EP0485440A4 - Inhibiting plant pathogens with an antagonistic microorganism(s) - Google Patents

Inhibiting plant pathogens with an antagonistic microorganism(s)

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Publication number
EP0485440A4
EP0485440A4 EP19900911608 EP90911608A EP0485440A4 EP 0485440 A4 EP0485440 A4 EP 0485440A4 EP 19900911608 EP19900911608 EP 19900911608 EP 90911608 A EP90911608 A EP 90911608A EP 0485440 A4 EP0485440 A4 EP 0485440A4
Authority
EP
European Patent Office
Prior art keywords
microorganism
yeast
group
nrrl
composition
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Withdrawn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
EP19900911608
Other languages
English (en)
Other versions
EP0485440A1 (fr
Inventor
Charles L Wilson
Edo Chalutz
Randy J Mclaughlin
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.)
Israel Ministry of Agriculture
US Department of Agriculture USDA
US Department of Commerce
Original Assignee
Israel Ministry of Agriculture
US Department of Agriculture USDA
US Department of Commerce
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 US07/395,681 external-priority patent/US5413783A/en
Priority claimed from US07/530,381 external-priority patent/US5041384A/en
Application filed by Israel Ministry of Agriculture, US Department of Agriculture USDA, US Department of Commerce filed Critical Israel Ministry of Agriculture
Publication of EP0485440A1 publication Critical patent/EP0485440A1/fr
Publication of EP0485440A4 publication Critical patent/EP0485440A4/en
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B7/00—Preservation or chemical ripening of fruit or vegetables
    • A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
    • A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
    • A23B7/154—Organic compounds; Microorganisms; Enzymes
    • A23B7/155—Microorganisms; Enzymes; Antibiotics
    • 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
    • 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
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A01N63/32—Yeast
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B9/00—Preservation of edible seeds, e.g. cereals
    • A23B9/16—Preserving with chemicals
    • A23B9/24—Preserving with chemicals in the form of liquids or solids
    • A23B9/26—Organic compounds; Microorganisms; Enzymes
    • A23B9/28—Microorganisms; Enzymes; Antibiotics
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
    • 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
    • C12R2001/645—Fungi ; Processes using fungi
    • 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
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida
    • 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
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the present invention relates to the biological control of plant diseases (e.g. either preharvest or postharvest diseases) in agricultural commodities such as fruit.
  • plant diseases e.g. either preharvest or postharvest diseases
  • this invention relates to: (1) methods for biologically controlling plant diseases (such as postharvest rots) on agricultural commodities using either, (a) at least one calcium salt and at least one microorganism which is an antagonist to plant pathogens, or (b) at least one microorganism which is an antagonist against plant pathogens but is not antibiotic; (2) compositions useful in such methods, and; (3) manufacturers produced by such methods. Additionally, this invention relates to a method for biologically controlling postharvest rots on agricultural commodities using strains of Pichia quilliermondii (anamorph Candida cruilliermondii) and a strain of Hanseniaspora uvarum.
  • plant diseases such as postharvest rots
  • a first aspect of the present invention relates to processes for inhibiting plant pathogen development on an agricultural commodity comprising: applying (in the context of the present invention, "applying 1 - is intended to be limited to the intentional and willful dispensing of the microorganism(s) onto the agricultural commodity, as opposed to the natural occurrence of a microorganism on an agricultural commodity) to an agricultural commodity at least one microorganism, the at least one microorganism being an antagonist against plant pathogens but not being antibiotic, wherein the at least one microorganism is applied in an amount effective to inhibit plant pathogen development on the agricultural commodity.
  • the most striking and novel aspect of this invention is the use of microorganisms which do not produce antibiotics to control the diseases of agricultural commodities. This method is of importance to the consumer because it avoids the potential adverse effects of antibiotics in the food supply, such as the development of antibiotic resistance in human pathogens.
  • a second aspect of the present invention relates to processes for inhibiting plant pathogen development on an agricultural commodity comprising: applying to the agricultural commodity at least one calcium salt and at least one microorganism which is an antagonist against plant pathogens (and preferably not antibiotic) ; wherein the at least one calcium salt and the at least one microorganism are applied to the agricultural commodity in an amount effective to inhibit plant pathogen development on said agricultural commodity.
  • compositions which maybe utilized in carrying out the aforementioned processes.
  • Such compositions include:
  • a composition comprising a mixture of, (1) at least one microorganism which is an antagonist against plant pathogens but is not antibiotic and, (2) a carrier for said at least one microorganism selected from the group consisting of a gel, gum, wax, oil, talc, starch and mixtures thereof;
  • a composition comprising a mixture of, at least one microorganism and a carrier for said at least one microorganism, wherein at least 99% by count of said at least one microorganism is antagonistic against plant pathogens but is not antibiotic; and/or, A composition comprising a mixture of, at least one calcium salt and at least one microorganism which is an antagonist against plant pathogens, and preferably is not antibiotic (preferably such a composition may: (a) consist essentially of the at least one calcium salt and the at least one microorganism, and/or; (b) have at least 99% by count of microorganisms therein be antagonistic to plant pathogens, and/or; (c) have at least 99% by count of microorganisms therein be nonantibiotic) .
  • a fourth aspect of the present invention relates to manufactures which may include:
  • a manufacture comprising an agricultural commodity having thereon a concentration of at least about 10 5 colony forming units per square centimeter of at least one microorganism which is an antagonist against plant pathogens but is not antibiotic;
  • a manufacture comprising an agricultural commodity having microorganisms thereon, wherein the majority of said microorganisms are at least one microorganism which is an antagonist against plant pathogens but is not antibiotic; .
  • a manufacture comprising an agricultural commodity having thereon a calcium salt and at least one microorganism which is an antagonist against plant pathogens (and preferably is not antibiotic) in a concentration of at least about 10 5 colony forming units per square centimeter; and/or
  • a manufacture comprising an agricultural commodity having a calcium salt and microorganisms thereon, wherein the majority of microorganisms on said agricultural commodity are at least one microorganism which is an antagonist against plant pathogens.
  • a fifth aspect of the present invention relates to a biologically pure culture of an isolate of Hanseniaspora uvaru having the identifying characteristics of isolate NRRL Y-18527.
  • microorganism(s) may for example be selected from the group consisting of: fungi (e.g. yeast) , bacteria, viruses and mixtures thereof.
  • NRRL Y-18313, NRRL Y-18314 and NRRL Y-18654 have been identified as Pichia quilliermondii (ana orph Candida cruilliermondii) and NRRL Y-18527 has been identified as Hanseniaspora uvarum (Nichaus) Shehata, Mra et Phaff.
  • the deposited materials have been accepted for deposit under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of patent procedure.
  • said depository affords permanence of the deposits and ready accessibility thereto by the public if a patent is granted, (2) the materials have been deposited under conditions that assure that access to the materials will be available during the pendency of the patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. All restrictions on the availability of progenies of the strain to the public will be irrevocably removed upon the granting of the patent.
  • plant diseases e.g. postharvest diseases
  • agricultural commodities e.g. fruits
  • agricultural commodities are subjected to an aqueous suspension comprising an isolate of yeast having the identifying characteristics of an isolate selected from the group consisting of: NRRL Y-18313, NRRL Y-18314, NRRL Y-18527, NRRL Y-18654 and mixtures thereof.
  • the organisms multiply and occupy the surfaces of wounded fruit, thereby preventing infection by plant (e.g. fruit-rot) pathogens.
  • Figure 1 is a bar graph of percent decay of three lots of grapes treated with NRRL Y-18314 and grapes in a control group, showing inhibition of Rhizopus rot.
  • Figure 2 is a line graph of the rot diameter area (mm) on apples infected with Botrytis cinerea v. time (days) , for: (1) control samples treated with water only, and; (2) samples treated with NRRL Y-18314.
  • Figure 3 is a line graph of rot diameter area (mm) on apples infected with Penicillium expansum v. time (days) for: (1) control samples treated with water only, and; (2) samples treated with NRRL Y-18314.
  • Figure 4 is a bar graph of percent infection showing relative effectiveness of yeast isolates in inhibiting Penicillium digitatum decay on grapefruit.
  • Figure 5A is a photograph of peanuts treated with both Aspergillus flavus NRRL Y-18314 in accordance with Example V.
  • Figure 5B is a photograph of peanuts treated with only Aspergillus flavus, according to Example V.
  • Figure 6A is a photograph of peanuts treated with both Aspergillus niger and NRRL Y-18314 as referred to in Example VI.
  • Figure 6B is a photograph of peanuts treated with only Aspergillus niger according to the process described in Example VI.
  • Isolates NRRL Y-18313 and NRRL Y-18314 were obtained from the surface of citrus fruits by repeatedly washing the fruit with water.
  • Isolate NRRL Y-18654 was obtained from the surface of a lemon by repeated washings.
  • Isolate NRRL Y-18527 was isolated from the surface of a grape.
  • the organisms are thereafter plated and grown on any nutritionally rich medium sufficient to support growth of microorganisms.
  • the medium is either nutrient yeast dextrose agar (NYDA) or yeast-malt extract agar (YM) .
  • Isolates NRRL Y-18313 AND NRRL Y-18314 have the following identifying characteristics: Colonies are cream white, slightly raised, shiny, round and smooth. No pseudohyphae were observed.
  • Isolate NRRL Y-18654 colonies are cream white, slightly raised, shiny, round with smooth edges.
  • Isolate NRRL Y-18527 has the following identifying characteristics as determined by the American Type Culture Collection: in liquid medium, cells appear lemon shaped and have bipolar budding. On solid medium, cells remain unicellular or non-filamentous. Colonies are white, dull with a slightly raised surface. Pseudomycelium is not produced. One round ascospore is produced per cell.
  • the incubation time is that time necessary for the organisms to reach a stationary phase of growth. Incubation time is preferably from about 40 to 60 hours for NRRL Y-18314 and NRRL Y-18313. Incubation time is preferably from about 24 to 48 hours for NRRL Y-18654. Growth of isolate NRRL Y-18527 is preferably achieved at a temperature of 25-28°C with an incubation time of 18 to 24 hours, such that cells are in the logrithmic phase of growth.
  • Isolates NRRL Y-18313, NRRL Y-18314, NRRL Y-18527 and NRRL Y-18654 may be grown in any conventional shake flask for small fermentation runs. For large scale operations, it is convenient to carry out the culture in a fermentation tank, while applying agitation and aeration to the inoculated liquid medium. Following incubation, the organisms are harvested by conventional sedimentary methodology; i.e. centrifugation or filtering. Cultures are stored on silica gel and frozen until use.
  • Isolates NRRL Y-18313, NRRL Y-18314, NRRL Y-18527 and NRRL Y-18654 are useful to control a variety of plant pathogens especially those which cause postharvest diseases in fruits.
  • Exemplary species of plant pathogens include, but are not limited to, Penicillium italicium Wehmer, Penicillium digitatum, Botyrtis cinerea, Rhizopus stolonifer. Geotrichum candidum, Penicillium expansu , and Alternaria alternata.
  • the microorganisms of the invention are useful in controlling plant pathogens on a variety of agricultural commodities including, but not limited to: fruits, vegetables (e.g. celery), cereals, grains, nuts, seeds, and silage.
  • fruits with which the present invention may be carried out include but are not limited to, citrus fruit, grapes, apples, pears, tomatoes, persimmons, strawberries, peaches, apricots, cherries and papayas.
  • Said citrus fruit may for example include: grapefruit, orange, lemon, kumquat, lime and pummelo.
  • Said nuts may for example include: peanuts, almonds and pecans.
  • Said grains may for example include: wheat, corn, sorghum, soybean and barley.
  • the microorganisms of the present invention may also be utilized with processed agricultural commodities including for example, raisins, prunes, figs, dried apricots and dates.
  • the microorganisms of the present invention may be applied to agricultural commodities in combination with a variety of additives, including carriers such as: (1) a gel or gum based carrier (e.g. xanthan gum) ; (2) a water based carrier (e.g. the microorganisms may be mixed/suspended in water. Other water based carriers include water plus wetting and/or spreading agents) ; (3) an oil based carrier (e.g.
  • “Fresh Mark” or “Fresh Wax 58P” which is a paste wax for peaches, plums and nectarines, containing - white oil, paraffin wax, petrolatum and oleic acid) both from Fresh Mark (Chemical Corporation, prlando, FL) ; (4) a wax based carrier (e.g.
  • a powdered carrier ingredient to provide the composition in powdered form, and in which the microorganism(s) are dispersed and thus diluted to a desired concentration in the powdered composition
  • examples of such powdered carrier ingredients are: starch (e.g. corn starch) and/or talc) , and; (6) and mixture of the foregoing.
  • Use with oil based carriers is preferred to use with water based carriers because the antagonist typically survives better in an oil based carrier.
  • compositions of the present invention may also include other additives including: (1) pesticides, such as fungicides (e.g. "TBZ" available from FMC Corporation) ; (2) one or more preservatives i.e. an environment enhancer such as compositions which hold moisture and/or help to maintain the microorganism(s) viable during storage and/or use, including e.g.
  • pesticides such as fungicides (e.g. "TBZ" available from FMC Corporation)
  • preservatives i.e. an environment enhancer such as compositions which hold moisture and/or help to maintain the microorganism(s) viable during storage and/or use, including e.g.
  • a gum for example a natural gum, such as guar gum, locust bean gum, karaya gum, tragacanth gum or preferably xanthan gum; (b) methyl cellulose; (c) silica gel, and; (d) mixtures of the foregoing preservatives; (3) surfactants and wetting agents, such as Tween 20 and Triton X-100 available from Rho and Hass Company; (4) additives which promote spreading of the compositions of the present invention; (5) additives which promote sticking of the compositions of the present invention to agricultural commodities; (6) nutrients for the microorganisms of the present invention, and; (7) mixtures of the aforementioned additives.
  • these additives When used, these additives should be used in an amount(s) which will not interfere with the effectiveness of the microorganism(s) of the present invention.
  • preparation of suitable compositions require only mixing of the microorganism(s) with the additives.
  • Typical preparation '* includes, adding together the microorganism(s) , preservative and powdered ingredient, and then mixing and/or grinding the constituents together.
  • the powdered composition may be used on an agricultural commodity, or the powdered composition may be used with liquid (e.g. water) and subsequently applied to an agricultural commodity.
  • the compositions of the present invention have excellent storage properties, do not require refrigeration, do not typically encounter contamination problems, and remain effective in typical fruit, vegetable and grain storage environments.
  • Concentrations of suspensions useful in the invention are any concentrations which inhibits the development of the targeted plant pathogen when applied to the fruit.
  • effective concentrations may vary depending upon such factors as: (1) the type of agricultural commodity; (2) the ripeness of the agricultural commodity; (3) the concentration of pathogens affecting the agricultural commodity; (4) the type of wound on the agricultural commodity; (5) temperature and humidity; and (6) the age of the plant pathogen.
  • Exemplary concentrations range from about 1 x 10 4 to 1 x 10 9 CFU/ml, most preferably, from about 1 x 10 7 to 1 x 10 9 CFU/ml.
  • the abbreviation 1 CFU is used herein to designate "colony forming units.”
  • the organisms of the invention may be applied to agricultural commodity using conventional methods such as dipping, spraying or brushing.
  • the organisms of the invention may be incorporated into waxes, wraps or other protective coatings used in processing the agricultural commodities.
  • the agricultural commodity may be treated anytime before or after harvest.
  • the preferred time of treatment is after harvest and prior to storage or shipment. In the case of some grapes, the preferred time of treatment is before harvest.
  • NRRL Y-18313, NRRL Y-18314, NRRL Y- 18527 or NRRL Y-18654 alone, or in combination.
  • the organisms may also be used in combination with other control agents useful to inhibit the development of plant pathogens on agricultural commodities. When used, these agents should be used in an amount, as readily determined by one skilled in the art, which will not interfere with the effectiveness of the microorganisms of the invention.
  • Y-18313, NRRL Y-18314, NRRL Y-18654 and NRRL Y-18527 on fruit may typically vary from 0 to 100 CFU/cm 2 .
  • Hanseniaspora uvarum, or its asexual form Kloeckera apiculata is commonly found as a natural component of the microbial flora that inhabit fruit surfaces (Kamra N., and Madan, M. , 1987, Microbios. Lett. 34:79; Stollarova, V., 1982, Biologica (Bratsil) 37:1115-1121).
  • the ability of these yeasts to control plant pathogens was unexpected since these yeast species have not previously been reported to have biological control properties.
  • One aspect of the present invention relates to applying the microorganism(s) of the present invention in concentrations significantly greater than the aforementioned natural/normal concentrations, e.g. at least about 10 5 CFU per cm 2 , or preferably at least about 10 6 CFU per cm 2 . It should noted in this regard, that another aspect of the present invention relates to an agricultural commodity having thereon a calcium salt and at least one antagonistic microorganism of the present invention in a concentration of at least about 10 5 CFU/cm 2 .
  • At least one calcium salt with the at least one microorganism of the present invention facilitates improved control of plant pathogens (notably, Rhizopus stolonifer of peaches, major rot pathogens of table grapes, Penicillium and Botrytis rot of apples and Penicillium rot of grapefruit) .
  • plant pathogens notably, Rhizopus stolonifer of peaches, major rot pathogens of table grapes, Penicillium and Botrytis rot of apples and Penicillium rot of grapefruit
  • the enhanced ability of the microorganism of the present invention to control plant pathogens in the presence of at least one calcium salt is especially unexpected in view of the fact that topical treatment of fruit with calcium chloride was shown not to reduce postharvest rot of apply by Conway; 1981-Plant Disease 66:402-403 and, Conway et al 1983 Phytopathology 73:1068-1011.
  • calcium chloride applied as a topical treatment would be useful as an agent for enhancing biological control of plant pathogens; calcium chloride would be more effective for enhancing biocontrol than other salts containing similar cations and anions, and; the effects of calcium chloride would be, exerted against such a wide variety of plant pathogens and, manifested with such a broad variety of biocontrol agents.
  • the at least one calcium salt and at least one microorganism may be applied to the agricultural commodity separately, or for ease of application may be applied as a mixture (e.g. also containing one or more of the aforementioned additives) .
  • Typical examples of the calcium salt include: calcium chloride, calcium carbonate, calcium propionate, and mixtures thereof.
  • calcium chloride may be utilized in concentrations of about 1 gm/100 ml, to about 10 gm/100 ml, preferably about 1 gm/100 ml to about 5 gm/100 ml and most preferably about 2 gm/100 ml.
  • Pichia guilliermondii NRRL Y- 18314 was evaluated using the following seven citrus cultivars: grapefruit (Citrus paradisi Macf. cv 'Marsh Seedless*); 'Shamouti' and 'Valencia' orange (C. sinensis Osbeck) ; lemon (C___. lemon L. Burm 'Eureka') ; Temple orange (Tanger hybrid, C_ s _ reticulata X C ⁇ . sinensis) ; Kumquat (Fortunella margarita) ; and pummelos, (C_ s _ grandis) .
  • a biologically pure culture of isolate NRRL Y-18314 was obtained using the following procedures: The surface of lemons was washed by placing the fruit in a 600 ml -beaker containing 200 milliliters (ml) of sterile water. The beakers containing the fruit were placed on a rotary shaker at 100 rpm for 10 minutes. One tenth ml of the wash water was then spread on a NYDA plate and allowed to incubate for 2,4 hours before colonies were selected. The same fruit received three separate washings and the same procedure were followed. Appearing colonies were isolated and purified using standard purification techniques. All cultures were stored on silica gel in a freezer until use. NRRL Y-18313 and NRRL Y-18654 may be obtained using similar procedures.
  • Isolate NRRL Y-18314 was grown in flasks containing nutrient yeast dextrose broth (NYDB) on a reciprocal shaker at 30°C for 48 hours, the culture was centrifuged at 7000 rpm for 10 minutes and the resulting pellet was suspended in water at various concentrations. Concentrations of the aqueous suspensions were adjusted on a spectrophotometer.
  • NYDB nutrient yeast dextrose broth
  • Freshly harvested fruit was wiped with 95% ethanol and placed on moist paper in 50 x 100 x 15 cm plastic trays, 24 fruits per tray. Two to four conical wounds. 3mm deep, were cut in the fruit peel. The wounds were brushed with an aqueous suspension of NRRL Y-18314. Concentrations of the aqueous suspensions ranged from 1 x 10 5 to 1 x 10 10 CFU/ml. One to two hours later, 20 microliters of an aqueous spore suspension of the targeted pathogen, 1 x 10 4 spores/ml, were pipetted into the wounds. Control fruits were inoculated with aqueous spore suspensions of the targeted pathogen only. Following incubation, the trays were covered with high density polyethylene sleeves and kept at room temperature for several days.
  • the number of inoculated sites on which decay developed was determined daily. Each treatment in each experiment consisted of at least 3 replicates of 6 fruits, 24 to 75 inoculation sites per treatment. Each experiment was repeated at least twice.
  • a Number of jjioculat ⁇ on sites per treatment is indicated in parentheses under the cultivar's/naroe.
  • isolate NRRL Y-18314 was highly effective in inhibiting Penicillium digitatum decay on citrus fruit in all cultivars tested.
  • the effectiveness of NRRL Y-18314 varied depending upon the sensitivity of the cultivars to the decay. When compared to its effectiveness on grapefruit, isolate NRRL Y-18314 was more effective on pummelo fruit but less effective on temple, lemon, orange, or kumquat fruits.
  • NRRL Y-18314 was effective in inhibiting Penicillium italicum decay on grapefruit, oranges and other citrus fruit cultivars. As in the case of Penicillium digitatum. NRRL Y-18314 more effectively controlled Penicillium italicum in grapefruits than in oranges. NRRL Y-18314 was also effective in inhibiting the development of Geotrichum candidum in citrus fruits. However, as shown in Table III, Geotrichum candidum was controlled to a lesser extent that the Penicillia decays, particularly in lemons.
  • Example II The ability of Pichia guilliermondii NRRL Y-18314 to inhibit Rhizopus rot development in grapes was demonstrated.
  • a biologically pure culture of NRRL Y-18314 was isolated and purified as described in Example I.
  • NRRL Y-18314 was incubated in 100 ml of NYDB in 250 ml Erlenmeyer flasks on a rotary shaker (100 rpm) at 28°C for 48 hours.
  • Freshly harvested grapes of the Perlette and Thompson Seedless cultivars were dipped momentarily in a suspension of the organism in NYDB.
  • the berries were treated as whole clusters with non-injured berries, as injured berries which had been removed from the stems by pulling and thereby causing a wound, or as injured single berries wounded by piercing non-injured berries with a needle. Control berries were dipped in sterile NYDB only.
  • the berries were dried and thereafter inoculated by dipping in an aqueous suspension containing spores of the targeted pathogen at a concentration of 1 x 10 4 spores/ml.
  • the berries were inoculated by placing a single decayed berry in the center of a group of non-injured berries; i.e. "nesting".
  • the treated berries were placed in polyethylene-covered cartons and held at room temperature for 5 days. Whole treated clusters were placed directly in commercial shipping cartons.
  • Decay incidence was determined by counting the number of infected berries.
  • Each treatment in each experiment consisted of at least three replicates of 20 berries or four replicates of five intact clusters placed in half of a shipping carton.
  • Example III The effectiveness of isolate of Pichia guilliermondii NRRL Y-18314 to inhibit Botrytis cinerea and Penicillium expansum rot was tested on apples. Golden Delicious apples were washed with 2% sodium hypochlorite to surface sterilize the fruit. After air drying, the apples were placed on styrofoam trays in plastic trays with lids. Water (100 ml) was added to each tray for humidity. The apples were wounded using a needle. Wound size was 4mm wide by mm deep. Three-day old shake cultures of NRRL Y-18314 growing no NYDB at a 1 X 10 9 CFU/ml concentration were added to the wounds, 50 microliters/wound. Apples were allowed to air dry.
  • Example IV The effectiveness of Pichia guilliermondii NRRL Y-18314, to inhibit Penicillium digitatum on grapefruit was compared to the effectiveness of eight previously identified isolates of D.hansenii.
  • ATCC American Type Culture Collection
  • the surface of the grapefruit was sterilized with 95% ethanol and placed on moist paper in 50 x 100 x 15 cm plastic trays, 24 fruits per tray. Thereafter, the surface of the fruit was wounded using a needle. Two to four conical wounds, 3 mm deep, were cut in the fruit peel. An aqueous suspension of an isolate was brushed onto the surface of the wound. Each isolate was tested on 48 sites of inoculations. One to two hours later, an aqueous suspension of Penicillium digitatum. 1 x 10 5 spores/ml, was added to the wounds, 20 microliters/wound. Controls were inoculated with water only. The percent of fruit infection was recorded 7 days after inoculation. The data was analyzed by analysis of variance and means were separated by Duncan's Multiple Range Test. Values followed by different letters are significantly different at a 1% level. The results are shown in Figure 4.
  • NRRL Y-18314 clearly exhibited superior control of Penicillium digitatum when compared to prior identified isolates of D. hansenii. After seven days of inoculation, total protection occurred in grapefruits inoculated with NRRL Y-18314 while as much as 25 to 65% infection occurred in fruits inoculated with isolates obtained from the ATCC.
  • EXAMPLE V The purpose of this example is to show the effectiveness of isolate NRRL Y-18314 at inhibiting Aspergillus flavus on peanuts.
  • the peanuts were produced in the following manner. A wound was cut in the surface of each nut. The NRRL Y-18314 was applied as described in Example 1. Similarly, the Aspergillus flavus was applied as described for the pathogen in Example 1. The treated nuts were incubated 14 days at 26° C.
  • Figure 5A is photograph of the peanuts treated with both Aspergillus flavus and NRRL Y-18314
  • Figure 5B is a photograph of peanuts treated only with Aspergillus flavus.
  • FIG. 5A shows only 11 (33%) of the wounds on which the pathogen grew (low to medium growth) compared with Figure 5B which shows extensive pathogen growth on 100% of the wounds.
  • EXAMPLE VI The purpose of this example is to show the effectiveness of isolate NRRL-Y-18314 at inhibiting Aspergillus niger on peanuts.
  • the peanuts were prepared in the following manner. A wound was cut in the surface of each nut.
  • the NRRL-Y-18314 was applied as described in Example 1.
  • the Aspergillus niger was applied as described in for the pathogen in Example 1.
  • the treated nuts were incubated 14 days at 26°C.
  • Figure 6A is a photograph of the peanuts treated with both Aspergillus niger and NRRL-Y-18314
  • Figure 6B is a photograph of peanuts treated only with Aspergillus niger. As shown in these photographs, the results show complete inhibition of the pathogen growth in the yeast-treated nuts (Figure 6A) compared with 100% infection in the non-treated control ( Figure 6B) .
  • EXAMPLE X The same procedures were followed as in Example IX except talc (28 grams) was used instead of either the corn starch or silica gel. The results are shown in Table VII in units of colony forming units per milliliter.
  • the purpose of this example is to show that CaCl 2 and CaC0 3 improve biocontrol, are more effective at improving biocontrol than other inorganic salts, and illustrate surprising and unexpected synergistic results.
  • Golden delicious apples were artificially wounded to a depth of 3 mm using a needle.
  • Each of a first portion of the apples was treated with 50 microliter aliquots of an aqueous solution consisting of NRRL Y-18314 in sterile distilled water at a concentration of 10 7 CFU/ml with each of the salts listed in Table VIII at a concentration of 2 grams/100 ml (with the exception of FeS0 4 which was utilized at a 5 millimolar concentration) .
  • a second portion of the apples was treated with a 50 microliter aqueous solution of each of the salts listed in Table VIII at a concentration of 2 grams/100 ml (with the exception of FeS0 4 which was utilized in a concentration of 5 millimolar) . Also a control was run using only sterile distilled water. Two hours after application of the above solutions, each of the apples was challenged with 20 microliters of a io 5 spore/ml suspension of Botrytis cinerea. The average percent fruit infection of four trials (8 to 10 replicates per trial) was measured 10 days after inoculation with the Botrytis cinerea. The results are shown in Table VIII.
  • NRRL Y-18314 and the calcium salts clearly provide synergistic infection reduction, as evidenced by the fact that CaCl 2 and CaC0 3 provided approximately 5% and approximately 18% infection reduction when used alone and the NRRL Y-18314 provided approximately 40% infection reduction when used alone. Therefore it may have been presumed that the additive effect would have been 45% or 58% respectively.
  • the combination of CaCl 2 with NRRL Y-18314 provided more than twice the expected value of 45% i.e. about 96.7%; and the combination of CaC0 3 with NRRL Y-18314 provided 72.5% which is significantly higher than the expected value of 58%.
  • the purpose of this example is to show that calcium salts provide improved biocontrol with a variety of yeast strains from different species.
  • Golden Delicious apples were wounded in accordance with the previous example. The apples were then treated with 50 microliters of a 10 8 CFU/ml suspension of the respective yeasts in sterile distilled water, with or without 2 grams/100 ml of CaCl 2 as referred to in Table IX. Two hours later the apples were challenged with 20 microliters of a suspension of 10 4 spores/milliliters of Botrytis cinerea. Seven days after inoculation-percent infection was observed. Results are shown in Table IX.
  • EXAMPLE XIII The purpose of this example is to demonstrate the effectiveness of NRRL Y-18314 and combinations thereof with CaCl 2 for controlling Penicillium rot.
  • Golden delicious apples were artificially wounded in accordance with Example XI.
  • the wounded apples were then treated with a 50 microliter suspension of NRRL Y-18314 in sterile distilled water, with or without CaCl 2 (concentration of 2 gram/100 ml) as noted in Table X.
  • Two hours later the apples were challenged with 20 microliters of a spore suspension of Penicillium expansum at the concentrations referred to in Table X. Seven days after inoculation lesion diameter was observed. Results are shown in Table X.
  • Table X The entries of Table X are the average of 5 replicates per treatment. It may be observed from the table that the isolate NRRL Y-18314, applied in the absence of CaCl 2 , did not facilitate significant reduction of decay (greater than 50%) in most treatments, as compared to the water control and CaCl 2 treatments without NRRL Y-18314. However, when NRRL Y-18314 was applied with CaCl 2 decay was reduced greater than 50% at all yeast concentrations and at all Penicillium spore concentrations tested. EXAMPLE XIV
  • Example II The purpose of this example is to illustrate the synergistic effects of combinations of various concentrations of CaCl 2 and microorganisms of the present invention.
  • Grapefruit was wounded as in Example I. The wounded grapefruit were then treated with 50 microliter aliquotes of the constituents identified in Tables XI and XII in sterile distilled water. Two hours later the apples were challenged with 20 microliters of a 10 4 spore/milliliter suspension of Penicillium digitatum. The grapefruit was incubated for 5 days at 24°C before observations were taken. Results of average percent fruit rot were as follows (data is the average of 2-3 trials per treatment) :
  • the purpose of this example is to show that yeast cells rather than the yeast culture broth provide biocontrol, and that washed yeast cells provide improved biocontrol over that achieved with the yeast cells and culture broth.
  • Peaches were artificially wounded and then treated with 50 microliters of washed yeast cells prepared by pelleting yeast cells from culture broth by centrifuging at 5,000 relative centrifugal force (RCF), the yeast cells were resuspended in sterile distilled water and repelleted by centrifugation as before and then resuspended with concentration adjustment in either water or culture broth to provide concentrations as specified in Table XIII. A portion of the peaches were treated with only culture broth (without yeast cells) .
  • EXAMPLE XVI Single Thompson seedless grapes were wounded by pulling from ste s. The grapes were then dipped in a suspension of the yeasts specified in Table XIV at concentration of io 8 -bo IO 9 cfu/ml and incubated at 220°C. At 5 and 6 days the percent fruit rot by naturally occurring organisms (e.g. Aspergillus niger and Rhizopus stolonifer ) was as follows (data is for 3 replicates of 20 berries per fruit treatment) ;
  • NYDB sterile culture broth, nutrient yeast dextrose broth.
  • n number or? fruit per treatment.

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Abstract

L'invention concerne la maîtrise biologique de maladies affectant les plantes (par exemple des maladies soit de pré-récolte soit de post-récolte) dans le domaine des denrées agricoles telles que les fruits. L'invention concerne plus particulièrement: (1) des procédés de maîtrise biologique de maladies affectant les plantes (tels que les rots de post-récolte) sur des denrées agricoles en utilisant soit (a) au moins un sel de calcium et au moins un micro-organisme lequel est un antagoniste contre des germes pathogènes affectant des plantes, soit (b) au moins un micro-organisme lequel est un antagoniste contre des germes pathogènes affectant des plantes mais n'est pas un antibiotique; (2) des compositions utiles dans lesdits procédés, et; (3) des produits obtenus à l'aide desdits procédés.
EP19900911608 1989-07-31 1990-07-31 Inhibiting plant pathogens with an antagonistic microorganism(s) Withdrawn EP0485440A4 (en)

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US38766989A 1989-07-31 1989-07-31
US387669 1989-07-31
US07/395,681 US5413783A (en) 1988-04-04 1989-08-18 Inhibiting plant pathogens with an antagonistic microorganism(s)
US395681 1989-08-18
US07/530,381 US5041384A (en) 1989-07-31 1990-05-30 Pichia guilliermondii (Anamorph Candida guilliermondii) useful for the biological control of postharvest rots in fruits
US530381 1990-05-30

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US5525132A (en) * 1991-04-11 1996-06-11 Daratech Proprietary Limited Yeasts as a biocontrol for microbial diseases of fruit
KR0145740B1 (ko) * 1991-05-23 1998-08-01 채영복 고정화 미생물 농약과 그의 제조방법
AU742005B2 (en) * 1993-03-31 2001-12-13 Dsm Ip Assets B.V. Yeast formulation for the preparation of baked products
US5591429A (en) * 1993-07-26 1997-01-07 The United States Of America As Represented By The Secretary Of Agriculture Composition containing 2-deoxy-D-glucose and Candida saitoana and a method of use for the biological control of postharvest diseases
AU7414394A (en) * 1993-09-22 1995-04-06 State Of Israel - Ministry Of Agriculture Fungicides and method for using same
US5633025A (en) * 1994-11-07 1997-05-27 The United States Of America As Represented By The Secretary Of Agriculture Bioactive coating for harvested commodities
US5711946A (en) * 1995-02-17 1998-01-27 The State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of Oregon State University Control of post-harvest fungal disease using saprophytic yeast
ES2089981B1 (es) * 1995-03-28 1997-04-16 Inst Recerca I Tecnologia Agroalimentaries Nueva cepa de la levadura candida sake (salto and ota) van uden and buckley y su utilizacion como agente de control biologico de las enfermedasdes fungicas de postcosecha en frutas.
CN117050586A (zh) * 2023-04-18 2023-11-14 华中农业大学 一种用于采后果实防腐保鲜的涂层剂、制备方法和应用

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AU6071490A (en) 1991-03-11
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