EP0938260A1 - Lutte biologique contre les infections fongiques des vegetaux - Google Patents

Lutte biologique contre les infections fongiques des vegetaux

Info

Publication number
EP0938260A1
EP0938260A1 EP97947585A EP97947585A EP0938260A1 EP 0938260 A1 EP0938260 A1 EP 0938260A1 EP 97947585 A EP97947585 A EP 97947585A EP 97947585 A EP97947585 A EP 97947585A EP 0938260 A1 EP0938260 A1 EP 0938260A1
Authority
EP
European Patent Office
Prior art keywords
plant
atcc
composition
growth
fruit
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97947585A
Other languages
German (de)
English (en)
Inventor
Pamela Gail Marrone
Sherry D. Heins
Denise C. Manker
Desmond R. Jimenez
Richard K. Bestwick
George J. Cinvestav del IPN VANDEMARK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Exelixis Plant Sciences Inc
Original Assignee
Exelixis Plant Sciences Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/746,893 external-priority patent/US5753222A/en
Application filed by Exelixis Plant Sciences Inc filed Critical Exelixis Plant Sciences Inc
Publication of EP0938260A1 publication Critical patent/EP0938260A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

Definitions

  • the present invention relates to a strain of B. subtilis for inhibiting growth of plant pathogenic fungi, and to methods for protecting a plant against fungal and bacterial infections.
  • Botrytis cinerea Pers Plants are susceptible to attack by a variety of phyto-pathogenic fungi.
  • Botrytis cinerea Pers is a particularly damaging plant phytopathogenic fungus.
  • plant diseases caused by Botrytis sp. are some of the most widely distributed and common diseases of greenhouse-grown crops, field crops, vegetables, ornamentals, and fruits throughout the world.
  • Botrytis, B. cinerea is the causal agent of several severe fruit diseases, including grey mold of strawberry (Fragaria ' ananassa Duchesne) and grapevine (Vitis vinifera L.) (Agrios, 1988).
  • Botrytis-related diseases cause losses not only in the field, but also in storage, in transit, and in the targeted wholesale and retail markets.
  • phytopathogenic fungi is of significant economic importance, since fungal growth on plants or parts of plants (e.g., seeds, fruits, blossoms, foliage, stems, tubers, roots, etc.) can inhibit production of foliage, fruit, or seeds, as well as reduce the quality and quantity of the harvested crop. Although most crops are treated with agricultural fungistats or fungicides, fungal damage to agricultural crops typically results in revenue losses to the agricultural industry of millions of dollars annually.
  • the present invention is based on the discovery of a new strain of Bacillus subtilis, designated herein as B. subtilis strain ATCC No. 55614, which is effective in inhibiting growth of plant pathogenic bacteria and fungi.
  • the invention also encompasses a method for protecting a plant against fungal or bacterial infection by applying to the plant or its environment B. subtilis strain ATCC 55614, a supernatant obtained from a culture of the isolate, or a bioactive extract thereof.
  • the invention is directed to a biological culture of B. subtilis strain ATCC 55614.
  • Various embodiments include a bioactive extract of B. subtilis strain ATCC 55614, a supernatant obtained from a culture of the isolate, and a composition containing one or more of the above.
  • the composition may contain B. subtilis strain ATCC 55614 in an impure state or in the form of a biologically pure culture, and is effective to inhibit fungal and bacterial growth.
  • the composition is effective to inhibit growth of Botrytis cinerea.
  • the composition is effective to inhibit growth of Fusarium.
  • the present invention is also directed to a method for producing a B. subtilis extract having antimicrobial activity.
  • the extract is isolated by extracting the cell culture medium of bacterial isolate ATCC 55614 to produce a crude extract, separating the crude extract on a solid support to produce separated fractions, screening the separated fractions for antifungal or antibacterial activity, and pooling the active fractions.
  • the invention provides a method of 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.
  • infectable surfaces of a plant susceptible to fungal or bacterial disease are coated with Bacillus subtilis strain ATCC 55614, a supernatant or an extract thereof.
  • the invention thus provides a method for protecting a plant against infection caused by various plant phytopathogenic fungi, e.g, Diplodia, Drechslera, Fusarium, Geotrichum, Sclerotinia, Sclerotium, Erysiphe, Podosphaera, Uncinula, Puccinia, Plasmopara and Stemphylium.
  • a method for inhibiting Botrytis cinerea or Fusarium infection in a plant includes a method for inhibiting growth of vegetative hyphae, and for inhibiting the formation of sclerotia by B. cinerea or Fusarium, by applying a composition containing Bacillus subtilis strain ATCC 55614 or an extract thereof, to a plant or its environment. Also provided is a method of inhibiting germination of conidia of B. cinerea or Fusarium.
  • the methods of the invention are useful for protecting fruit-bearing plants, vegetable plants, flowering plants, and their associated post-harvest crops, against fungal or bacterial infection.
  • FIGS. 1 A and IB are computer generated photographs of PDA plates containing B. cinerea alone (Fig. 1 A; negative control), and B. cinerea in combination with B. subtilis isolate ATCC 55614 (Fig. IB);
  • Figs. 2A and 2B show computer generated photomicrographs of conidial germination on a control PDA plate (Fig. 2 A) and on a PDA plate containing a streak of B. subtilis ATCC 55614 (Fig. 2B);
  • Figs. 3 A and 3B show the percentage of total harvest weight due to diseased fruit (y-axis), at harvest time (Fig. 3A), and after storage at 40°C for 7 days under 95% RH (Fig. 3B), in harvests 1, 2 and 3 (x-axis) for fruit receiving various treatments (indicated);
  • Figs. 4A and 4B show the weight of healthy fruit (y-axis), at harvest time (Fig. 4A), and after storage at 40°C for 7 days under 95% RH (Fig. 4B), in harvests 1, 2 and 3 (x-axis) for fruit receiving various treatments (indicated); and
  • Figs. 5 A and 5B are computer generated photographs of PDA plates plated with Fusarium sp., and a commercial biocontrol agent, MYCOSTOP (Streptomyces griseoviridis strain 61) and Fusarium sp. and B. subtilis strain ATCC 55614, respectively.
  • B. subtilis strain ATCC 55614" includes mutants and variants thereof, particularly mutants and variants effective to inhibit growth of soil or air-borne fungal pathogens from the division Deuteromycota (e.g., Botrytis sp.).
  • the microorganism i.e., B. subtilis strain ATCC 55614
  • B. subtilis strain ATCC 55614 can be used in an impure state in combination with other materials which will not substantially interfere with the phytopathogenic fungi disease-suppressing characteristics of B. subtilis strain ATCC 55614, or in the form of a biologically pure culture.
  • a “biologically pure culture” is meant a culture of the microorganism that does not include other materials (i) which are normally found in soil in which the microorganism grows, and/or (ii) from which the microorganism is isolated.
  • the term “culturing” refers to the propagation of organisms on or in media of various kinds.
  • Whole broth culture refers to a liquid culture containing both cells and media.
  • Supernatant refers to the liquid broth remaining when cells grown in broth are removed by centrifugation, filtration, sedimentation or other means well known in the art.
  • biological control is defined as control of a pathogenic organism by the use of a second organism.
  • Known mechanisms of biological control include enteric bacteria that control root rot by out-competing fungi for space on the surface of the root.
  • 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 may be administered so it produces the toxin in situ.
  • a "bioactive" extract of B. subtilis strain ATCC 55614 is one which possesses the antimicrobial properties of B. subtilis strain ATCC 55614, i.e., is capable of inhibiting growth of a microorganism.
  • B. subtilis strain ATCC 55614 inhibits the growth of both bacteria and fungi.
  • a "microbial-suppressing amount" of 5. subtilis strain ATCC 55614 or an active compound produced thereby is an amount sufficient to suppress growth of a phytopathogenic microorganism by at least about 50% in comparison to microbial growth for an untreated plant.
  • the microbial-suppressing amount will be sufficient to suppress from about 60%-80% of fungal or bacterial growth occurring on an untreated plant, and will depend upon various factors such as soil condition, climate, plant type, planting conditions and the like.
  • an “effective amount” is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations. In terms of treatment and protection, an “effective amount” is that amount sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of fungal or bacterial disease states.
  • Plant as defined herein, includes any and all portions of a plant, including the root system, the shoot, including the stem, nodes, internodes, petiole, leaves, flowers, fruit, and the like, either prior to or post-harvest. Plant is also meant to include any cell derived from a plant, including undifferentiated tissue (e.g. , callus) as well as plant seeds, pollen, progagules and embryos.
  • undifferentiated tissue e.g. , callus
  • flowering plant any angiosperm.
  • angiosperms include nearly all of the plants that have been domesticated for agriculture, such as wheat, maize, beans, rice, oats, potatoes and soybeans, as well as ornamentals.
  • Ornamental flowering plant is meant to include flowering ornamentals such as orchids, petunias, zinnias, asters, begonia, geranium, lily, African violet and rose.
  • “Fruit-bearing plant” encompasses fruit-producing plants such as strawberry, raspberry, grapevine, tomato, pepper, cucumber, squash, melon, cantaloupe, watermelon, apple, peach, plum, nectarine, pear, sweetsop, cherimoya, banana, avocado, currant, persimmon, papaya, mango, guava and kiwifruit.
  • a “vegetable plant” is one which produces vegetables; examples of such plants include bean, beet, carrot, potato, spinach, celery, broccoli, cauliflower, cabbage and lettuce.
  • the present invention is based upon the discovery of a unique strain of B. subtilis, a spore-forming, aerobic, flagellate bacterium, which exhibits potent antifungal and antibacterial properties against a wide range of bacteria and filamentous and non-filamentous fungi.
  • B. subtilis strain ATCC 55614 The isolation and identification of B. subtilis strain ATCC 55614, will now be described.
  • the microorganism of the present invention a strain of Bacillus subtilis
  • a strain of Bacillus subtilis can be obtained from soil or from plant hosts.
  • Preferred sources of the bacterium are rhizosphere soil and root tissue from a raspberry plant.
  • One particularly preferred plant host is the raspberry cultivar, cv Meeker.
  • bacteria associated with the sample is isolated and cultured as follows.
  • the sample is typically washed with sterile water, soaked in phosphate buffered saline (PBS) solution, or surface sterilized.
  • PBS phosphate buffered saline
  • the tissue is typically chopped up and streaked onto agar medium, typically with a wire loop.
  • a number of different common agar media can be used to culture the bacteria, such as nutrient glucose agar (NDA; Difco, Detroit, MI), yeast extract- dextrose-calcium carbonate, nutrient-broth yeast extract agar (NBY), and Kings' medium B agar (KB), potato dextrose agar (PDA), the recipes for which are provided in Schaad (Schaad, 1988, page 3). Colonies typically appear on the medium in about 1-5 days.
  • NDA nutrient glucose agar
  • NBY nutrient-broth yeast extract agar
  • KB Kings' medium B agar
  • PDA potato dextrose agar
  • a streak test is conducted by first streaking single colonies of bacterial isolates on suitable agar media, such as PDA. The sample is incubated for about 2-5 days, followed by addition of a plug of fungal pathogen to the previously incubated culture, at a specified distance from the bacterial streaks. The resulting culture is then examined for areas in which growth of pathogen is inhibited.
  • Botrytis e.g., Botrytis cinerea
  • Additional exemplary fungal pathogens against which antifungal activity can be preliminarily assessed, such as by spot testing, include Fusarium, Diplodia, Drechslera, Fusarium, Geotrichum, Sclerotinia, Sclerotium, Erysiphe, Podosphaera, Uncinula, Puccinia, Plasmopara and Stemphylium.
  • screening of bacterial isolates obtained as described above resulted in the initial identification of several isolates capable of inhibiting mycelial growth of 5. cinera (Example 2).
  • the tests were performed on two different media, potato dextrose agar (PDA) and 25% tryptic soy agar (TSA).
  • PDA potato dextrose agar
  • TSA tryptic soy agar
  • strain ATCC 55614 Based upon its ability to severely inhibit growth of B. cinerea on both PDA and 25% TSA media, and its ability to effectively inhibit growth of Fusarium, this isolate, designated strain ATCC 55614, was chosen for further evaluation. The results of relevant in vitro screening assays are described in Examples 2 and 3. To summarize the results presented therein, strain ATCC 55614 was effective in inhibiting the growth of the fungal pathogen Botrytis cinerea by about 70% on both PDA and TSA media in comparison to untreated controls. Another screening assay that can be used to identify isolates effective to inhibit growth of various pathogenic fungi is a spot test or overlay test (Gross and Devay, 1977; Gross, et al, 1977).
  • agar-containing plate e.g., PDA
  • the colonies are then typically removed with a sterile swab and the plate is exposed to chloroform vapors for an extended period of time (e.g., 20 minutes) followed by dissipation of the chloroform vapors to kill remaining bacterial cells.
  • the plate is oversprayed with a spore suspension of fungal pathogen, and then examined for areas in which growth of fungal pathogen is inhibited.
  • Representative fungi against which antifungal activity can be preliminarily assessed, such as by spot testing, include but are not limited to those described above, i.e., Botrytis, Diplodia, Drechslera, Fusarium, Geotrichum, Sclerotinia, Sclerotium and Stemphylium.
  • a bacterial isolate is considered to be antagonistic against the growth of a given microbial pathogen if growth of the target phytopathogenic microbe is palliated ameliorated, stabilized, reversed, slowed or delayed, and preferably, if the progression of fungal or bacterial disease states inhibited by at least about 50 percent in comparison to microbial growth in the absence of the bacteria.
  • bacterial strain ATCC 55614 was characterized as Bacillus subtilis. Diagnostic tests for determining the identity of a bacterial isolate are described generally below.
  • Bacteria belonging to the species B. subtilis possess a number of characteristic features, which have been described in detail in Schroth, et al. (1983) and in Schaad (1988) and are representative of B. subtilis strain ATCC 55614.
  • subtilis Characteristic features of 5. subtilis are described briefly below.
  • LOP AT tests Levan formation, Oxidase test,
  • Potato rotting ability, Arginine dehydrolase production, and Tobacco hypersensitivity are useful for determining a number of identifying features of a bacterial isolate.
  • Bacteria belonging to the species B. subtilis will, on the whole, exhibit LOP AT characteristics as described in Schaad.
  • microplate testing method examines the ability of bacteria to utilize or oxidize a preselected panel of different carbon sources, as described in GN MICROPLATE INSTRUCTIONS FOR USE, BIOLOG, Hayward, CA September 1993.
  • bacterial isolate strain ATCC 55614 was identified as B. subtilis, as described in Example 4.
  • B. subtilis 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 Depository Authority on September 21, 1994.
  • B. subtilis strain ATCC 55614 is effective in inhibiting not only the growth of vegetative hyphae, but also in inhibiting sclerotia formation, e.g., by B. cinerea (Examples 3 and 5, respectively).
  • the data was also calculated as a ratio of mycelial growth to the number of sclerotia. If the reduction in sclerotia for the treated sample was simply a result of a reduced number of mycelia, the ratios for the control and B. subtilis-XxQdXQ ⁇ sample would be expected to be similar. Based upon the mean 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 sclerotia for a given number of mycelia (smaller denominator), and thus a greater value for the overall ratio in comparison to the control.
  • Bacillus isolate of the invention is effective in inhibiting not only the growth of vegetative hyphae, but also in inhibiting sclerotia formation, e.g., by B. cinerea.
  • B. subtilis isolate ATCC 55614 is effective in inhibiting germination of conidia of a fungal pathogen, e.g., B. cinerea, as supported by the illustrative results presented in Example 6.
  • Conidia which are produced by mycelium, are the primary source of inoculum in the field. The conidia (or spores) landing on or near a plant, germinate to produce mycelia, which then produce conidiophores with conidia, leading to growth of the fungus and the development of disease.
  • composition of the invention may contain B. subtilis strain ATCC 55614, a culture supernatant or an extract thereof.
  • the isolate is typically at least partially purified away from cellular components, to provide an extract having antimicrobial activity as described in section II above.
  • a bioactive extract produced by B. subtilis strain ATCC 55614 is obtained generally as follows.
  • B. subtilis strain ATCC 55614 is grown in potato dextrose broth, or Antibiotic Medium 3 (Difco Laboratories (Detroit, MI) as stationary cultures for a period of several days.
  • the culture medium is then extracted with a suitable solvent, e.g., ethyl acetate, chloroform, acetone, to remove various contaminating materials and obtain a bioactive extract.
  • the resulting bioactive extract can be further separated by any of a number of common separation techniques, such as adsorption chromatography.
  • Suitable solid support materials for chromatographic-based separations include gel filtration supports such as DEAE, C-18, for reverse phase applications, and XAD-type non-ionic supports. Recovered fractions are assayed for antifungal activity, such as by spot testing as described previously. Fractions exhibiting bioactivity are then pooled, and may be concentrated into dry form.
  • the resulting semi-purified preparation containing a bioactive component may then be used directly for treating fungal or bacterial infection, such as in topical applications (e.g., to inhibit fungal or bacterial growth in plants).
  • compositions containing B. subtilis strain ATCC 55614 may contain the isolate (i) in an impure state (e.g. , culture broth) in combination with other materials which will not adversely impact the phytopathogenic -disease suppressing characteristics of the isolate, nor adversely affect the health of the target plant or surrounding environment, (ii) as a biologically pure culture, (iii) a supernatant, or (iv) as an extract.
  • the composition may include bioactive component mutants and variants of B. subtilis strain ATCC 55614, or a mixture thereof, particularly mutants and variants exhibiting phytopathogenic disease- suppressing characteristics that are substantially the same or improved over B. subtilis strain ATCC 55614.
  • Bacillus subtilis strain ATCC 55614 is grown using conventional techniques as described previously. Cultivation of B. subtilis can be carried out in either liquid or in solid nutrient media at a temperature of about 220-3 OOC. If limited amounts of the microorganism are desired, surface cultures and bottles may be employed.
  • the microorganism is typically grown in a nutrient medium containing a carbon source, such as, an assimilable carbohydrate, and a nitrogen source, e.g., an assimilable nitrogen compound or proteinaceous material.
  • a carbon source such as, an assimilable carbohydrate
  • a nitrogen source e.g., an assimilable nitrogen compound or proteinaceous material.
  • Representative carbon sources include glucose, brown sugar, sucrose, starch, lactose, and the like.
  • Representative nitrogen sources include yeast, soybean meal, cornmeal, milk solids, and such.
  • Trace metals e.g., zinc, magnesium, cobalt, iron may optionally be added to the fermentation media.
  • a vegetative inoculum in a nutrient broth culture is typically prepared, e.g. , by inoculating the broth culture with an aliquot from a soil, root tissue, or slant culture, and transferred aseptically to a large vessel or tank.
  • the active ingredient portion of a composition in accordance with the invention can contain from about 0.001% to about 50% by weight, and preferably from about 0.01% to about 30% by weight, of Bacillus subtilis endospores.
  • composition of the present invention may contain one or more biologically inert components, e.g., carrier materials such as talc, gypsum, kaolin, attapulgite, wood flour, and/or binders such as ethylene glycol, mineral oil, polypropylene glycol, polyvinylacetate, nutrients, and plant growth hormones.
  • carrier materials such as talc, gypsum, kaolin, attapulgite, wood flour, and/or binders such as ethylene glycol, mineral oil, polypropylene glycol, polyvinylacetate, nutrients, and plant growth hormones.
  • the antimicrobial composition can be formulated into a variety of formulations including powder, aqueous, flowable, dry flowable, or granular applications.
  • the composition may also be microencapsulated.
  • Powder formulations are generally prepared by mixing together the dry components, including any carrier and/or other additive(s), until a homogeneous mixture is formed. A binder, if employed, may then be added and the entire mass mixed again until it has become essentially uniform in composition.
  • a liquid formation may, for example, include organic solvents such as xylene, methanol, ethylene glycol and mineral oil. Additional components may include surface active agents, e.g., calcium dodecylbenzenesulfonate, polyglycol ether, ethoxylated alkyl phenol or alkyl aryl sulfonates.
  • the liquid formulation may be a aqueous-based suspension. Such formulations will typically contain about 10 6 - 10 9 colony forming units (CFU) of B. subtilis strain ATCC 55614 per ml of aqueous carrier.
  • an aqueous carrier may optionally contain a wax such as paraffin wax.
  • the carrier can be an inorganic or organic material.
  • Representative examples include attapulgite, montmorillonite, bentonite, wood flour, starch, cellulose, bran, etc.
  • the formulation may also include a binder such as mineral oil, lignosulfonate, polyvinyl alcohol or sucrose, to maintain granular integrity.
  • composition of the invention can be used together with one or more additional fungicidal or pesticidal materials.
  • fungicidal or pesticidal materials include, for example, organochlorine compounds such as lindane (1,2,3,4,5,6-hexachlorocyclohexane (gamma isomer)); organophosphoric esters such as diazinon (0,0-diethyl 0-2-isopropyl-6-methylpyrimidin-4-yl phosphorothioate), isazofos (0-5-chloro- 1 -isopropyl- 1 H- 1 ,2,4-triazol-3-yl- 0,0-diethyl phosphorothioate), thiofanox
  • 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 percent of its original viability after storage of the composition for a period of up to about 36 months.
  • the method of the present invention comprises applying to plants an effective amount of the biocontrol composition described herein.
  • the composition can be utilized for either field or post harvest application.
  • the composition can be applied to any portion of a plant including its foliage, fruit, root system, or may be employed as a seed dressing.
  • the composition is generally applied to seed at a rate of from about 125 to about 2000 grams, and preferably at a rate of from about 250 to about 750 grams per 100 kilograms of seed.
  • B. subtilis strain ATCC 55614 is used to coat infectable surfaces of a plant susceptible to fungal- or bacterial-promoted disease.
  • the isolate will typically be formulated either as a liquid (for spray applications), an aerosol, or powder (for dusting infectable plant surfaces) as described above.
  • the strain may be used for systemic treatment of plant fungal diseases, for uptake by the root system.
  • the isolate is typically formulated as granules, usually for convenience in application. Granule formulations are typically activated by application of water, and release of active compound typically occurs over an extended period of time, such as from 2-12 weeks.
  • plant protecting compositions in accordance with the invention exhibit potent protection against phytopathogenic fungi, particularly Botrytis, Phytophthora, Pythium, Rizoctonia, Alternaria, Monilinia and Fusarium, and also against the gram-negative bacterium, Erwinia.
  • a method for forming a fungal- or bacterial-resistant transgenic plant.
  • 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 plant-compatible promoter effective to drive expression of the chimeric gene, and encodes a product effective to confer resistance to a fungal or bacterial pathogen (e.g., Botrytis, Fusarium) in transformed plant cells.
  • a fungal or bacterial pathogen e.g., Botrytis, Fusarium
  • subtilis strain ATCC 55614 is first identified, e.g., by mutagenesis of B. subtilis. Prior to mutagenesis, the ability of wildtype B. subtilis strain ATCC 55614 to antagonize growth of B. cinerea is confirmed. Mutagenis of B. subtilis is then carried out according to standard protocols. Following mutagenesis, mutants are assayed for their ability to inhibit vegetative mycelial growth essentially as described above and in Example 2. Putative mutants deficient in antagonism against B. cinerea are then used to identify a genetic locus attributable to the antagonistic phenotype of B. subtilis ATCC 55614.
  • 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 (e.g., Botrytis, Fusarium) to the plant. Additionally, the transgene may be introduced into a prokaryotic or eukaryotic organism to form a recombinant organism capable of producing the above-described antifungal compound secreted by B. subtilis strain ATCC 55614. V. Utility
  • B. subtilis strain ATCC 55614 is effective in inhibiting growth of plant pathogenic fungi including but not limited to Botrytis, Phytophthora, Pseudomonas, Erwinia, Alternaria, Trichoderma, Monilinia, Puccinia, Rhizoctonia, Phythium and Plasmopara and Fusarium, and is also effective for preventing the development of plant diseases associated with these pathogens.
  • the bioactive strain can be used to treat plant diseases such as seedling damping off and root rot disease, vascular wilt diseases, or any disease state caused by attack of one of the above-described fungal or bacterial plant pathogens (e.g., Table 13).
  • plant diseases such as seedling damping off and root rot disease, vascular wilt diseases, or any disease state caused by attack of one of the above-described fungal or bacterial plant pathogens (e.g., Table 13).
  • the composition of the invention is useful for preventing Tiotry/w'-related diseases such as grey mold of strawberry and raspberry, bunch rot of grapes, grey mold rot of vegetables such as bean, beet, carrot, and cucumber, tip-end rot of lettuce, pepper, tomato, and squash, dry eye rot disease in apple, and grey mold or blight of numerous ornamentals such as begonia, geranium, lily, African violet and rose.
  • the composition can also be used to treat vascular wilt diseases induced by Fusarium, such as wilt diseases of tomato, peas, bananas and cotton.
  • composition and method of the invention may be employed to protect commercial raspberry cultivars such as Canby, Chilcotin, Amity, and Willamette against grey mold. These cultivars are particularly susceptible to damage by grey mold caused by Botrytis. Similarly, the composition and method described herein can be used to protect commercial varieties of strawberry susceptible to attack by grey mold, e.g., Chandler, Pajaro, and Selva.
  • Example 7 greenhouse tests conducted in support of the invention (Example 7) revealed the effectiveness of B. subtilis strain ATCC 55614 in reducing losses in greenhouse grown strawberries (cv Pajaro) due to grey mold caused by B. cinerea. The details of the study are summarized briefly below.
  • Example 7 strawberry plants inoculated with B. cinerea were subjected to various treatment regimes.
  • the treatments included 5 foliar applications of (i) Rovral 50 VP, a commercially available fungicide used for controlling Botrytis fruit mold on strawberries, (ii) potato dextrose broth, (iii) B. subtilis strain ATCC 55614, and (iv) an untreated control series.
  • the plants, including the flowers, were treated prior to inoculation.
  • Test results were evaluated based upon a number of factors, including numbers of healthy fruits, weight of healthy fruit recovered, weight of diseased fruit, and percent of total harvest weight attributable to diseased fruit.
  • foliar treatment with a composition containing B. subtilis strain ATCC 55614 was effective in controlling fruit losses due to grey mold, as indicated by the quantity of diseased fruit expressed as a percentage of total harvest weight, for both treated and untreated (control) fruit. Additionally, treatment with B. subtilis strain ATCC 55614 did not adversely impact the yield (i.e., weight) of healthy strawberry fruit.
  • the antifungal composition described herein can also be applied to grape varieties such as White Riesling and Pinot Noir, Chardonnay, Zinfandel and Chenin blanc for preventing grey mold infection. These varieties are all susceptible to attack by B. cinerea (Pscheidt, 1990, 1991; 1993 PACIFIC NORTHWEST PLANT DISEASE CONTROL HANDBOOK; English, et al, 1989; Gubler, et al, 1987).
  • Bacillus isolate of the invention is effective in inhibiting both (i) the growth of vegetative hyphae, and (ii) sclerotia formation, e.g., by B. cinerea (Examples 3 and 5, respectively).
  • B. cinerea on long lived perennial crops (e.g., grape and raspberry)
  • sclerotia that overwinter on dead debris.
  • the sclerotia germinate in the spring and initiate infection of the new growing season (Weller, 1988).
  • application of a composition containing B. subtilis strain ATCC 55614, an extract or bioactive metabolite thereof, may be used for reducing the source of inoculum overwintering on dead, necrotic, or senescent tissue.
  • B. subtilis isolate ATCC 55614 is also effective for inhibiting the germination of conidia of B. cinerea (Example 6).
  • the compositions and methods described herein can be used as a preventative measure for reducing Botrytis-related losses.
  • the antifungal composition may also serve as a control for disparate genera of phytopathogenic fungi that are members of the classes Ascomycot ⁇ and Deuteromycot ⁇ (Fungi imperfecti).
  • Representative genera include Verticillium sp., Fusarium sp., Macrophomina sp., Thielaviopsis sp. , and genera of fungi that are causal agents of downy mildew diseases.
  • an antifungal composition comprising an extract produced by B. subtilis strain ATCC 55614 may be used to treat human fungal diseases in which the disseminated disease propagule is a conidia, for example, Aspergillus sp., Histoplasma sp., and Tinea sp.
  • a conidia for example, Aspergillus sp., Histoplasma sp., and Tinea sp.
  • PDA potato dextrose agar
  • PB potato dextrose broth
  • TSA tryptic soy agar
  • MEA malt extract agar
  • Botrytis cinerea B. cinerea
  • ATCC American Type Culture Collection
  • Rockville, MD Accession number 11542
  • Bacteria were isolated from rhizosphere soil and from root tissue of raspberry plants (cv. Meeker) located at Washington State University - ARS (Vancouver, WA).
  • Soil and root materials were placed in a test tube.
  • Five volumes of phosphate saline buffer (PSB, pH 7.3; Wollum, 1982) were added and the tube was vortexed for 1 minute.
  • a dilution series (10 "1 to 10 "8 ) was made using PSB.
  • One hundred 11 of each dilution was plated onto petri dishes containing PDA (ATCC MEDIA HANDBOOK, 1984). Plates were incubated at 200C in a growth chamber employing a 16 hour day length.
  • Single colonies of bacterial isolates were streaked in a straight line approximately 12 mm from the edge of a 100 mm ' 15 mm petri dish (plate) containing PDA.
  • the streaked plate was incubated for 2 days in a growth chamber as in Example 1 above. Following the 2 day incubation period, a 5 mm plug of B. cinerea hyphae grown on PDA was added to each plate approximately 55 mm from the edge of the bacterial streak closest to the center of the plate. Control plates lacking bacterial streaks were similarly inoculated to assess the growth of B. cinerea 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 growth of B. cinerea.
  • Putative antagonistic isolates were identified and retested on PDA plates, as well as on plates containing 25% tryptic soy agar (TSA).
  • Bacterial isolates identified in the initial screen as having antifungal activity were then retested using 10 replica plates of each PDA and 25% TSA, to determine the relative ability of each isolate to inhibit growth of B. cinerea.
  • the 12 isolates shown to inhibit growth of B. cinerea were at least partially tested for their ability to inhibit growth of Fusarium sp.
  • the tests were carried out essentially as described above for antagonism against B. cinerea, using PDA plates onto which a 5 mm hyphal plug of Fusarium sp. was introduced.
  • strain ATCC 55614 was essentially as effective as a known commercial biocontrol agent, "MYCOSTOP" (Streptomyces griseoviridis strain 61) in inhibiting growth of Fusarium sp.
  • isolate ATCC 55614 was selected for further analysis based on its ability to severely inhibit growth of B. cinerea on both PDA and 25% TSA plates. This isolate was designated isolate ATCC 55614.
  • Figures 1A and IB show computer generated photographs of PDA plates on which are plated B. cinerea and Isolate ATCC 55614 (Fig. IB), and a negative control plate containing only B. cinerea (Fig. 1A). The images were obtained 14 days after application of a 5 mm hyphal plug of B. cinerea.
  • the isolate identified above as having activity against B. cinerea was further characterized using conventional methods for identifying plant pathogenic bacteria (Schaad, 1988). Specifically, the identify of the isolate was determined using GC-FAME analysis and the Biolog Microtiter System. The Biolog Microtiter system is based upon an isolate's ability to utilize or oxidize a preselected panel of 96 different carbon sources using the GN MICROPLATEO test panel (Biolog, Hayward, CA), according to the manufacturer's recommended protocol (GN MicroplateO, Instructions for Use, Biolog, Inc., Hayward, CA, 1993). Wells in which no reaction occurred remained colorless; positive results were indicated by the appearance of a purple color. The test results were processed using the MICROLOG 30 computer software program available through Biolog, Inc. (Hayward, CA), which automatically cross-references the pattern of purple wells to an extensive library of species.
  • isolate ATCC 55614 was identified as an antimycotic-producing strain of Bacillus subtilis (B. subtilis).
  • Bacterial isolate B. subtilis ATCC 55614 has been deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852, and assigned an ATCC designation number 55614. The deposit was received by the ATCC on September 21, 1994.
  • ATCC American Type Culture Collection
  • Sclerotia counting Due to the possibility that any reduction in the number of sclerotia counted in the assay was a consequence of the reduction in growth of vegetative mycelia, the ratio of mycelial growth to the number of sclerotia was calculated for each condition.
  • Range (#) indicates the total number of sclerotia present on a plate, while the column with the heading Range represents the ratio of mycelial growth to number of sclerotia.
  • Conidia were produced in culture by growing B. cinerea on malt extract agar (MEA) for 5 days at 25°C with a 12 hr day length, followed by exposing the plates to ultraviolet (UV) light for 3 days (Leifert, et al, 1993). The plates were then flooded with 10 ml double deionized H 2 O and the conidia were removed from conidiophores by gently scraping the plates with a rubber policeman. The liquid was pipetted from the plates and filtered 4 times through a double layer of cheesecloth. The resulting suspension was vortexed for 1 minute to separate the conidia into individual propagules. The density of conidia per ml of double deionized H 2 O was determined using a hemocytometer, and the suspension was diluted to achieve a stock concentration of 1.0 ' 10 5 conidia/ml.
  • UV ultraviolet
  • 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 on each plate for germination. Exemplary photomicrographs of conidial germination obtained in this experiment are presented in Figures 2 A and 2B.
  • the image in Figure 2 A illustrates complete germination of conidia on control PDA plates.
  • the image in Figure 2B presents an example of inhibition of germination of conidia that were placed 1 cm away from the streak of B. subtilis.
  • B. subtilis ATCC 55614 The efficacy of B. subtilis 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 the control of Botrytis fruit mold on strawberries.
  • Rovral 50 WP IPRODIONE
  • Strawberry plants (cv. Pajaro) were obtained as rooted crowns from BHN Research (Watsonville, CA). The plants were potted into 6" plastic pots (one plant/pot) filled with BlackGold 100% organic soil (BlackGold, Inc., Hubbard, OR). Plants were watered, fertilized regularly with Peters 20:20:20 fertilizer and grown under an 18 hour light/6 hours dark cycle.
  • Treatments included foliar applications of (i) Rovral 50 VP at 1.5 lb/100 gallons water, (ii) no treatment (control), (iii) PDB alone, or (iv) Bacillus subtilis strain ATCC 55614 (5 x 10 "8 ) CFU/ml in PDB).
  • the treatments were administered by spraying plants with a hand sprayer containing one of the above compositions. Plants were sprayed to run-off (approximately 75 ml/plant). Treatments (i) through (iv) were applied 5 times during the test. The first application was at 10% flower bloom and the next four were at two week intervals thereafter. The fifth treatment was applied one day before the first harvest. D. Artificial Greenhouse Inoculation of Strawberry Plants with B. cinerea
  • Plants were inoculated with B. cinerea by spraying to run-off with a 4 X 10 "3 conidia ml suspension (approximately 75 ml/plant) in three spraying sessions. The first spraying was administered on the day that the first inflorescence was observed, and the next two were administered at two week intervals thereafter.
  • All ripe berries were harvested from all plants, once per week, for three consecutive weeks. The first harvest was the day following the last treatment.
  • Treatments were conducted on "blocks" of 5 plants each and each treatment was replicated 4 times (A, B, C, and D), resulting in 20 plants per treatment.
  • the y-axis in Fig. 3 A shows the percent of harvest weight due to diseased fruit at each of three harvests (1, 2 and 3) indicated on the x-axis.
  • the mean weight (and percent of total) of diseased fruit treated with Rovral and B. subtilis ATCC 55614 was significantly less than the control treatment.
  • the data segregated into two subgroups The mean weight (and percent of total) of diseased fruit from samples treated with Rovral or Bacillus was significantly less than the weight and percentage of diseased fruit from samples that had received either no treatment (control), or samples treated only with PDB. No significant differences were observed within the two subgroups.
  • the mean percentage of diseased fruit was significantly less in samples treated with Rovral than in samples receiving any of the other three treatments.
  • the y-axis in Fig. 3B shows the percent of harvest weight due to diseased fruit at each of three harvests (1, 2 and 3) indicated on the x-axis.
  • the mean weight (and percent of total) of diseased fruit treated with Rovral or Bacillus was significantly less than corresponding measurements from samples receiving control treatments.
  • the mean weight (and percent of total) of diseased fruit subjected to PDB and control treatments were significantly higher than corresponding measurements from samples receiving Rovral or Bacillus treatments.
  • the mean percentage of diseased fruit from the third harvest was significantly less in samples treated with Rovral than in samples receiving any of the other three treatments. No consistent significant differences were observed between control and PDB treatments in both freshly-analyzed and stored fruit.
  • the y- axis in Fig. 4 A shows the weight of healthy fruit (in grams) at each of three harvests (1, 2 and 3) indicated on the x-axis.
  • the y-axis in Fig. 4B shows the weight of healthy fruit (in grams) at each of three harvests (1, 2 and 3) indicated on the x-axis.
  • 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.
  • ATCC 55614 was grown in potato dextrose broth (PDB) (Difco) for three days. To test whole broth cultures, the strain was grown to approximately 1 x 10 6 to 6 x 10 6 CFU/mL and aliquots were taken from these cultures. Supernatant was obtained by density centrifugation of the culture at 5,200 rpm for 20 minutes.
  • PDB potato dextrose broth
  • Trichoderma harzianum strain T-14
  • Trichoderma harzianum strain T-14
  • a number four cork borer was used to make four wells in PDA plates.
  • ATCC 55614 was applied to one of the four wells.
  • Two discs of number four cork borer- sized Trichoderma mycelial plugs were added to each plate in between two wells on each side of the plate. Results were recorded 24 hours later. The size of the cleared zone between the bacterium and the mycelium was recorded.
  • ATCC 55614 produced a 4 mm zone.
  • EXAMPLE 9 Fungicidal Activity of ATCC 55614 Using Whole Plants The ability of ATCC 55614 to control late blight (P. infestans) infection was tested on whole tomato plants. Tomato plants (Ace and Patio varieties) were purchased from Ace hardware and transplanted into 6 packs having three plants per pack. ATCC 55614 was grown in Trypticase Soy Broth (TSB) (Difco) for 72 hours and reached a concentration of 5 X 10 6 CFU/mL. One plant of each variety of tomato plant was sprayed to runoff with a whole broth culture or supernatant of ATCC 55614 and then air-dried at approximately 21 °C. Two control plants were untreated. All plants were then sprayed to runoff with a P.
  • Tomato plants Ace and Patio varieties
  • Tomato plants Ace and Patio varieties
  • ATCC 55614 was grown in Trypticase Soy Broth (TSB) (Difco) for 72 hours and reached a concentration of 5 X 10 6
  • ATCC 55614 of the present invention was utilized.
  • 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 testing.
  • ATCC 55614 was grown in either half-strength TSB or in potato dextrose broth (PDB) and the broth or supernatant tested without freezing. Whole broth cultures and supernatants were sprayed onto the strawberries until runoff, then allowed to air dry.
  • PDB potato dextrose broth
  • B. cinerea spores were grown on potato dextrose agar in a petri plate and scraped into de-ionized water to form a liquid inoculum.
  • the B. cinerea inoculum measuring approximately 5.8 x 10 5 cells per mL was sprayed onto the berries until runoff, and the berries allowed to air dry.
  • test #1 the berries were placed inside a cardboard container with plastic wrap lid at 25°C.
  • test #2 all berries were place uncovered in an incubator at approximately 16°C. Results are shown in Table 12.
  • ATCC 55614 frozen supernatant was completely effective at inhibiting B. cinerea infection on live strawberry plants.
  • the whole broth culture of ATCC 55614 was completely effective at preventing B. cinerea infection, regardless of the medium used.
  • Supernatant from ATCC 55614 grown in TSB was partially effective but, when grown in PDB, was 100% effective against B. cinerea.
  • ATCC 55614 against a number of fungal pathogens
  • the strain was grown in potato dextrose broth. Cells were cultured to 5 x 10 6 cells/mL. Replicates of three test plants and three control plants per pathogen were utilized.
  • the test plants were each sprayed with a whole broth culture of ATCC 55614 to run-off with a hand-held sprayer. When the foliage had dried, each test plant was sprayed a second time. After the second application of the bacterial strain culture had dried, the test plants and the control plants were inoculated with the appropriate fungal pathogen. Plants were incubated under conditions conducive to disease development.
  • ATCC 55614 provided complete control of 5. cinerea. ATCC 55614 was also highly active against grape downy mildew and leaf rust, and had slight activity against Phytophthora in this test. EXAMPLE 12
  • a 250 mL culture of ATCC 55614 was grown for 3.5 days in PDB as previously described.
  • Peaches were purchased from a local grocery store (Safeway) and were surface sterilized with a 10% Clorox solution, rinsed with deionized water and air dried.
  • Whole fermentation broths of ATCC 55614 (8.7 x 10 6 CFU/mL) were sprayed with a hand-held sprayer on two peaches until runoff (approximately 50 mL per two peaches). The peaches were allowed to air dry. Monilinia spores were scraped from a petri plate and suspended in deionized water to a concentration of 1.09 x 10 5 spores/mL.
  • the peaches were then sprayed with the spore suspension until runoff and allowed to air dry. Two peaches were untreated and two peaches were sprayed with Monilinia only. The peaches were placed in a polypropylene container in an incubator in the dark at 18°C for four or six days. The amount of brown rot on each peach was measured, as show in Table 15.
  • ATCC 55614 suppressed Monilinia brown rot compared to the untreated controls and Monilinia only peaches. After six days, the control peaches were all heavily infected with brown rot, while only one of the ATCC 55614 peaches showed infection. Furthermore, the one infected ATCC 55614 peach had a lesion which was much smaller than those in the untreated or Monilinia only controls.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Virology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Agronomy & Crop Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Plant Pathology (AREA)
  • Dentistry (AREA)
  • Environmental Sciences (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une souche unique de Bacillus subtilis qui permet d'inhiber la croissance des champignons et des bactéries phytopathogènes, ainsi que des méthodes permettant de traiter ou de protéger les végétaux des infections fongiques et bactériennes.
EP97947585A 1996-11-18 1997-11-18 Lutte biologique contre les infections fongiques des vegetaux Withdrawn EP0938260A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US08/746,893 US5753222A (en) 1996-11-18 1996-11-18 Antibiotic-producing strain of bacillus and methods for controlling plant diseases
US746893 1996-11-18
US5252997P 1997-06-27 1997-06-27
US52529P 1997-06-27
PCT/US1997/021149 WO1998021964A1 (fr) 1996-11-18 1997-11-18 Lutte biologique contre les infections fongiques des vegetaux

Publications (1)

Publication Number Publication Date
EP0938260A1 true EP0938260A1 (fr) 1999-09-01

Family

ID=26730713

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97947585A Withdrawn EP0938260A1 (fr) 1996-11-18 1997-11-18 Lutte biologique contre les infections fongiques des vegetaux

Country Status (8)

Country Link
EP (1) EP0938260A1 (fr)
JP (1) JP2001503642A (fr)
AU (1) AU720981B2 (fr)
BR (1) BR9713812A (fr)
CA (1) CA2271118A1 (fr)
IL (1) IL129837A0 (fr)
NZ (1) NZ335648A (fr)
WO (1) WO1998021964A1 (fr)

Families Citing this family (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030068303A1 (en) * 2001-05-11 2003-04-10 Selvig Thomas A. Biologic-chemical fungicide compositions and methods of use
US7097830B2 (en) * 2001-09-04 2006-08-29 Council Of Scientific And Industrial Research Synergistic bioinoculant composition comprising bacterial strains of accession Nos. NRRL B-30486, NRRL B-30487, and NRRL B-30488 and a method of producing said composition thereof
US7211428B1 (en) * 2004-05-18 2007-05-01 Council Of Scientific And Industrial Research Strain of Bacillus as a bioinoculant
US8329446B2 (en) 2008-02-26 2012-12-11 Monaghan Mushrooms Ltd. Green mold inhibitor
US8524223B2 (en) 2008-07-11 2013-09-03 University Of Yamanashi Bacillus subtilis KS1 as a plant disease control agent
HU231053B1 (hu) 2011-09-08 2020-03-30 Szegedi Tudományegyetem Rézrezisztens, fengicin hipertermelő Bacillus mojavensis törzs növényi kórokozók elleni védekezésre, alkalmazása és az ezt tartalmazó készítmények
ES2402726B1 (es) * 2011-10-28 2014-03-13 Investigaciones Y Aplicaciones Biotecnologicas, S.L. Nueva cepa de bacillus subtilis destinada a luchar contra las enfermedades de las plantas.
CN104507319B (zh) 2012-05-30 2018-08-03 拜尔农作物科学股份公司 包含生物防治剂和选自脂质膜合成抑制剂、黑素生物合成抑制剂、核酸合成抑制剂或信号转导抑制剂的杀真菌剂的组合物
WO2013178662A1 (fr) 2012-05-30 2013-12-05 Bayer Cropscience Ag Compositions comprenant un agent de lutte biologique et un insecticide
ES2689879T3 (es) 2012-05-30 2018-11-16 Bayer Cropscience Ag Composición que comprende un agente de control biológico y un fungicida seleccionado de inhibidores de la biosíntesis del ergosterol
CN104470359B (zh) 2012-05-30 2017-05-24 拜尔农作物科学股份公司 包括一种生物防治剂和一种杀虫剂的组合物
ES2698061T3 (es) 2012-05-30 2019-01-30 Bayer Cropscience Ag Composición que comprende un agente de control biológico y fluopicolida
US20150289514A1 (en) 2012-05-30 2015-10-15 Bayer Cropscience Ag Composition comprising a biological control agent and a fungicide selected from inhibitors of the respiratory chain at complex iii
BR122019010603B1 (pt) 2012-05-30 2020-06-30 Bayer Cropscience Ag composição compreendendo um agente de controle biológico e um fungicida, seus usos, semente resistente a fitopatógenos, e método para reduzir o dano total das plantas e partes de plantas
EP2854551A1 (fr) 2012-05-30 2015-04-08 Bayer Cropscience AG Compositions contenant un agent de lutte biologique et un fongicide appartenant au groupe constitué des inhibiteurs des complexes i ou ii de la chaîne respiratoire
JP6285423B2 (ja) 2012-05-30 2018-02-28 バイエル・クロップサイエンス・アクチェンゲゼルシャフト 生物農薬および殺虫剤を含む組成物
CN104507317B (zh) 2012-05-30 2019-11-15 拜尔农作物科学股份公司 包含生物防治剂和杀真菌剂的组合物、及其用途、试剂盒
US10306889B2 (en) 2012-05-30 2019-06-04 Bayer Cropscience Ag Compositions comprising a biological control agent and an insecticide
AU2013269662B2 (en) 2012-05-30 2016-12-15 Bayer Cropscience Ag Compositions comprising a biological control agent and an insecticide
KR20150119022A (ko) 2013-02-11 2015-10-23 바이엘 크롭사이언스 엘피 고제로틴 및 생물학적 방제제를 포함하는 조성물
CA2920729A1 (fr) 2013-08-12 2015-02-19 Bio-Cat Microbials Llc Compositions comprenant des souches de bacillus et leurs procedes d'utilisation pour supprimer les activites et la proliferation d'agents pathogenes fongiques de vegetaux
US9485994B2 (en) 2013-11-08 2016-11-08 The Regents Of The University Of California Synergy-based biocontrol of plant pathogens
WO2015160618A1 (fr) 2014-04-16 2015-10-22 Bayer Cropscience Lp Compositions comprenant de la ningnanmycine et un agent de lutte biologique
MX2017003452A (es) 2014-09-17 2017-07-28 Bayer Cropscience Lp Composiciones que comprenden celulas recombinantes de bacillus y otro agente de control biologico.
AU2019218391A1 (en) * 2018-02-12 2020-08-13 Boost Biomes, Inc. Microbial compositions for the prevention or reduction of growth of fungal pathogens on plants

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5047239A (en) * 1984-05-01 1991-09-10 The United States Of America As Represented By The Secretary Of Agriculture Biological control of fruit rot
US4764371A (en) * 1984-05-01 1988-08-16 The United States Of America As Represented By The Secretary Of Agriculture Postharvest biological control of stone fruit brown rot by bacillus subtilis
GB8805394D0 (en) * 1988-03-07 1988-04-07 Agricultural Genetics Co Antibiotic
JP2673718B2 (ja) * 1989-02-10 1997-11-05 呉羽化学工業株式会社 細菌による植物病害防除方法
SU1738200A1 (ru) * 1989-09-25 1992-06-07 Украинский Научно-Исследовательский Институт Садоводства Штамм бактерий BacILLUS SUвтILIS дл получени препарата против возбудител серой гнили земл ники
JP2948936B2 (ja) * 1991-03-14 1999-09-13 森永製菓株式会社 フザリウム属に属するカビが産生するカビ毒の防除方法
JP3132195B2 (ja) * 1992-10-26 2001-02-05 昭和電工株式会社 新規微生物および植物病害防除剤

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9821964A1 *

Also Published As

Publication number Publication date
IL129837A0 (en) 2000-02-29
AU5263298A (en) 1998-06-10
BR9713812A (pt) 2000-03-14
NZ335648A (en) 2000-10-27
WO1998021964A1 (fr) 1998-05-28
AU720981B2 (en) 2000-06-22
CA2271118A1 (fr) 1998-05-28
JP2001503642A (ja) 2001-03-21

Similar Documents

Publication Publication Date Title
US6004774A (en) Biological control of plant fungal infections
AU720981B2 (en) Biological control of plant fungal infections
WO1998021964A9 (fr) Lutte biologique contre les infections fongiques des vegetaux
US11771090B2 (en) Fungal endophytes for improved crop yields and protection from pests
US5527526A (en) Use of streptomyces bacteria to control plant pathogens
Rytter et al. Biological control of geranium rust by Bacillus subtilis.
US5344647A (en) Strain of microorganism having antimicrobial activity
Karabulut et al. Integrated control of postharvest diseases of sweet cherry with yeast antagonists and sodium bicarbonate applications within a hydrocooler
Agostini et al. Population dynamics and survival of strains of Colletotrichum gloeosporioides on citrus in Florida
US5549889A (en) Nematocidal and fungicidal Streptomyces dicklowii biopesticide
Jackson et al. In vitro screening for the identification of potential biocontrol agents of Allium white rot
US20080050344A1 (en) Biological control of plant diseases
Fiddaman et al. Screening of bacteria for the suppression of Botrytis cinerea and Rhizoctonia solani on lettuce (Lactuca sativa) using leaf disc bioassays
US6312940B1 (en) Bacillus species for reducing fusarium head blight in cereals
CA2323019C (fr) Bacteries et levures pour reduire la brulure de l'epi des cereales et selection de ces dernieres
Stirling et al. Isolation and selection of bacteria and yeasts antagonistic to preharvest infection of avocado by Colletotrichum gloeosporioides
MXPA99004568A (en) Biological control of plant fungal infections
Wu et al. Biological control of grape ripe rot and bitter rot
AU2001267948A1 (en) Biological control of plant diseases
EP0353689A2 (fr) Procédé pour lutter contre les maladies foliaires causées par des champignons pathogènes avec des bactéries fongicides et cultures pures de bactéries fongicides
KR100506721B1 (ko) 바실러스 리케니포미스 ｎ1 및 이를 포함하는 식물병원성 진균 방제용 미생물 제제
CN1241909A (zh) 植物真菌侵染的生物学控制
KR100411186B1 (ko) 아미노글리코시드계 항생제를 이용한 식물병 방제제
CA2011705C (fr) Methode et compose pour la lutte biologique contre les maladies des vegetaux
Schisler et al. Bacillus species NRRL B-30212 for reducing Fusarium head blight in cereals

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19990615

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE ES FR GB IT LI NL PT

17Q First examination report despatched

Effective date: 20010904

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20030130