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WO1997002749A1 - Bacillus licheniformis produisant des agents antifongiques et leurs utilisations dans la lutte contre des champignons phytopathogenes - Google Patents

Bacillus licheniformis produisant des agents antifongiques et leurs utilisations dans la lutte contre des champignons phytopathogenes Download PDF

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WO1997002749A1
WO1997002749A1 PCT/US1995/008536 US9508536W WO9702749A1 WO 1997002749 A1 WO1997002749 A1 WO 1997002749A1 US 9508536 W US9508536 W US 9508536W WO 9702749 A1 WO9702749 A1 WO 9702749A1
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plant
antifungal
spp
substance
microorganism
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Carlos A. Neyra
Lakshmi Sadasivan
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Rutgers State University of New Jersey
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Rutgers State University of New Jersey
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Priority to AU29650/95A priority Critical patent/AU2965095A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • 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
    • 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
    • A01C—PLANTING; SOWING; FERTILISING
    • A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
    • A01C1/08—Immunising seed
    • 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

Definitions

  • This invention relates to the control of fungal diseases of plants, and more specifically to strains of Bacillus licheniformis producing antifungal compounds and uses thereof for controlling a wide variety of phytopathogenic fungi in soils, on seed and on plants of agronomic importance.
  • Rhizobacteria have long been known for their role in promotion of plant growth and biological control of plant pathogenic microorganisms. It is believed that plant growth-promoting rhizobacteria act by displacing or antagonizing plant pathogenic microorganisms. Many of these rhizobacteria produce antibiotics, the production of which is strongly correlated with inhibition of various pathogenic microorganisms in vitro and disease suppression in vivo .
  • Pseudomonas cepacia and Bacillus species, have been reported to produce antibiotics and antifungal compounds that effectively control or suppress phytopathogenic fungi both in vitro and in vivo .
  • Bacteria of the genus Bacillus produce a variety of peptide antibiotics that are antibacterial and/or antifungal.
  • Bacillus species including B . subtilis, B . pumilus and B . cereus , have been shown to be antagonistic to plant pathogenic fungi and bacteria. For example, phytopathogen-antagonistic strains of B .
  • subtilis have been reported to produce two peptide antibiotics: bacilysin, a dipeptide that inhibits yeast and bacteria; and fengycin (fengymycin) , a lipopeptide antagonistic against phytopathogenic fungi such as Rhizoctonia solani .
  • bacilysin a dipeptide that inhibits yeast and bacteria
  • fengycin a lipopeptide antagonistic against phytopathogenic fungi
  • Rhizoctonia solani a lipopeptide antagonistic against phytopathogenic fungi such as Rhizoctonia solani .
  • Bacillus cereus produces mycocerein, another antifungal peptide ( akayama et al. (1984), Antimicrobial Agents and Chemotherapy __ : 939-940) .
  • Bacillus genus is divided into three groups, for purposes of identifying and classifying Bacillus species (Claus & Berkeley (1986) "Genus Bacillus Cohn 1872 174 AL “, pp. 1104-1139 in Bercrev's Manual of Systematic Bacteriology, Volume II (Sneath, Mair, Sharpe and Holt, eds.), Williams & Wilkins Co., Baltimore) .
  • Most Bacillus species reported to be useful as biological control agents belong to the "subtilis” group, which includes B . subtilis, B . cereus, B . pumilus and B . licheniformis , among others.
  • Bacillus licheniformis is industrially valuable for its ability to produce secreted products, such as thermally stable alpha amylases (See, e.g., Saito (1973) , Arch, of Biochem. & Biophys. 155: 290- 298; Yuuki et al. (1985), J. Biochem. 98.: 1147-1156).
  • Bacillus licheniformis is known for its ability to survive and proliferate in extreme environments, including high temperature and anaerobic conditions (B . licheniformis is a facultative anaerobe) .
  • Bacillus licheniformis is widely known to produce antibiotics such as bacillomycin, bacitracin, licheniformin and proticin (See Katz & Demain (1977) , Bacteriol. Rev. 41: 449-474).
  • antibiotics such as bacillomycin, bacitracin, licheniformin and proticin
  • See Katz & Demain (1977) Bacteriol. Rev. 41: 449-474.
  • the ability of this species to produce antifungal compounds, as well as its usefulness as a biological agent for the control of plant diseases has remained largely unexplored (See Loeffler et al. 1986, supra.).
  • Bacillus licheniformis can grow anaerobically and at elevated temperatures, it is capable of inhabiting a wide variety of rhizospheres, including waterlogged soils and/or soils exposed to extremes of heat, such as in semi-arid or arid regions, or tropical and sub-tropical areas. For this reason, it would be advantageous to identify strains of Bacillus licheniformis that are antagonistic to plant pathogenic fungi and that produce antifungal compounds. It would be a further advantage to identify such Bacillus licheniformis strains having an antagonistic effect on a wide variety of phytopathogenic fungi. Such organisms could be used for biological control of fungal plant pathogens on a broad range of plant species, against a large variety of pathogens. Insofar as is known, Bacillus licheniformis strains having such characteristics have heretofore not been identified.
  • One object of the present invention is to provide a microorganism having the identifying features of Bacillus licheniformis , which is antagonistic to a wide variety of fungal species, for use as a biological control agent of fungal plant diseases on horticulturally and agronomically important plants.
  • a microorganism having the identifying features of Bacillus licheniformis which is antagonistic to plant-colonizing fungi and which produces at least one antifungal compound, is provided for use in the biological control of phytopathogenic fungi on seeds and plants.
  • this microorganism possesses the identifying characteristics of Bacillus licheniformis strain PR1- 36a.
  • Bacillus licheniformis strain PRl-36a produces an antifungal principle that is active against a diverse array of fungal species. Another object of the present invention is to provide an antifungal principle produced by fungal- antagonistic strains of Bacillus licheniformis , for use in control of fungal plant diseases. According to another aspect of the present invention, an antifungal principle is provided, which is obtained from Bacillus licheniformis, preferably B . licheniformis strain PR1- 36a. This antifungal substance comprises one or more active components which exhibit antifungal effects on plant-colonizing fungal species.
  • microbial inoculants comprising fungal-antagonistic strains of Bacillus licheniformis , preferably strain PRl-36a. Methods are also provided for using such microbial inoculants for biological control of one or more fungal plant diseases.
  • methods are provided for producing an antifungal principle from fungal-antagonistic strains of Bacillus licheniformis , preferably B . licheniformis strain PRl-36a. Methods are also provided for using such antifungal principles for the control of fungal plant diseases.
  • antifungal compound-producing Bacillus licheniformis strains for control of fungal plant diseases provides numerous advantages with respect to other antifungal bacterial inocula currently available.
  • the diffusibility of the antifungal principle produced by Bacillus licheniformis contributes to its ease of purification, which is an additional advantage of B . licheniformis over other microbial inoculants.
  • the antifungal principle is stable for long periods of time, which is yet another advantage of the antifungal Bacillus licheniformis strains of the present invention.
  • X axis wavenumber (cm' 1 ) ; Y axis — absorbance.
  • FIGURE 2A Proton ('H) NMR spectrum (inset is expanded view of portion of larger spectrum) .
  • FIGURE 2B carbon ( 13 C) NMR spectrum. From top to bottom: 0-200
  • Bacillus licheniformis is antagonistic to a variety of plant pathogenic fungi, including several common pathogens of turf grass and other graminaceous crops, such as corn (Zea mays L.) . It has further been discovered in accordance with the present invention that Bacillus licheniformis appears to exert its antagonistic effect by producing one or more substances having antifungal activity. Insofar as is known, Bacillus licheniformis strains producing substances antagonistic to phytopathogenic fungi have not been identified.
  • the detailed description and examples set forth hereinbelow describe the isolation, identification and characterization of a Bacillus licheniformis strain, PRl-36a, , which is antagonistic to various soil-borne plant pathogenic fungi and which produces one or more antifungal principles against a broad spectrum of plant- and rhizosphere-associated fungi.
  • the term "antagonistic” as used herein refers broadly to the ability of one organism to negatively affect another organism, by mechanisms including, but not limited to, inhibiting growth of that organism, competing for nutrients, producing toxic substances and/or physical displacement of the organism.
  • the antifungal principle is also described and exemplified hereinbelow.
  • the antifungal principle may comprise one or more components.
  • antifungal principle, agent or compound refers to the plural as well as the singular.
  • antifungal refers broadly to the ability of an organism or agent to exert an inhibitory effect on a fungus. Such an effect may be classified as fungicidal, fungistatic, sporocidal, sporostatic, or a combination thereof.
  • PRl-36a isolate of Bacillus licheniformis is described and exemplified herein, such description is for the purpose of illustration, and is not intended to limit the invention to that strain. It will be appreciated by those skilled in the art that similar strains of B.
  • licheniformis having the identifying characteristics and antifungal properties described for PRl-36a may be isolated and purified from the rhizosphere of various plant species, according to the methods set forth herein by which strain PRl-36a was obtained.
  • Bacillus licheniformis strain PRl-36a was isolated from a rhizosphere of perennial ryegrass (Lolium perenne L.). The isolation of antifungal bacterial species from this rhizosphere and the purification and classification of B. licheniformis strain PRl-36a is described in detail in Example 1.
  • isolate PRl-36a was identified as an endospore forming Bacillus licheniformis strain able to grow aerobically at 50°C and anaerobically at 37°C.
  • B . licheniformis strain PRl-36a exhibited strong antagonism against filamentous fungi such as Rhizoctonia solani and Magnaporthe poae (see Example 1). Furthermore, imbibing corn kernels with B. licheniformis strain PRl-36a and sowing them in sand amended with inoculum of the corn pathogen, Fusarium ro ⁇ eum , resulting in the suppression of Fusarium seedling blight in controlled environmental chambers.
  • B. licheniformis strain PRl-36a produces a diffusible antifungal principle that can be obtained by acid precipitation of the culture filtrate followed by ethanol extraction, as described in detail in Example 2 herein.
  • a crude ethanolic extract (CEE) prepared from culture filtrates of the bacterium was found to be fungistatic against a broad spectrum of phytopathogenic fungi (See Table 3 of Example 2) , particularly filamentous fungi, causing malformation and swelling of the mycelium.
  • the CEE was stable at room temperature for more than six months.
  • the extract was resolved by thin-layer chromatography * (TLC) into one ninhydrin-positive band whose Rf value was 0.79 in an ethanol:water (2:1) solvent system.
  • the CEE was resolved by thin-layer chromatography into four biologically active ninhydrin-positive bands, having Rf values of 0.18, 0.21, 0.45 and 0.48. These ninhydrin-positive bands were all tested to be antifungal against
  • the biologically active ninhydrin-positive band having an Rf value of 0.46 in the chloroform:methanol:water (65:24:4) solvent system was analyzed by infrared (IR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, as described in Example 2 herein. Both analyses suggest that the compound associated with this band comprises a short peptide (4-6 amino acid residues) , and a lipid component.
  • IR infrared
  • NMR nuclear magnetic resonance
  • Bacillus licheniformis strain PRl-36a The analysis of the crude ethanolic extract from Bacillus licheniformis strain PRl-36a suggests that Bacillus licheniformis also contains one or more such peptide- and/or lipid- containing compounds, and that at least one of these compounds, or a combination thereof, is fungistatic against numerous filamentous and other phytopathogenic fungi.
  • Bacillus licheniformis is considered an innocuous rhizosphere microorganism whose presence in conjunction with various grasses and crop species is either non-deleterious or beneficial. Accordingly, the B. licheniformis strain PRl-36a described herein also demonstrated no phytotoxic properties, but instead has been found to be a plant growth promoting organism. This plant growth promoting ability may be due in part to its ability to protect seeds and plants from fungal colonization. For example, corn seeds inoculated with strain PRl-36a were protected from Fusarium blight when tested in a controlled environment (see Example 2) .
  • Bacillus licheniformis described herein demonstrates several desirable physiological properties, such as the ability to grow anaerobically and/or at elevated temperature, production of desiccation-resistant endospores, rapid growth rates in common media, plant growth promoting capabilities and production of antifungal substances against common phytopathogens, particularly those causing diseases in turf grasses and other cereal crops, such as corn. All of these properties render this microorganism very suitable for use as a biological control agent for plant diseases of many types, under a variety of environmental settings and constraints.
  • the microorganism is grown to stationary phase in a defined medium that favors the production of the antifungal substance or substances.
  • a suitable culture medium for growing the microorganisms of the invention is set forth in Example 2.
  • Other suitable growth media will be readily apparent to those of skill in the art. Cultures may be grown in batch culture as described in Example 2, or the culture may be scaled up and performed in small or large fermentors, according to standard methods. After growth to stationary phase (e.g., 3 days growth) cells are separated from the medium by centrifugation, and a bacterial pellet is harvested, which contains a mixture of spores and vegetative cells.
  • the pellet can be resuspended in a liquid medium for use as a source of bacterial inoculum, or may be otherwise prepared for storage or mixing with soil and planting media.
  • Inocula comprising antifungal Bacillus licheniformis strain prepared as described above may be further prepared in a variety of ways for delivery to crop plants for which biological control of one or more fungal diseases is desired.
  • consistent dosage and viability of the B. licheniformis inoculum is achieved by entrapping bacterial cells and endospores in various biopolymers, such as polyacrylamide, xanthan or carob gums, or alginate, according to known methods (see, e.g., Dommergues et al. , Appl.
  • the viability and stability of a microbial inoculant comprising the B. licheniformis of the invention is improved by entrapping the Bacillus in the biopolymer matrix produced by flocculating rhizobacterial species, such as Azospirillum or Rhizobium .
  • a method of producing a microbial inoculant for delivery of agriculturally beneficial microorganisms, such as the Bacillus licheniformis of the invention, as a co- flocculent with another flocculating bacterium, such as Azospirillum or Rhizobium is described in detail in co-pending U.S. Application Serial No. 08/024,328, which is incorporated by reference herein, in its entirety and is also described briefly below. According to the methods described in U.S. Application Serial No.
  • a microbial inoculum comprising a flocculated form of bacterium, such as Azospirillum or Rhizobium , in which is entrapped another agriculturally beneficial microorganism such as the Bacillus licheniformis of the invention, is prepared as follows.
  • a liquid growth medium (flocculation growth medium) is prepared, containing minimal salts medium, 8 mM fructose and 0.5 mM KN0 3 .
  • the minimal salts medium contains the following ingredients: 0.2 g/1 MgS0 4 • 7 H 2 0; 0.1 g/1 NaCl; 0.02 g/1 CaCl 2 ; 0.06 g/1 Fe(EDTA) ; 0.02 g/1 Na 2 Mo0 4 • 2 H 2 0; 0.01 g/1 MnS0 4 • H 2 0; 4.9 g/1 KOH; 5.0 g/1 malic acid; 0.02 g/1 yeast extract (Difco) in a 10 mM phosphate buffer, final pH adjusted to 6.8.
  • Pre-inocula of the flocculating bacterium are harvested from log-phase cultures grown in nutrient broth (NB, Difco, comprising peptone and beef extract having a low C:N ratio) , and inoculated into the flocculation growth medium.
  • NB nutrient broth
  • pre-inocula of B. licheniformis is inoculated into the flocculation growth medium. Cultures are incubated at 25-37°C and harvested after overnight (or longer) incubation. After such time, Azospirillum shift from a vegetative growth state to the flocculating physiology forming large macroscopic floes in which the other microorganism, B.
  • Co- flocs of the flocculating bacterium containing B. licheniformis are easily harvested by decanting the culture medium from floes, which have settled to the bottom of the culture container. Such floes may thereafter be stored or used as wet floes, or may be dried, e.g., by air drying. Alternatively, culture medium may be removed from the co-flocs by coarse filtration methods and thereafter used or stored as wet floes, or dried.
  • additional microorganisms such as the B. licheniformis of the invention, may be mixed with the flocculating bacterium subsequent to the flocculation step. If inoculum produced in this manner, the additional microorganisms are not as securely entrapped in the biopolymer matrix of the flocculated bacteria; hence, the co-flocculation procedure described above is preferred.
  • Flocculated inocula comprising B. licheniformis provide a high-density inoculum containing 10 n -10 12 cell/gm of floe. These inocula may be stored for long periods of time with no appreciable loss of cell viability.
  • microbial inoculants comprising the antifungal Bacillus licheniformis of the invention for biological control of plant pathogens includes several simple techniques, such as coating seeds with the inoculant, mixing the inoculant into soil or germination media and spraying the inoculant onto various portions of plants. Such methods are familiar to agronomists and do not call for sophistication in processing and equipment.
  • seeds are coated with a microbial inoculant comprising B. licheniformis for the purpose of controlling seed-borne fungal pathogens during germination and seedling development and also for the purpose of extending the storage life of seeds.
  • a microbial inoculant comprising B. licheniformis
  • the application of the B. licheniformis strains of the invention to seed is useful as a general seed cleaning method, by which to control seed-borne fungi that are not only plant pathogenic, but which may be toxic to livestock consuming such seed as feed (e.g., toxin- producing fungi such as those producing aflatoxins) .
  • Seeds may be treated with the B. licheniformis of the invention by dipping them directly into a solution containing the microorganism (concentration of 10 7 -10 8 cells/ml) or by otherwise coating the seeds with the microorganism. Such coating may be facilitated by providing additives to the microbial inoculant, such as various adhesives, or by providing the inoculant as a co-flocculent with a flocculating bacterium as described above.
  • a microbial inoculant comprising B. licheniformis may also be used to inoculate planting media, such as soil, potting mixtures, or other germination mixtures prior to planting seeds. This is accomplished by mixing the inoculum with planting mixtures or by tilling the inoculum into field soils, either alone or along with fertilizers or other pre- planting soil treatments. Such methods are well known to agronomists.
  • a microbial inoculant comprising the B. licheniformis of the invention may also be applied to selected portions of plants to control fungal plant disease.
  • inoculants may be sprayed on the leaf portions of turf grass sod to control various fungal diseases or turf grass.
  • Other methods of applying a microbial inoculant comprising the B. licheniformis of the invention for control of various plant diseases will be apparent to those of skill in the art.
  • Bacillus licheniformis strain PRl-36a was found to be an effective antagonist against a wide variety of fungi, including several fungal species that cause diseases of turf grass and cereal crop which can lead to significant economic loss.
  • Bacillus licheniformis strains such as PRl-36a, which produce broad-spectrum antifungal compounds, are contemplated for use in control of a wide variety of economically significant phytopathogenic fungi of turf grass, cereal crops and other graminacious species.
  • certain fungal pathogens against which B. licheniformis is active e.g., Fusarium
  • non-grass crop species e.g., tomato
  • the Bacillus licheniformis strains of the invention are also contemplated for utility in biological control of those diseases. Fungi that may be biologically controlled by antifungal compound-producing B.
  • licheniformis strains of the invention include, but are not limited to: Altemaria spp. , Aspergillus spp. (e.g., A. flavus , A. niger) , Curvularia spp. , Diplodia maydis , Fusarium spp. (including F. moniliforme , F. oxysporum, F. roseum) , Helminthosporeum spp. (including H. maydis) , Magnaporthe poae , Penicillium spp. , Rhizopus spp. , and Rhizoctonia solani . Plant or crop species that may be biologically protected by the B.
  • licheniformis strains of the invention include, but are not limited to: (1) turf grasses and field grasses, such as perennial ryegrass, bluegrass, fescue, and bent grass, among others; (2) cereal crop species, such as corn, sorghum, rice, wheat, rye, millet and barley; (3) broadleaf crops, such as soybeans, alfalfa, beans, peanuts, cotton, tobacco and potato; and (4) vegetable and floral crops, such as tomatoes, cucumbers, peppers, beets, carrots and roses.
  • turf grasses and field grasses such as perennial ryegrass, bluegrass, fescue, and bent grass, among others
  • cereal crop species such as corn, sorghum, rice, wheat, rye, millet and barley
  • broadleaf crops such as soybeans, alfalfa, beans, peanuts, cotton, tobacco and potato
  • vegetable and floral crops such as tomatoes, cucumbers, peppers, beets, carrots and roses
  • the ethanolic extract of B. licheniformis strain PRl-36a culture filtrate which contained the antifungal principles, was found to be active against numerous filamentous fungi and retained activity for several months. Accordingly, the active antifungal principles of the B. licheniformis strains of the invention may also be utilized for control of such fungal plant pathogens, thereby improving health, promoting growth and increasing yield of horticulturally and agronomically important plants.
  • the antifungal principle may be isolated from cultures of Bacillus licheniformis strains such as PRl-36a, according to known methods, such as those described by McKeen et al. (1986) , Phytopathology, 1__.
  • the one or more antifungal principles present in such an ethanolic extract may be used without further separation in formulations for pest control, according to standard methods. Concentrations of the antifungal principle suitable for obtaining a fungistatic effect fall in the range of 10-50 ⁇ g of evaporated ethanol extract per ml.
  • the antifungal principle may be formulated with any standard solvents, dispersion media, wetting agents, powders, grannules, etc., for application to soils and plant parts. To the extent they are compatible with the antifungal principle produced by the B. licheniformis of the invention, any and all such solvents and additives are contemplated for use in the practice of the invention. Furthermore, it is also understood that the antifungal principle present in the ethanolic extract described herein may be subjected to further treatment (e.g., removal of ethanol, lyophiiization or other drying measures, etc.) to the extent that such additional treatments do not interfere with the activity of the antifungal principle or components thereof.
  • further treatment e.g., removal of ethanol, lyophiiization or other drying measures, etc.
  • the antifungal principle in the ethanolic extract of B. licheniformis strains such as PRl-36a may also be subjected to further separation and purification according to standard methods. Such methods include, but are not limited to, various chromatography procedures, such as thin-layer chromatography, gas chromatography or high-performance liquid chromatography (HPLC) , using standard preparative protocols. Individual components of the antifungal principle may then be used separately in formulation of liquid- or solid-based fungicides for control of plant colonizing fungi on a wide variety of plant species, as described hereinabove for microbial inoculants comprising B. licheniformis strains of the invention.
  • the present invention provides an antifungal compound-producing Bacillus licheniformis which is antagonistic against numerous soil-borne and/or plant pathogenic fungi.
  • the type isolate, PRl-36a possesses the typical characteristics of B. licheniformis , including endospore formation, aerobic growth at elevated temperatures and the ability to grow anaerobically.
  • the Bacillus licheniformis of the invention produces a physiologically active agent, which is a diffusible antifungal principle obtainable by acid precipitation of the culture filtrate followed by ethanol extraction.
  • the broad spectrum of antifungal activity against several agronomically important phytopathogenic fungi renders antifungal compound-producing strains of Bacillus licheniformis particularly useful as biological control agents to reduce the impact of plant disease, thus improving seed germination, seedling establishment and plant growth.
  • filamentous fungi in particular, the use of this microorganism may be extended for the purpose of general seed cleaning to improve germination and/or to reduce the incidence of toxin-producing fungi which are detrimental to livestock consuming seeds of such crops.
  • the ability of this microorganism to form spores, its thermophilicity and anaerobic growth capability render it extremely suitable for use under a variety of environmental settings.
  • the diffusible nature of the antifungal principle enables easy purification of the active principle, and the stability of the antifungal principle provides a further advantage for production of chemical formulations for control of fungal plant pathogens.
  • a process is described for isolating and identifying bacterial species that show fungal-suppressive activities against plant pathogenic fungi of turf grass and other grass species.
  • the procedure involves the isolation and culture of bacteria from the rhizosphere supporting the host plant species for which antifungal bacteria are desired. In the present case, a turf grass rhizosphere was selected.
  • Rhizosphere soil samples from this grass were serially diluted in water and plated on various specific and non-specific media to isolate bacteria showing potential for antifungal properties.
  • the media used for the isolation were Plate Count Agar (PCA) , Crystal Violet Nutrient Agar (CVA) , King B agar for Pseudomonas spp. and MacConkey agar for Enterobacter spp. and were purchased from Difco Laboratories, Detroit, MI.
  • Dextrose Agar Dextrose Agar
  • the endospore forming isolates were initially recognized as Bacillus spp . and were classified morphologically and biochemically in accordance with the Bergey's Manual of Systematic Bacteriology.
  • Isolation of antifungal bacteria Screening for antifungal bacteria from the rhizosphere of perennial ryegrass, using various media, yielded several different classes of bacteria showing antagonism towards contaminating fungi on the plates. About 100 different bacterial isolates were obtained, of which 38% belonged to the endospore forming Bacillus spp. , 18% were Pseudomonas spp. and 10% were Enterbacter spp. Since the Bacillus group was predominant in the rhizosphere tested, the study was initiated using the Bacillus isolates to determine the antifungal properties against three different known phytopathogens that affect turfgrasses. As shown in Table 1, twenty-eight isolates were strongly antagonistic against R . solani and M . poae but less effective against P. ultimum . Only five of the 33 bacillus isolates tested were ineffective against all three pathogens. Strain identification and classification.
  • Table 2 shows the morphological and biochemical tests performed on the isolates according to an established protocol for the identification of Bacillus species given in the Bergey's Manual of Systematic Bacteriology. All isolates were endospore forming, strongly catalase positive and VP positive and indicative of Bacillus spp . Isolate #PRl-36a was further characterized biochemically and it was able to grow at 50°C aerobically and anaerobically at 37°C. Other properties included lysozyme sensitivity and inability for tyrosine degradation. Although it did not change the pH indicator color in the propionate utilization test, small colonies of the isolate did grow on the medium after 48 hours of incubation.
  • Bacillus licheniformis strain PRl-36a is deposited with the American Type Culture Collection (Rockville, MD) and assigned ATCC accession number 55606. TABLE 1
  • STRAIN PRl-36a In this Example we describe the recovery and physical characterization of antifungal substances from B. subtilis strain PRl-36a. The range of antifungal activity of a crude ethanolic extract of these substances is also described.
  • the antibiotic medium contained 20 g of Difco-Bacto dextrose, 5 g of DL- glutamic acid, 1.02 g of MgS0 4 7 H,0, 1 g of K 2 HP0 4 , 0.5 g of KCI, and 1 ml of trace element solution (0.5 g of MnS0 4 , 0.16 g CuS0 4 5 H 2 0, and 0.015 g of FeS0 4 • 7H 2 0 in 100 ml of water) per liter.
  • the pH of the medium was adjusted to 6.0-6.5.
  • the cultures were grown in batch cultures in 500 ml conical flasks containing 200 ml of the medium.
  • the flasks were incubated at 30°C in a gyrotory shaker, (New Brunswick Scientific) , at a speed of 200 rpm for 3 days.
  • the supernatant of the culture was obtained by centrifugation for 20 minutes at 16,500 g at 4°C.
  • the antifungal compounds were precipitated by adjusting the pH to 2.5 with concentrated HCl.
  • the precipitated material was later harvested by centrifugation for 10 minutes at 10 minutes at 16,500 g.
  • the pellet containing the active fraction was then suspended and extracted in 50 ml of 80% ethanol three times.
  • the 80% ethanolic extract was later evaporated to dryness in a rotary evaporator at 55°C.
  • the dried material designated as Crude Ethanol Extract (CEE) was stored in vials at room temperature as well as at 4°C until further use.
  • Antimicrobial spectra of the crude ethanol extract The antimicrobial activity of the CEE was performed on PDA plates for fungi and yeast. For testing of antibacterial and anti-yeast activity,
  • Luria Bertani (LB) agar plates were used (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) .
  • the antimicrobial activity was determined by paper disc method in which 100 ⁇ g of the CEE were placed on sterile sensi-discs of 9.52 mm diameter (Schleicher & Schuell Inc., N.H.). The discs were placed on respective plates of actively growing fungal cultures. For antagonism against bacteria, the plates were spread with bacteria and immediately the discs impregnated with the CEE was placed and the plates were incubated at 30°C for 2 to 7 days. Hyphal morphology of the mycelial mat from the CEE treatment were examined under microscope. Minimum Inhibitory Concentration (MIC) .
  • MIC Minimum Inhibitory Concentration
  • Minimum inhibitory concentration of the CEE was determined by incorporating serial dilutions of CEE ranging from 10 ug per ml to 100 ⁇ g per ml in PDA. The plates were poured with different concentrations and plugs of mycelia from different test fungi were placed on the plates and incubated at 30°C for two weeks. Fresh outgrowth from mycelial plug was assessed in the plates containing different concentrations of CEE. Thin layer chromatography of the CEE.
  • Bioautography The chromatograms obtained after thin-layer chromatography of the CEE in chloroform/methanol/water (65:25:4) were included in malt agar seeded with Aspergillus niger as the sensitive organism. Plates were incubated for 2 days at 28°C, after which the presence of an active antifungal compound was detected by a zone of growth inhibition (method of Besson & Michel (1991) , Microbios., j>!5: 15-21). Spectroscopy. Infrared spectroscopy was performed according to established methods. Proton- and ,3 C-nuclear magnetic resonance spectroscopy were also performed according to standard methods.
  • Bioassay for seedling blight of corn Sweet corn kernels obtained from Abbot and Cobb Co. (Seed Lot # 60591 PKF) were imbibed in water (untreated control) and in aqueous suspension of B. licheniformis PRl-36a for 6 hours. B. licheniformis cells were harvested from tryptic soy broth culture grown overnight. Five seeds per treatment were used for testing the disease suppression in a controlled environmental chamber. Seeds were sown individually in 2" pots filled with sand mixed with soil in the ratio of 3:1. A 4 cm deep hole was made in each soil surface and 1 ml of a weak old mycelial suspension of Fusarium roseum were pipetted into each hole and the holes were half filled with soil. One kernel was dropped into each hole and covered with soil. The experiment was conducted in a growth chamber set at 20°C.
  • Plant bioassay with Bacillus licheniformis Plant bioassay with Bacillus licheniformis .
  • the biologically active ninhydrin-positive band having an Rf value of 0.46 in the chloroform:methanol:water (65:25:4) system (hereinafter referred to as the "0.46 Rf component"; see Table 5) was further analyzed by infrared spectroscopy and by l E and 13 C nuclear magnetic resonance spectroscopy.
  • the IR spectrum of the 0.46 Rf component is shown in Figure 1.
  • the IR spectrum indicates that the compound associated with this component comprises a peptide, as reflected by the bands at 3,400 cm" 1 for amino- and hydroxy1-groups of amino acids, as well as a pair of strong bands at 1,650 and 1,520 cm" 1 , indicative of a peptide bond.
  • the bands appearing at about 2,800-3,000 cm "1 are indicative of aliphatic side chains, which could be associated with amino acid residues or with lipids.
  • Other functionalities indicated by the IR spectrum include the presence of an aromatic group, possibly associated with a tyrosine residue.
  • the l E and 13 C NMR spectra obtained for the 0.46 Rf component are shown in Figure 2. These spectra indicate that the component contains a lipid moiety, as reflected by a close aggregation of CH 2 groups. The NMR spectra also indicate that the compound contains a peptide moiety in which the peptide chain is probably short (i.e., approx. 4-6 amino acids) . Additionally, as indicated by the ⁇ NMR (which shows a pair of symmetrically coupling aromatic protons) and the 13 C NMR (which shows at least one peak characteristic of a tyrosine residue) , one of the amino acid residues of the peptide may be tyrosine.
  • the 0.46 Rf component of the CEE appears to comprise a lipopeptide.
  • the peptide moiety is relatively short, and the molecular weight of the entire component is probably low.

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Abstract

Bacillus licheniformis produisant un principe antifongique actif contre une variété importante de champignons pathogènes de plantes. On a obtenu le microorganisme à partir de la rhizosphère du ray-grass vivace (Lolium perenne L.). L'isolat typique, la souche PR1-36a, présente un antagonisme particulier contre les champignons filamenteux, y compris Rhizoctonia et Magnaporthe. Ce microorganisme s'est avéré efficace en tant qu'agent de lutte biologique contre la maladie des plantules du maïs provoquée par Fusarium. Le principe antifongique produit par le microorganisme peut se diffuser et être purifié par précipitation acide du liquide de culture, suivie par une extraction à l'éthanol. Ce principe antifongique est fongistatique contre plusieurs espèces différentes de champignons et présente une activité particulièrement puissante contre des champignons filamenteux, dont la plupart sont des pathogènes sérieux de plantes. L'invention concerne des procédés de production et d'utilisation de souches antifongiques de Bacillus licheniformis, ainsi que de ses principes antifongiques, dans le traitement de sols, de semis, de semences et de parties de plantes afin de lutter contre différentes maladies provoquées par des champignons.
PCT/US1995/008536 1995-07-07 1995-07-07 Bacillus licheniformis produisant des agents antifongiques et leurs utilisations dans la lutte contre des champignons phytopathogenes Ceased WO1997002749A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000043497A1 (fr) * 1999-01-20 2000-07-27 Universidad San Pablo Ceu Bacteries productrices de gibberellines et stimulant la croissance vegetale
EP1166632A3 (fr) * 2000-06-22 2003-04-23 T.S. Plant Science Institute Co., Ltd. Méthode de contrôle de maladies de semence
KR100506721B1 (ko) * 2002-09-25 2005-08-08 학교법인 동아대학교 바실러스 리케니포미스 ｎ1 및 이를 포함하는 식물병원성 진균 방제용 미생물 제제
EP2179652A1 (fr) 2008-09-10 2010-04-28 ABiTEP GmbH Gesellschaft für AgroBioTechnische Entwicklung und Produktion Produit antibactérien destiné au traitement d'un incendie dans des vergers et d'autres maladies bactériennes des plantes
WO2017093977A1 (fr) * 2015-12-04 2017-06-08 Universidad Eafit Procédé d'induction d'une activité antimicrobienne dans des bactéries sporogènes aérobies
JP2017525356A (ja) * 2014-08-16 2017-09-07 ディーシーエム シュリラム リミテッド バチルス属の新規細菌およびその使用
CN111808776A (zh) * 2020-07-28 2020-10-23 德州八虎生物科技有限公司 一株耐盐碱空气芽孢杆菌及其活菌制剂的制备方法与应用
CN114853850A (zh) * 2022-06-08 2022-08-05 河南农业大学 一种带正电荷的抗真菌肽qm-18及其应用

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APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, Volume 39, issued 1993, A. GALVEZ et al., "Isolation and Physico-Chemical Characterization of an Antifungal and Antibacterial Peptide Produced by Bacillus Licheniformis A12", pages 438-442. *
ARCHIVES OF MICROBIOLOGY, Volume 162, issued 1994, M. LEBBADI et al., "Purification of Amoebolytic Substances from Bacillus Licheniformis M-4", pages 98-102. *
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000043497A1 (fr) * 1999-01-20 2000-07-27 Universidad San Pablo Ceu Bacteries productrices de gibberellines et stimulant la croissance vegetale
ES2147160A1 (es) * 1999-01-20 2000-08-16 Univ San Pablo Ceu Bacillus pumilus b3 (cect 5105) y bacillus licheniformis b12 (cect 5106) como productores de giberelinas y estimulantes del crecimiento vegetal.
EP1166632A3 (fr) * 2000-06-22 2003-04-23 T.S. Plant Science Institute Co., Ltd. Méthode de contrôle de maladies de semence
US6823623B2 (en) 2000-06-22 2004-11-30 Takii & Company, Limited Method of controlling seed disease
KR100506721B1 (ko) * 2002-09-25 2005-08-08 학교법인 동아대학교 바실러스 리케니포미스 ｎ1 및 이를 포함하는 식물병원성 진균 방제용 미생물 제제
EP2179652A1 (fr) 2008-09-10 2010-04-28 ABiTEP GmbH Gesellschaft für AgroBioTechnische Entwicklung und Produktion Produit antibactérien destiné au traitement d'un incendie dans des vergers et d'autres maladies bactériennes des plantes
JP2017525356A (ja) * 2014-08-16 2017-09-07 ディーシーエム シュリラム リミテッド バチルス属の新規細菌およびその使用
EP3180420A4 (fr) * 2014-08-16 2017-12-27 DCM Shriram Ltd. Nouvelle bactérie du genre bacillus et ses utilisations
WO2017093977A1 (fr) * 2015-12-04 2017-06-08 Universidad Eafit Procédé d'induction d'une activité antimicrobienne dans des bactéries sporogènes aérobies
CN111808776A (zh) * 2020-07-28 2020-10-23 德州八虎生物科技有限公司 一株耐盐碱空气芽孢杆菌及其活菌制剂的制备方法与应用
CN114853850A (zh) * 2022-06-08 2022-08-05 河南农业大学 一种带正电荷的抗真菌肽qm-18及其应用
CN114853850B (zh) * 2022-06-08 2023-05-02 河南农业大学 一种带正电荷的抗真菌肽qm-18及其应用

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