. These results indicate that the Bacillus isolate of the present invention inhibits the growth of vegetative mycelium. Not only for B. cinerea but also for inhibition of sclerotium formation .   In addition, the B. subtilis isolate ATCC 55614 shows the exemplary results shown in Example 6. As supported by conidia of fungal pathogens (eg, B. cinerea) Effective against harm. Conidia produced by mycelium are the primary species in the field It is a source of bacteria. Conidia (or spores) that land on or near the plant Sprouts and the mycelium (which in turn produces conidiophores with conidia and provides fungal growth and And lead to the development of the disease).   As detailed in Example 6, conidia from B. cinerea were isolated and And used to prepare conidia suspensions. The suspension is B. subtilis ATCC 5561 4 to both agar plates with and without agar plates, then Incubated. The extent of germination was then evaluated by light microscopy (FIG. 2A And 2B). The results are summarized in Table 3. Micrographs and ratios shown in FIGS. 2A and 2B When compared to the control sample, the presence of B. subtilis ATCC 55614 It can be seen that about 85% germination was inhibited. III.B.subtilis Isolation of bioactive extract of strain ATCC55614   A.Separation of bioactive components   As discussed above, the compositions of the present invention comprise B. subtilis strain ATCC 55614, a culture thereof. Product supernatant or extract thereof. Extract of B. subtilis strain ATCC 55614, or Is isolated in a composition containing a biologically active extract produced by the isolate. The product is typically at least partially purified from cellular components and is described in Section II above. An extract having antimicrobial activity is provided as described.   The bioactive extract produced by B. subtilis strain ATCC 55614 generally comprises: It is obtained as follows.   Typically, B. subtilis strain ATCC 55614 is potato dextrose broth, or In Antibiotic Medium 3 (Difco Laboratories (Detroit, MI)) Grow for days. The culture medium is then added to a suitable solvent (eg, ethyl acetate, chloropho , Acetone) to remove various contained substances and obtain a bioactive extract You. The resulting bioactive extract can be combined with any of a number of common separation techniques, such as adsorption chromatography. It can be further separated by chromatography. Chromatographic separation Suitable solid support materials include gel filtration supports such as DEAE, C-18 for reverse phase applications And XAD type non-ionic supports. The collected fraction has antifungal activity Is assayed, for example, by the spot test described above. Then physiological activity The active fraction can be stored and concentrated to a dry form.   The resulting semi-purified preparation containing the bioactive ingredient is then, for example, topically applied. And can be used directly to treat fungal or bacterial infections (eg, in plants). To inhibit fungal or bacterial growth). IV.B.subtilis Composition containing strain ATCC 55614   A.Antifungal formulation   The composition containing B. subtilis strain ATCC 55614 provides (i) a phytopathogenic disease control property of the isolate. No adverse effects or adverse effects on the health of the target plant or surrounding environment Impure (eg, culture broth) in combination with other substances that do not give In (ii) biologically pure culture, (iii) supernatant, or (iv) extract And may include isolates. The composition comprises a mutant and modified version of B. subtilis strain ATCC 55614. Variant bioactive ingredient or mixture thereof, substantially equivalent to B. subtilis strain ATCC 55614 Or certain variants exhibiting improved phytopathogenic disease control properties beyond that And variants. To prepare such a composition, Bacillus subtilis Strain ATCC55614 is grown using the conventional techniques described above. Culture of B. subtilis Can be performed in nutrient media, either liquid or solid, at a temperature of about 22 ° C to 30 ° C. If a limiting amount of microorganisms is desired, surface cultures and bottle cultures Can be used.   Microorganisms typically comprise a source of carbon (eg, assimilable carbohydrate), a source of nitrogen (eg, (Nitrogen compounds) or proteinase substances. Typical carbon sources include glucose, brown sugar, sucrose, starch, lactose, etc. . Typical nitrogen sources include yeast, soy flour, corn flour, and solid milk. I will. Trace elements (eg zinc, magnesium, cobalt, iron) Can be added to the fermentation medium.   When large numbers of microorganisms are required, plant inoculants in nutrient broth cultures are generally Is a broth culture with an aliquot from, for example, soil, root tissue or slope culture And aseptically transferred to large containers, or tanks It is.   Bacillus used herein in plant treatment ingredients for certain applications  The optimal amount of subtilis strain ATCC 55614 endospores can be readily determined by one skilled in the art. . In general, the active ingredient portion of the composition of the present invention will comprise the endospores of Bacillus subtilis. From about 0.001% to about 50%, and preferably from about 0.01% to about 30% by weight. Possess obtain.   The compositions of the present invention may comprise one or more biologically inert components (eg, a carrier material). Quality (eg, talc, gypsum, kaolin, attapulgite, wood flour) and / or Or binders (eg, ethylene glycol, mineral oil, polypropylene glycol, Polyvinyl acetate), nutrients and plant growth hormone).   Antimicrobial component is powder, aqueous, flowable, dry flowable, or granule-adapted Can be formulated into various formulations including: The composition may also be microencapsulated You. Generally, the powder formulation is dried with any carrier and / or other additives. Prepared by mixing with the ingredients until a homogeneous mixture is formed. Use If used, the binder is then added and the whole is present in the composition. It can be mixed again until qualitatively uniform.   For example, the liquid former may be an organic solvent such as xylene, methanol, ethylene glycol. It may include coal and mineral oil. An additional component is a surfactant such as dodecyl Calcium benzenesulfonate, polyglycol ether, ethoxylated alkyl It may include phenol or alkylaryl sulfonate. Or liquid processing The compost can be an aqueous-based suspension. Such formulations typically contain 1 ml of aqueous About 10 per carrier⁶~Ten⁹Colony forming unit (CFU) of B. subtilis strain ATCC55614 Including. In addition to the water content, the aqueous carrier may also be Such waxes may be included.   For granule applications, the carrier can be inorganic or organic. Typical Examples are attapulgite, montmorillonite, bentonite, wood flour, starch, Examples include cellulose and bran. The formulation also helps maintain the integrity of the granules. Such as mineral oil, lignosulfonate, polyvinyl alcohol or sucrose It may contain various binders.   Further, the compositions of the present invention may be combined with one or more additional fungicidal or pesticidal agents. Can be used. Illustrative insecticides for inclusion in the composition include, for example, organic chlorinated Compounds such as lindane (1,2,3,4,5,6, -hexachlorocyclohexane (γ isomer Body)); organic phosphorus esters such as diazinon (0,0-diethyl 0-2-isopro Pill-6-methylpyrimidin-4-yl phosphorothioate), isazophos (isaz ofos) (0-5-chloro-1-isopropyl-1H-1,2,4-triazol-3-yl-0,0-die Tyl phosphorothioate), thiofanox (1- (2,2-dimethyl) 1-methylthiomethylpropylideneaminooxy) -N-methylformamide) Carbamates such as carbofuran (2,3-dihydro-2,2-dimethylbenzo) Furan-7-ylmethyl carbamate), mercaptodimethu (mercaptodimethu) r) (3,5-dimethyl-4- (methylthio) phenol methyl carbamate), benzyl Ocarb (2,2-dimethyl-1,3-benzodioxol-4-ylmethylcarbamate) : Repellents such as anthraquinone, thioanthraquinone, benzanthrone, di Ram (bis (dimethyldithiocarbamate) zinc) and diphenylguanidine It is.   Generally, the endospore component of Bacillus subtilis ATCC 55614 of the composition of the present invention comprises about For periods up to 36 months, at least about its original viability after storage of the composition Typically maintain 50 percent, and preferably at least about 70 percent . B.How to prevent fungal or bacterial growth   The method of the present invention provides an effective amount of a biocontrol composition described herein for a plant. Use. This composition is used for either field or post-harvest applications I can do it. The composition may be applied to any part of the plant, including its leaves, fruits, root system. You. Alternatively, it can be used as a seed dressing. When used as a seed dressing, The composition generally comprises from about 125 grams to about 2000 grams per 100 kilograms of seed. In percentage, preferably about 250 to 750 grams per 100 kilograms of seed And applied to sowing.   One approach to preventing fungal or bacterial growth in plants And B. subtilis strain ATCC 55614 is resistant to fungal or bacterial It is used to coat infectious surfaces of sensitive plants. Stations like this For point-of-care applications, this isolate was prepared as described above in liquid (for spray application). ), As a spray, or with a powder (to spread on infectious plant surfaces) It is typically prescribed for either. Alternatively, the strain is a plant fungal disease For overall processing, it can be used for uptake by the root system. In this case , The isolate is typically formulated as a granule, usually for convenience of application. Granule formulations are typically activated by the application of water and the release of the active compound is Typically, it occurs over an extended period of time, such as 2 to 12 weeks. In the example below As can be seen, the plant protection composition according to the invention is useful for phytopathogenic fungi, in particular Botryt is, Phytophthora. Pythium. Rizoctonia. Alternaria. Monilinia and Fusari It shows strong protection against um and also against the gram-negative bacterium Erwinia.   According to another aspect of the present invention, a fungal or bacterial resistant transgenic plant is A method for forming is provided. Transgenic plants are B. cerevisiae. subtilis strain A Includes chimeric genes corresponding to genes that are attributable to the antagonistic phenotype of TCC 55614. A chimeric gene is a plant-compatible protein that is effective to drive the expression of the chimeric gene. A fungal or operable plant cell that is operably linked to a motor and transformed. Is effective in conferring resistance to bacterial pathogens (e.g. Botrytis, Fusarium) Encodes a unique product.   In short, in performing this method, B. subtilis strain ATCC 55614 Loci resulting from the antagonistic phenotype of subtilis mutagenesis Identified first. Prior to mutagenesis, B. Wild type B. antagonizes the growth of cinerea subtili The ability of s strain ATCC 55614 is confirmed. Then, B. Subtilis mutagenesis is standard This is performed according to the protocol. Following mutagenesis, the mutant is essentially as described above and As described in Example 2, the ability to inhibit vegetative mycelium growth was assessed. Essay. Then, B. putative deficiency in antagonism to cinerea The mutant is subtilis used to identify loci that contribute to the antagonistic phenotype of ATCC 55614. Used.   Upon identification, cloning, and sequencing of the gene, the transgene was then It can be used to form transgenic plants. Where the transgene Expression confers plant resistance to fungal pathogens (e.g., Botrytis, Fusarium) It is effective in doing. In addition, the transgene is According to subtilis strain ATCC 55614 Prokaryotes that form recombinant organisms capable of producing the above-described antifungal compounds that are secreted Or eukaryotes. V.Use   Experiments performed in support of the present invention are described in subtilis strain ATCC 55614 is phytopathogenic Fungi (Botrytis, Phytophthora, Pseudomonas, Erwinia, Alternaria, Trichoder ma, Monilinia, Puccinia, Rhizoctonia, Phythium and Plasmopara and Fusa rium) (including but not limited to rium); It is effective in preventing the development of plant diseases associated with these pathogens. And demonstrate.   This biologically active strain can be used for seedling and root rot, vascular wilt, or Any disease caused by an attack of one of the fungal or bacterial plant pathogens (Eg, Table 13).   The composition according to the present invention comprises: strawberry and raspberry gray mold, grape bunch rot, beans Moldy rotten, lettuce, of vegetables like radish, sugar beets, carrots, and cucumber Terminal rot of pepper, tomato and pumpkin, dry eye rot of apple And many like begonias, geraniums, lilies, African violets and roses To prevent Botrytis-related diseases, such as gray mold and blight of ornamental plants Useful. The composition also includes tomato, pea, banana, and cotton Used to prescribe Fusarium-induced vascular closure wilt, such as wilt Can be   The compositions and methods of the present invention can be used to convert gray mold to Canby, Chilcotin, Amity and And can be used to protect commercial raspberry varieties such as Willamette. this These varieties are particularly susceptible to gray mold damage caused by Botrytis . Similarly, the compositions and methods described herein can be used to treat gray mold, e.g., Chandler. Protects the commercial value of strawberries susceptible to attacks by Pajaro, Pajaro and Selva Can be used to   As an example, a greenhouse test performed in support of the present invention (Example 7) cinerea Reducing greenhouse-grown strawberry (cv Pajaro) loss by resulting gray mold In B. The efficacy of subtilis strain ATCC 55614 was revealed. Details of this study , Are briefly summarized below.   In performing the tests described in Example 7, B.I. Strawberry plant inoculated with cinerea The products were subjected to different treatment regimes. This treatment removes (i) strawberry Botrytis fruit mold. Rovral 50 VP, a commercial fungicide used to control, (ii) potato Strauss broth, (iii) B.I. subtilis strain ATCC 55614, and (iv) a series of untreated Five foliar treatments of the control were included. Treat plants (including flowers) before inoculation Was.   The test results were calculated based on the number of healthy fruits, the weight of healthy fruits recovered, and the weight of diseased fruits. And many factors, including the percentage of total recovered weight that contributes to diseased fruit Was evaluated based on Considering the results provided in Tables 4, 5, 6, and 7, B . Subtilis leaf treatment with a composition containing strain ATCC 55614 comprises treatment and untreated ( Control) For fruit, disease disease expressed as a percentage of total recovery Effective in controlling fruit loss by gray mold, as indicated by fruit quality Was. Further, B. Treatment with subtilis strain ATCC556l4 yields healthy strawberry fruit High (ie, weight) did not adversely affect.   The antifungal compositions described herein may also be used to prevent gray mold infection. , White Riesling and Pinot Noir, Chardonnay, Zinfandel, and Cheninb It can be applied to grape varieties such as lanc. All of these varieties are B. cinerea (Psch eidt, 1990,1991,1993 PACIFIC NORTHWEST PLANT DISEASE CONTROL HANDBOOK; E nglish et al., 1989; Gubler et al., 1987).   Experiments performed in support of the present invention also demonstrate that the Bacillus isolate of the present invention Propagation of hyphal hyphae, and (ii), for example, B. cinerea (Examples 3 and 5, respectively) It is effective in inhibiting both sclerotium formation by A.   B. cinere in long-lived perennial crops such as grapes and raspberries One component of the disease cycle of a is the production of fungi that overwinter on the dead debris. Fungus departs in spring It sprouts and initiates a new growing infection (Weller, 1988). Therefore, B. subtilis strain A Application of a composition containing TCC556l4, an extract or a biologically active metabolite thereof, Reduce the source of overwintering inoculum in necrotic or senescent tissue Can be used for   Another important component of the disease cycle of B. cinerea is the production of conidia. Conidia Serves as the main source of inoculum under outdoor conditions. Actually implemented in support of the present invention The test is subtilis isolate ATCC55614 also inhibits conidia germination of B. cinerea (Example 6). By inhibiting conidial germination The compositions and methods described herein reduce botrytis-related losses. Can be used as a preventive measure.   Due to its effectiveness in inhibiting conidial germination, antifungal compositions also plant pathogenicity, a member of the ta and Deuteromycota classes (Fungi imperfectl) It can serve as a control for different genera of fungi. Typical genus is Vert icillium sp., Fusarium sp., Macrophomina sp., Thielaviopsis sp., and Includes fungi that are causative agents of downy mildew pathogens.   In addition, B. Antifungal composition comprising an extract produced by subtilis strain ATCC 55614 In other words, disseminated disease embryos are conidia, such as Aspergillus sp., Histoplasmasp. And Tineas sp. For the treatment of human fungal diseases.   The following examples illustrate but do not limit the invention in any way.                               Materials and methods   Where indicated, chemicals and reagents (eg, potato dextrose cold) Heaven (PDA), potato dextrose broth (PDB), trypsin soy agar (TSA), And malt extract agar (MEA)) were obtained from Sigma Chemical Company, St. Louis, MO. Was.   A.Fungal culture   Pure culture of Botrytis cinerea (B. cinerea) is available from American Type Culture Col. lection (ATCC; Rockville, MD; accession number 11542) and 25 ° C on a PDA Maintained at                                  Example 1 Collection / isolation of new bacterial strains   Bacteria were harvested at the strawberry plant at Washington State University-ARS (Vancouver, WA). (Cv. Meeker) was isolated from rhizosphere soil and root tissue.   Soil as well as root material were placed in test tubes. 5 volumes phosphate buffered saline (PSB, pH 7.3; Wollum, 1982) was added and the tubes vortexed for 1 minute. Rare A series (10^-1From 10^-8To) were made using PSB. 10011 of each dilution, PD Plated in Petri dish containing A (ATCC MEDIA HANDBOOK, 1984). Proliferate plate Incubated at 20 ° C. using 16 hours light length in the chamber.                                  Example 2 Screening of bacterial isolates for antifungal activity   Single colony of bacterial isolate in 100 mm 15 mm Petri dish (plate) containing PDA Was streaked in a straight line of about 12 mm from the edge of. The plate inoculated with the streak was used in Example 1 above. For 2 days in a growth chamber. 2 days incubation After that, a 5 mm plug of mycelium of B. cinerea continuously grown on the PDA was For the plate, added about 55 mm from the edge of the bacterial streak closest to the center of the plate . To assess the growth of B. cinerea in the absence of bacteria, a control lacking bacterial streaks Troll plates were similarly seeded. All plates as in Example 1 above Incubated.   Each bacterial isolate was first tested twice for its ability to inhibit the growth of B. cinerea. Identify putative antagonist isolates, PDA plates and 25% trypsin soy agar (TS Retested on plates containing A). First screenini with antifungal activity Bacterial isolates identified, then 10 PDA of each PDA as well as 25% TSA Using a replica plate of each relative, each isolate inhibits the growth of B. cinerea Retested to determine performance.   Of the 1211 bacterial isolates tested for antagonism to the growth of B. cinerea , 12 cell isolates compared B. cinerea growth under control conditions, B. cin erea was identified as inhibiting mycelial growth by at least 50%.   Twelve isolates that showed growth inhibition of B. cinerea were at least partially sp. was tested for its ability to inhibit growth. Introduce 5mm hypha plug of Fusarium sp. Basically, using the inserted PDA plate for the antagonism test against B. cinerea This was performed as described above.   Of the 12 isolates, only one (later B. subtilis strain, ATCC Accession No. 55614) Designated severely restricted the growth of Fusarium. Based on in vitro test results ATCC 55614 strain is a known commercially available strain for inhibiting the growth of Fusarium sp. MYCOSTOP (Streptomyces griseoviridis strain 61) It was equally effective.   The results are shown in FIGS. 5A and 5B.                                  Example 3 Effect of isolate on mycelial necrosis   Ability to severely inhibit B. cinerea growth on both PDA and 25% TSA plates Of the 12 previous isolates for further analysis. This simple The eluate was called isolate ATCC 55614.   The effect of the isolate on B. cinerea mycelium growth was assayed as described above and the results were The results are summarized in Table 1 below. Antagonism of Botrytis determines the extent of fungal growth And reported as distance from the agar plug containing the inoculum. table As can be determined from 1, higher numbers indicate a greater degree of fungal growth and And a lesser degree of antifungal activity.                                    table 1 Botrytis cinerea In vitro antagonism of proliferation ¹ 1.a. Growth assayed 7 days after seeding 5 mm mycelial plug   b. Growth conditions = 20 ° C, 16 hours light period   c. Average and SD calculated with n = 10 2. Mean is significantly different from control (α = 0.01), Dunnett's test.   The data shown in Table 1 shows that on the PDA and TSA plates, This shows that the growth of B. cinerea was inhibited by 71.3% and 69.6%, respectively.   FIGS. 1A and 1B show PDA plates plated with B. cinerea and isolate ATCC 55614. (Figure 1B) and a negative control plate containing only B. cinerea (Figure 1 The computer-made photograph of A) is shown. The image shows the application of a 5 mm hyphal plug from B.cinerea Obtained after 14 days.   The maintenance of healthy B. cinerea hyphae was greatly affected by bacterial isolates. Mycelium Necrosis is evident only after about 5 days, and the hyphae closest to the bacteria die and continue. In a linear fashion, such as the necrosis of hyphae further from the bacterial streak. Necrosis of the thread continues. In plates containing the isolate ATCC 55614, observed after 14 days The growth of healthy mycelium with fluffy white characteristics of healthy B. cinerea mycelium (Fig. It was always slight (about 3 mm).                                  Example 4 Isolate characterization and Species identification   The isolate identified above as having activity against B. cinerea is isolated from a plant pathogen Further characterization using conventional methods for identifying bacterium (Schaad, 1988) .   Specifically, the identity of the isolates was determined by GC-FAME analysis and Biolog Microtiter Syste ructions for Use, Biolog, Inc., Hayward, CA, 1993). Utilizing a preselected panel of different carbon sources or based on the ability of the isolate to oxidize. Anti Unresponsive wells remained colorless; positive results are indicated by the appearance of purple Processed using a computer software program and the purple well pattern This program automatically cross-references a broad library of species. You.   Based on these tests, isolate ATCC 55614 was isolated from Bacillus subtilis (B. subtili s) was identified as an antifungal-producing strain.   B. subtilis ATCC 55614, a bacterial isolate, is available from American Type Culture Collecti. on (ATCC), deposited at 12301 Parklawn Drive, Rockville, MD2085l, ATCC designated number No. 55614 was assigned. The deposit was accepted by the ATCC on September 21, 1994.                                  Example 5 Sclerotium assay   The effect of B. subtilis ATCC 55614 on sclerotium production was also tested. PDA plate (fine Seed with a 5 mm hypha plug containing B. cinerea as above, both with and without bacterial isolate did. 21 days after seeding on PDA plates, sclerotia were counted.   From the observation that the sclerotium formation by B. cinerea was significantly reduced in TSA medium, The experiment was not performed on TSA plates.   Sclerotium counting: Some reduction in the number of sclerotium counted in the assay Due to the possible consequence of reduced vegetative mycelium growth, The ratio was calculated for each condition.   The results of these studies are summarized in Table 2 below. Range exists on the plate The column with the heading “Range” indicates the total number of sclerotia, Represents the ratio of mycelial growth.                                   Table 2 Antagonistic effect on sclerotium proliferation ¹ 1. a.PDA plates with 5 mm mycelium plugs were counted 21 days after incubation. Number of sclerotia     b. Growth conditions = 20 ° C, 16 days     c. Mean and standard deviation were calculated for n = 5. 2. The mean value is significantly different from the control in the Dunnett's test (α = 0. 01).   The data indicate that the bacterial isolate B. subtilis ATCC 55614 is a negative control It shows that the number of sclerotia is reduced as compared with the Further, B. fungus by subtilis Inhibition of nucleus production cannot be the result of reduced hyphal growth. Because in the sclerotia The growth rate reached is between the negative control and B. significant between subtilis treatment This is because there is a difference (for example, when comparing the values in the last column).   Thus, the in vitro assay described herein is based on B.C. subtilis ATCC 55614 Not only inhibits the growth of vegetative hyphae, but also Blocks sclerotium formation by cinerea Has the ability to harm.                                 Example 6 Conidium assay   B. B. cinerea in another aspect of the disease cycle. effect of subtilis ATCC 55614 That is, the production of conidia). Conidia is one of the inoculants of the disease under farm conditions Act as a secondary source.   In culture, B. cinerea on malt extract agar (MEA) for 5 days at 25 ° C for 12 hours Grow long and then expose the plate to ultraviolet light (UV) for 3 days Produced conidia (Leifert et al., 1993). The plate was then transferred to 10 ml Submerge in heavy deionized water, and gently condense using a rubber policeman. The conidia were removed by scraping the plate. Remove the liquid from the plate Transferred with pets and filtered four times through two layers of cheesecloth. The resulting suspension is Vortexed for hours to separate conidia into individual breeders. 1 ml of double deionized water The concentration of the conidia is determined using a hemocytometer and the suspension is^FiveConidia / m Diluted to achieve a concentration of l.   Plate 10 μl conidia suspension (approximately 1000 conidia) on PDA plate, Incubate at 25 ° C. for 12 hours in the dark. Another 10 μl of the suspension was subti lis ATCC 55614 streaks were plated on PDA plates grown for 2 days. Conidia The suspension was placed 1 cm away from the bacterial streaks and incubated similarly.   Germination was assessed after 12 hours incubation at 28 ° C. in the dark. Pre The conidia were observed under a light microscope (100x), and 100 conidia were transferred to each plate for germination. Was evaluated. Exemplary micrographs of conidial germination obtained for this experiment Shown in FIGS. 2A and 2B. The image in Figure 2A shows conidia on a control PDA plate. Shows complete germination. The image in FIG. 1cm from the subtilis streak culture Fig. 3 shows an example of inhibition of germination of conidia placed apart.   The results of these experiments are summarized in Table 3 below.                                   Table 3 Conidium germination assay ¹ 1. Approximately 1000 conidia are placed on potato dextrose agar and The roll was placed 1 cm away from the streak two days ago. Plate at 25 ° C Incubated overnight. 100 conidia per plate are used for conidia germination Assayed at random. The experiment was repeated five times.   The results in Table 3 show B. cinerea inhibits conidia germination subtilis ATCC 55614 Shows efficacy.                                 Example 7 Greenhouse testing   Greenhouse test due to gray mold caused by Botrytis cinerea, Bacillus subtili in controlling the loss of greenhouse grown strawberries (cv. Pajaro) s Performed to determine the efficacy of ATCC 55614.   B. The efficacy of subtilis ATCC 55614 was compared to: (i) foliar application of PDB only ( Negative control), and (ii) Rovral 50 WP ("IPRODIONE"; Rhone-P olenc, Paris, France) (commercially available, Botrytis fruit mold on strawberries) Are widely used to control fungicides).   A.Strawberry cultivation   Strawberry plant (cv. Pajaro) roots from BHN Research (Watsonville, CA) Got it as a head. Put the plants in 6 plastic pots (1 plant / pot), and Filled the pot with BlackGold 100% organic soil (BlackGold, Inc., Hubbard, OR) . Water the plants, regularly fertilize Peters 20:20:20 chemical fertilizer, and 18 hours light / Cultivated under a 6 hour dark cycle.   B.Bacterial culture   B. subtilis ATCC 55614 single colony, PDB (1 liter / flask) Used to inoculate two 2L flasks containing Transfer the flask to a rotary shaker And grown at 225 rpm for 48 hours at 28 ° C. The cell density was determined using a spectrophotometer. It was measured by absorbance at 600 nm. Based on this reading, culture was^-8Fine Adjusted with PDB to a final cell density of vesicles / ml.   C.processing   Treatments were (i) Rovral 50 VP with 1.5 1b / 100 gallon water, (ii) untreated (control ), (Iii) PDB only, or (iv) P.B. subtilis strain ATCC 55614 (5 × 10^-8) CFU / m l Includes foliar application.   By spraying the plant with a hand sprayer comprising one of the above compositions, Was administered. Plants were sprayed and drained (about 75 ml / plant). Processes (i) to (iv) were tested Was applied 5 times during. The first treatment is at 10% flowering, and the next four are two weeks thereafter Performed at intervals. Fifth treatment applied one day before first harvest .   D.B . Artificial greenhouse sowing of strawberry plants using cinerea   Plants were treated with B.I. cinerea 4 × 10^-3Conidia / ml suspension (about 75 ml / plant ) And inoculated by draining. The first spray, the first flowering Dosing on the day observed, and the next two doses at two week intervals thereafter did.   E. FIG.harvest   Ripe berries were harvested from all plants once a week for three consecutive weeks. the first The harvest was after the last treatment.   F.sample size   The treatment was performed on "blocks" each consisting of 5 plants. And each place Process four times (A, B, C, and D), resulting in 20 plants per treatment Was.   G. FIG.Parameter evaluation   The following parameters were evaluated after each harvest: (i) healthy fruit number, (ii) healthy Fruit weight (g), (iii) pathological fruit weight (g), and (iv) total yield due to pathological fruit Percentage of harvest.   After evaluating the above parameters; add healthy fruit for individual plots Placed in tic trays (so that the fruits do not come into contact with each other). Then, pull the tray Covered with plastic wrap and stored at 4 ° C. for 7 days. 4 ℃, 95% relative humidity (RH) Processing mimicked storage conditions in the wholesale / retail distribution network.   After storage at 4 ° C for 7 days, 95% RH healthy fruits are evaluated as described above for: Done: (i) healthy fruit weight (g), (ii) pathological fruit weight (g), (iii) due to pathological fruit Percentage of total harvest to make.   The following equation was used to determine the quota of total yield due to diseased fruit . H.result   Exemplary results of the greenhouse test are shown in Tables 4, 5, 6, and 7, and FIGS. 3A, 3B, 4A, And 4B.   Table 4 shows the percentage of the total harvest attributable to (T = 0) pathological fruits at the time of harvest. In 2, 3, and 3, the control fruit, Rovral or B. subtilis ATCC 5 Shown for fruit treated with 5614. These results are also shown graphically in FIG. You. The Y axis in FIG. 3A is the disease of each of the three harvests (1, 2, and 3) shown on the X axis. It shows the ratio of the harvest amount due to the target fruit.Table 4 Diseased fruit as a percentage of yield at harvest   At the first harvest, Rovral and B.A. Pathological fruits treated with subtilis ATCC 55614 Actual average weight (and overall percentage) was significantly lower than control treatment . At the second harvest, the data separated into two subgroups. Rovral and B . Average weight of diseased fruit (and total percentage) from samples treated with subtilis Indicates untreated (control) samples or samples processed with PDB only Was significantly lower than the weight and percentage of diseased fruit from S. Significant within these two subgroups No significant difference was seen. At the third harvest, the average percentage of diseased fruit is any other Significant in samples treated with Roval than in samples treated with 3 Was low.   Table 5 below shows the control after storage at 4 ° C for 7 days under 95% RH (T = 7). Fruit and Rovral or B. harvest of fruits treated with subtilis ATCC 55614 The percentage of total yield due to diseased fruit in 1, 2, and 3 is shown. these The results are also shown graphically in FIG. 3B. In FIG. 3B, the Y axis corresponds to 3 shown on the X axis. The percentage of yield attributable to diseased fruit at each of the 1st harvest (1, 2, and 3) is shown.                                   Table 5 Diseased fruit as a percentage of harvest after storage for 7 days (4 ° C, 95% RH)   At the first harvest, the average weight of diseased fruit treated with Rovral or Bacilius (And total percentage) are from the corresponding measurements of the control treated samples It was significantly lower. In the second harvest, the disease treated with PDB and control The average weight (and total percentage) of the offending fruit was Rovral or Bacillus treated It was significantly higher than the corresponding measurement result of the sample. In the case of the sample inspected immediately after harvest Similarly, the average percentage of diseased fruit from the third crop is the percentage of samples treated with Rovral. Was significantly lower than samples that received any of the other three treatments. harvest Immediately between the control and PDB treatments for both the fruit and the stored fruit analyzed No significant differences were observed.   Table 6 below shows control fruit and Rovral or B. subtilis ATCC 5561. For the fruit treated in 4 the harvest at harvest (T = 0) at harvest 1, 2 and 3 Display the weight of all fruits. These results are also graphically illustrated in FIG. ing. The y-axis in FIG. 4A is the health of each of the three crops (1, 2 and 3) shown on the x-axis. Shows total fruit weight (grams).                                   Table 6 Healthy fruit weight at harvest   In the first crop, there was a significant difference in the average weight of healthy fruits that received each of the four treatments. No differences were observed. In the second crop, the data is split into two subgroups Was. Average weight of healthy fruit from samples treated with Bacillus was control, PD Significantly greater than the average weight of healthy fruit from B or Rovral treated samples, There were no significant differences between treatments in any of the subgroups. In the third harvest , There were no other significant differences.   Table 7 below shows control fruit and Rovral or B. subtilis ATCC 5561. 95% for 4 days at 4 ° C. for fruits treated in 4 at harvest 1, 2 and 3 The weight of healthy fruit after storage at RH (T = 7) is indicated. These results are also shown in FIG. Is graphically illustrated in FIG. The y-axis in FIG. 4B is the three axes shown on the x-axis. The healthy fruit weight (gram) of each of the harvests (1, 2 and 3) is shown.                                   Table 7 Healthy fruit weight after 7 days storage (4 ℃, 95% RH)   At the first harvest, no significant difference in healthy fruit weight was observed between the different treatments Was. At the second harvest, control and PDB treatments were significantly more significant than Bacillus treatments. Rovral treatment yielded significantly lower weight than Bacillus treatment, resulting in lower healthy fruit weight yields. Yield of fruit. No significant differences were observed between the four treatments at the third harvest .   In summary, the above results show that Bacillus ATCC 55614 treatment had three yields. Loss due to gray mold for the first two crops (total yield of diseased fruit) This shows that control was as effective as Rovral.   For healthy fruits, Bacillus treatment is significantly more significant than control or PDB treatment No lower yields were produced, and Bacillus treatment was significantly more significant than Rovral treatment There was no ineffectiveness.                                 Example 8 ATCC Fungicidal activity of 55614 culture   ATCC 556143 is a fungus, Phytophthora infestans, Pythium ultimum, Botryti s Effective against Cinerea, Rhizoctonia solani, and Alternaria solani The following experiment was performed to determine if Petri plate on agar plate (PDA-potato dextrose agar, Difco). The above fungal culture , Liquid YPG-1 medium (0.4% yeast extract, 0.1% KH_TwoPO_Four, 0.05% MgSO_Four・ 7H_TwoO, 1.5% glue (Course) for 3 days. Pathogen spore suspension (concentration is about 2 × 10⁶Spores / mL ) Was spread on agar. ATCC 55614, potato dext Grow in Roth Broth (PDB) (Difco) for 3 days. Test whole broth culture About 1 × 10⁶~ 6 × 10⁶Proliferate to CFU / mL and extract from these cultures. A recoat was taken. Supernatant should be density centrifuged at 5,200 rpm for 20 minutes Obtained by   Two 7 mm holes were made in each petri dish. For each 7mm hole, 100μL volume test (Either supernatant or whole broth) was added. Each test was performed in duplicate . No microbial test sample was added to the control plate. Inhibition zone (around each hole Was measured after 3-10 days. Phylophthora, Bortrytis. Table 8 shows the results for Rhizoctonia, Alternaria and Pythium. You. Control plates showed no zones of inhibition.                                   Table 8  The same experiment as above was performed using different media (dextrose, bactopeptone, yeast). Extract, malt extract), an important plant pathogen, the gram-negative bacterium Pseudo Including monas Syringae. Table 9 shows the results.                                   Table 9   By repeating similar experiments, the fungus Trichoderma har, an important pathogen of mushrooms, ATCC 55614 against zianum was tested. Trichoderma harzianum, T14 strain, Ca Obtained from mpbell (Davis, CA). Using a No.4 cork punch, PDA plate Four wells were prepared. ATCC 55614 was applied to one of the four wells. Play a No. 4 cork punch size Trichoderma hypha plug from two discs Was added to each plate so that it was located between the two wells on each side of the plate. The result, 24 o'clock Measured shortly after. The size of the fate zone between the bacteria and the hypha was measured. ATCC 55614 is 4 A mm zone resulted.   ATCC 55614, Erwinia herbicola, a gram-negative bacterium, and M, a fungus The following experiments were performed to determine in vitro efficacy against onilinia fructicola. went. Bacterial strains were cultured as described above. Monilinia fructicola culture, V-8 Agar (20g agar, 4g CaCO_Three, 200mL V-8 juice) for 8 days at room temperature in the dark Propagated. Add the spores to the surface of the culture plate by adding sterile distilled water and using a sterile needle. The spores were collected by rubbing the surface with. Spore concentration 3.3 × 1 0⁶It was adjusted to spores / mL and 400 μl was added to 4 mL of potato dextrose soft agar. The mixture is poured onto the surface of a potato dextrose agar culture site and softened. The agar was solidified. Erwinia herbicola, fermented overnight in half strength TSA, And 1 × 10^FiveCells were spread on TSA agar plates. Sterilized No.4 cork perforator 5 wells were made on each plate using 100 μl of ATCC 556 Fourteen three-day cultures were added to each well. Incubate the plate in the dark at room temperature. Bait and the growth-free zone around each well was measured. Table 10 shows the results. About.                                   Table 10                 Bacterial control of M. fructicola and E. herbicola Example 9 Fungicidal activity of ATCC 55614 using whole plants   ATCC 55614 for controlling late blight (P. infestans) infection Activity was tested on whole tomato plants. Tomato plants (Ace and patio varieties) From Ace hardware and 6 packs with 3 plants per pack Transplanted to ATCC 55614 in Trypticase Soy Broth (TSB) (Difco) for 72 hours Breed and then 5 × 10⁶The CFU / mL concentration was reached. One plant of each variety of tomato plants , Sprayed with whole broth culture or supernatant of ATCC 55614, followed by approximately 21 Air dried at ℃. Two control plants were not treated. All plants of After 1.55 × 10^FiveSpraying was performed with CFU / mL P. infestans broth until effluent. Plants are Air dry at about 21 ° C, lightly spray with deionized water, seal in a transparent plastic bag, And incubated at about 16 ° C. 15 days after the treatment, the blight infestation amount was measured. Was. Table 11 shows the results.                                   Table 11       Mild blight and bacterial chip infection 15 days after ATCC 55614 treatment   These results indicate that the antibiotic-producing strains according to the present invention produce late burn and bacterial debris. Indicates that it is valid.                                Example 10               B.cinerea Fungicidal activity of ATCC 55614 supernatant against water   For testing the efficacy of the bacterial strain ATCC 55614 supernatant according to the invention against B. cinerea For this, fresh strawberries collected on the test day were used. For test number 1, ATCC 55 614 frozen supernatants were used. ATCC 55614, potato dextrose as described above Grow in broth. The supernatant was frozen 1 to 1.5 months before the test. Examination number In issue 2, ATCC 55614 was converted to semi-strength TSB or potato dextrose broth (PDB )) And the broth or supernatant was tested without freezing. Whole broth Cultures and supernatant were sprayed onto strawberries until they flowed out and air-dried.   B. cinerea spores grow on potato dextrose agar in Petri plates. And diffused into deionized water to provide a liquid inoculum. B. cinerea inoculum (about 5.8 × 10^Five(Measured as cells / mL) is sprayed on the strawberries until they flow out, and Strawberries were air dried. In Test No. 1, strawberries were put at 25 ° C with plastic wrap lid In a cardboard container. In Test No. 2, about 16 ° without covering all strawberries Placed in an incubator at ° C. Table 12 shows the results.                                   Table 12   ATCC 55614 frozen supernatant is used to control B. cinerea infection in living strawberry plants It was completely effective. In addition, all broth cultures of ATCC 55614 However, it was completely effective in preventing B. cinerea infection. ATCC 5561 grown in TSB The supernatant from 4 was partially effective, but when grown in PDB, It was 100% effective.                                Example 11 Activity of ATCC 55614 against fungal pathogens   To test for ATCC 55614 activity against a number of fungal pathogens, Grow in todextrose broth. 5 × 10 cells⁶Cultured to cells / mL . Duplicates of 3 test and 3 control plants per pathogen were used. test Plants are each fed with a whole broth culture of ATCC 55614 until spilled by manual spray. Sprayed. After the leaves had dried, each test plant was sprayed a second time. Fine After drying of the second application of the strain culture, the test plants and control plants are treated with appropriate fungi. The sexual pathogen was inoculated. Plants were incubated under conditions that allowed disease onset. Further In addition, a positive control is used to determine the appropriate Fungal Utilized by testing known pesticides against pathogens. Each plant is 0% control to 100% control (0 = disease level of untreated control; Estimate disease control ratio using a scale up to 100 = plants without visible lesions) Was evaluated by: Fungal pathogens, resulting diseases, host plants and plants Table 13 shows the trawl pesticides. Table 14 shows the results.                                   Table 13                                  Table 14 ^yPi = P.infestans, As = A.solani, PV = P.viticola, Un = U.necator, Bc = B.cinerea, Sn = S.nodorum, Pr = P.recondita f.sp. tritici (%)^ZDisease index = diseased tissue ratio in untreated inoculated plants   ATCC 55614 provided complete control of B.cinerea. ATCC 55614 also It is highly active against Ubeto and rust, and in this study Phyt ophthora showed some activity.Example 12 ATCC 55624 activity against red rot, Monilinia fructicola   A 250 mL culture of ATCC 55614 was grown in PDB for 3.5 days as described above. regional Purchase peaches from the grocery store (Safeway) and surface sterilize with 10% Clorox solution to remove After rinsing with ionized water, it was air-dried. ATCC 55614 whole fermentation broth (8.7 × 10⁶CFU / mL ) Was sprayed with a manual sprayer to two peaches. (Two peaches Peach was air-dried. Scoop Monilinia spores from Petri dish, 1.0 9 × 10^FiveThe cells were suspended in deionized water to a spore / mL concentration. Then, peach, spore The suspension was sprayed until flowing off and then air-dried. Do not process two peaches, 2 Each peach was sprayed with Monilinia only. Put peaches in a polypropylene container And placed in a dark incubator at 18 ° C. for 4 or 6 days. Red rot in each peach Was measured as shown in Table 15.                                   Table 15                   Monilinia suppression of ATCC 55614 in peaches   On both days 4 and 6, ATCC 55614 contained untreated control and MonCC Monilinia red rot was suppressed compared to peach treated with ilinia alone. Six days later, All control peaches were heavily infected with red rot but one ATCC 55614 treatment Only peaches from the lab showed infection. In addition, one infected ATCC 55 6 14 peaches are much better than untreated or Monilinia only treated controls Showed small lesions.     ATCC Comparison of Monilinia fructicola inhibition of 55614 and chemical fungicides   Using a similar protocol as described above, the ATCC 55614 culture and And Monilinia spores to increase the effect of ATCC 55614, a commercial fungicide Benl                                   Table 16 It is effective in controlling nia infection.   Although the present invention has been described with reference to specific methods and embodiments, the present invention has been described in detail. It is recognized that various modifications and changes may be made without departing from the scope. You.

[Procedure amendment] [Date of submission] August 23, 1999 (August 23, 1999) [Content of amendment] Claims 1. A method of protecting or treating plants from fungal and bacterial infections, applying to the plant an effective amount of antibiotic-producing Bacillus subtilis strain ATCC 55614, or a soluble extract or culture supernatant obtained therefrom, the Including, methods. 2. The infection is Phytophthora infestans, Botrytis cinerea, Pseudomonas syri ngae, is Erwinia herbicola, Alternaria solanl, Trichoderma harzianom , Monil inia fructicola, Puccinia recondita, Rhizoctonia solani, provoked by microorganisms selected from the group consisting of Pythium ultimum and Plasmopara viticola The method of claim 1. 3. 2. The method of claim 1, wherein the antibiotic producing Bacillus subtilis strain is applied to the plant as a whole broth culture . 4. 4. The method of claim 3, wherein said culture is diluted. 5. 2. The method according to claim 1, wherein the culture supernatant is applied to the plant. 6. The method according to claim 5 , wherein the culture supernatant is refrigerated or frozen before application to the plant . 7. The method according to claim 5, wherein the culture supernatant is diluted. 8. It said antibiotic producing bacteria strains, wet powder, granules, aqueous stream animal, or applied as a dry stream animal, or microencapsulated in a suitable substance, the method according to claim 1. 9. 2. The method of claim 1, wherein the antibiotic producing bacterial strain is applied in combination with a chemical pesticide . 10. B. In culture medium. B. subtilis strain ATCC 55614 comprising culturing B. subtilis strain ATCC 55614 . A method for producing a subtilis composition. 11. B. A method for isolating a biologically active extract produced by S. subtilis strain ATCC 55614, comprising: step of culturing the subtilis strain ATCC 55614, Contact and B. The method includes the step, be isolated from the culture medium bioactive extract produced by subtilis strain. 12. B. A method for isolating a biologically active extract produced by B. subtilis strain ATCC 55614 , comprising the steps of: step culturing subtilis strain ATCC 55614; (ii) extracting bioactive components present in the cell culture medium, the crude extract that forms producing step; a (iii) crude extract on a solid support It separated, step to produce separated fraction; subscription the separation fraction for and (iv) antimicrobial activity - comprising the step, of training method. 13. 4. The method of claim 3, further comprising: (v) pooling the active fractions identified in step (iv).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12R 1: 125) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR) , GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG) ), AP (GH, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU , AZ, BA, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, ID, IL, IS, JP, E, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD , SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Manker, Denise Sea. 3037 (72) Inventor Jimenez, Desmond Earl. USA 95695, Woodland, Pendergast Street 9 (72) Inventor Bestwick, Richard Kay. United States Oregon 97223, Portland, S.E. Wu. Canby 6680 (72) Inventor Bandemark, George Jay. Mexico, Guanajuato, Irapuato, Apartard Postal 629, Sin Vestab del Ipeene

Claims (1)

[Claims] 1. Biologically pure culture of Bacillus subtilis strain ATCC Deposit No. 55614. 2. Composition comprising Bacillus subtilis strain ATCC 55614, its supernatant or biological activity extraction object. 3. A method for producing a Bacillus subtilis composition having antibacterial activity, comprising: Process of: (A) Bacillus subtilis strain ATCC in a culture medium effective to support fungal cell growth Culturing a culture of 55614 (ATCC No. 55614), A method comprising: 4. A culture medium for the biologically active extract produced by the Bacillus subtilis strain? 4. The method of claim 3, further comprising the step of isolating from the lipase. 5. The isolation step comprises: (I) extracting a biologically active component present in the cell culture medium to produce a crude extract; That (Ii) separating the crude extract on a solid support to produce a separated fraction; (Iii) screening the separated fraction for antimicrobial activity; and (Iv) pooling the active fractions identified in (iii), 5. The method of claim 4, comprising: 6. A method for protecting a plant from fungal or bacterial infection, comprising the following steps: The plant or its environment contains Bacillus subtilis strain ATCC 55614 (ATCC No. 55614). Applying an effective amount of the composition, its supernatant or extract. 7. The infection is Botrytis, Fusarium, Phytophthora, Pseudomonas, Erwinia Alternaria, Trichoderma, Monilinia, Puccinia, Rhizoctonia, Phythium, And caused by an organism selected from the group consisting of: Plasmopara 7. The method according to 6. 8. 7. The method according to claim 6, wherein the supernatant is refrigerated or frozen before application to the plant. 8. The method according to claim 7. 9. The method according to any one of claims 6 to 8, wherein the supernatant is diluted. 10. The composition is applied as a wet powder, granules, aqueous fluid, dry fluid 10. Any of claims 6 to 9 wherein said microcapsules are or are microencapsulated in a suitable substance. Or the method of claim 1. 11. The method according to claim 6, wherein the plant is a fruit-bearing plant. 12. 7. The method of claim 6, wherein said plant is a vegetable plant. 13. The method according to claim 6, wherein the plant is an ornamental horticultural plant. 14． The plant according to claim 11, wherein the fruit is a strawberry or a strawberry. the method of. 15. A method for inhibiting Botrytis cinerea infection in a plant, comprising the steps of: Applying the composition described above to the plant in an amount effective to inhibit the growth of Botrytis cinerea. A method comprising the steps of: 16. A method for inhibiting Fusarium infection in a plant, comprising the following steps: 3. The composition of claim 2 in an amount effective to inhibit Fusarium growth to said plant. A method comprising the step of applying. 17． A plant treated with the composition of claim 2. 18. The composition is applied in combination with at least one chemical pesticide The method of claim 6, wherein: