CA2064730A1 - Inhibiting plant pathogens with an antagonistic microorganism(s) - Google Patents

Abstract

The present invention relates to the biological control of plant diseases (e.g. either preharvest or postharvest diseases) in agricultural commodities such as fruit. More particularly, 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.

Description

WO91/01~1 PCT/US90/~2 INHIBITING PLANT PATHOGENS WITH AN
ANTAGONISTIC MICROORGANISMfS) Cross-Reference to Related Applications The present application is a continuation-in-part of application serial number 07/387,669 filed July 31, 1989 which is a continuation-in-part of application serial number 07/177,236 filed April 4, 1988.
Field of the Invention The present invention relates to the biological control of plant diseases (e.g. either preharvest or postharvest diseases) in agricultural commodities such as ~ruit. More particularly, this invention relates to:
(l) method~ 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 WO91/01~1 PCT/US90/04290 .

using strains of Pichia ~uilliermondii (anamorph Candida quillie~mond i) and a strain of Hanseniaspora uvarum.

Description of Prior Art Postharvest diseases of fruit cause 15 to 25% losses yearly in the fruit industry worldwide. Fungicides, the major weapon in combatting these diseases, are often ineffective and pose hazards to humans and the environment. Therefore, a critical need exists for new methods to control postharvest diseases.
Recently, it has been shown that the postharvest treatment of fruit with antagonistic microorganisms is an e~ecti~e approach to the control of postharvest rots.
Remarkable success was shown in the control of brown rot in peaches caused by Monilinia fructicola (Wint.). Honey with Bacillus subtilis. Pusey et al. [Plant Dis.
86:753-756 ~1986)]. De Matos was able to reduce mold incidence from 35% to 8~ when a species of Trichoderma was inoculated with Penicillium diaitatum into lemon peel. De Matos, Ph.D. Dissertation, University of California, Riverdale, (1983). Singh and Deverall demonstrated biocontrol with bacterial antagonists to the citrus pathogens Alternaria citri Pierce, Geotrichum WO9t/01~1 PCT/US90/04290 ~Trans. Br. Mycol. Soc. 83:487-490 (1983)]. Dipping wounded citrus fruit in suspensions of bacterial cells, particularly a strain of Bacillus subtilis (Ehrenber) Cohn, delayed decay by the three rot pathogens.

Summary of the Invention 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" 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 WO91/01~1 PCT/US90/04290 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.
A third aspect of the instant invention pertains to 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 WO91/01~1 PCT/US90/04290 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 o~ microorganisms therein be nonantibiotic).
A foùrth aspect of the present invention relates to manufactures which may include:
~ manufacture comprising an agricultural commodity having thereon a concentration of at least about lO5 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 WO91/01~1 PCT/US90/04290 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 lO5 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 aspsct of the present invention relates to a biologically pure culture of an isolate of Hansenias~ora uvarum having the identifying characteristics of isolate NRRL Y-18527.
The aforementioned microorganism(s) may for example be selected from the group consisting of: fungi (e.g.
- 20 yeast), bacteria, viruses and mixtures thereof.
In regard to a preferred embodiment of the present invention, we have discovered new strains of yeast that are highly effective in controlling a variety of plant WO91/~l~l PCT/US90/04290 (e.g. fruit-rot) pathogens which affect a wide variety of agricultural commodities. Four isolates of the new strains have been deposi~ed with the culture collection at The Northern Regional Research Center, U.S. Department of Agriculture, Peoria, Illinois 61604, under the acquisition numbers NRRL Y-18313, NRRL Y-18314, NRRL Y-18654 and NRRL Y-18527. NRRL Y-18313, NRRL Y-18314 and NRRL Y-18654 have been identified as Pichia auilliermondii (anamorph Candida auilliermondii) and NRRL
Y-18527 has been identified as Hanseniaspora uvarum (~ichaus) Shehata, Mrak et Phaff. The deposited materials have been accepted for deposit under the Buda~es~ Treaty on the International Recognition of the Deposit o~ Microorganisms for the purposes of patent procedure. Further, (1) 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 WO91/01~1 PCT/US90/04290 strain to the public will be irrevocably removed upon the granting of the patent.
Accordingly, it is an object of the present invention to provide novel biological control agents which pose no risk to the consumer and are highly effective in controlling a variety of plant pathogens causing preharvest and postharvest diseases on a variety of agricultural commodities (e.g. fruits).
It is also an object of the invention to provide a method of biologically controlling plant diseases (e.g.
postharvest diseases) on agricultural commodities (e.g.
fruits) which does not require the use of fungicidal treatments.
In a preferred embodiment of our invention, 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. In effect, the organi~ms multiply and occupy the surfaces of wounded fruit, thereby preventing infection by plant (e.g. fruit-rot) pathogens.

WO91/01~1 PCT/US90/042 BRIEF DESCRIPTION OF THE DRAWINGS
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 inhibitiQn 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 onlyl 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 diaitatum decay on grapefruit.
Figure 5A is a photograph of peanuts treated with both Asperaillus flavus NRRL Y-18314 in accordance with Example V.
Figure 5B is a photograph of peanuts treated with only Asperaillus flavus, according to Example V.
Figure 6A is a photograph of peanuts treated with both Asperaillus niaer and NRRL Y-18314 as referred to in Example VI.

W091/01~1 PCT/US90/04290 Figure 6B is a photograph of peanuts treated with only As~eraillus niaer according to the process described in Example VI.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
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 o~ microorganisms. Preferably, 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 aré
cream white, slightly raised, shiny, round and smooth.
No pseudohyphae were observed.
No ascospores were produced after one week on corn Meal agar, V-8 Juice agar, YM or acetate. On solid YM, cells are unicellular in liquid culture after one day.
Small globose cells are observed mainly in chains or clusters, many with one bud. Isolate NRRL Y-18654 WO91/01~ PCT/US90/04290 colonies are cream white, slightly raised, shiny, round with smooth edges.
Isolate NRRL Y-18S27 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.
~iochemical and physiological tests of the isolates were as ~ollows:
Carbon Assimilation: NRRL Y-18314 Y-18313 Y-18654 Glucose + + +
Galactose + + +
L-sorbose + + +
Maltose + + +
Sucrose + + +
Cellobiose + + +
Trehalose + + +
Lactose Melibiose + - +
Raffinose + + +
Melezitose + + +
Inulin + + +
Soluble Starch w w +
D-xylose + + +
L-arabinose + + +
D-arabinose + + +
D-ribose + + +
L-rhamnose + w +
D-glucosamine + w +

w = weak Ethanol w w +
Erythritol w Glycerol + + +
Adonitol (Ribitol) + + +
Dulcitol ~Galactitol) + + +
D-mannitol + + +
D-sorbitol (glucitol) + + +
a-methly-D-glucoside + + +
Salicin + + +
Inositol Lactic acid w +
Citric acid + + +
Succinic acid + + +
Nitrogen assimilation: NRRL Y-18314 Y-18313 Y-18654 NH4N03 + + +
KN03 + + + (w~ak) N0 w Et~ylamine + + +
Fermentation:NRRL Y-18314 Y-18313 Y-18654 Glucose + + +
Galactose w +
Maltose - - -Sucrose + + +
Lactose Ra~finose - - ~f Nelibiose Inulin w - -Cellobiose - - -Melezitose Starch Trehalose w = weak Carbon Assimilation: NRRL Y-18527 Glucose +
Galactose L-sorbose Maltose Sucrose Cellobiose +

WO91/01~1 PCT/US90/04290 Trehalose Lactose Melibiose Raf f inose Melezitose Inulin Soluble Starch NT*
D-xylose L-arabinose D-arabinose D-ribose NT
L-rhamnose D-glucosamine NT
Ethanol Erythritol NT
Glycerol Adonitol (Ribitol) Dulcitol (Galactitol) D-mannitol NT
D-sorbitol (glucitol) a-meth~l-D-glucoside Salicin +
Inositol NT
Lactic acid NT
Citric acid NT
Succinic acid NT
Nitrogen assimilation: NRRL Y-18527 NH~N03 +
KNO3 +

Ethylamine +
35 Fermentation: NRRL Y-18527 Glucose +
Galactose Maltose Sucrose Lactose Raffinose Melibiose Inulin Cellobiose +
Melezitose Starch Trehalose +
w = weak NT = not tested Growth of the organisms is effected under aerobic conditions at any temperature satisfactory for growth of the organisms; i.e. from about 10C to about 30C. The preferred temperature range is about 20C to 25C. The pH of the nutrient medium is about neutral; i.e. 6.7 to 7.2. 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 ~rom about 24 to 48 hours for NRRL Y-18654.
Growth of isolate NRRL Y-18527 is preferably achieved at a temperature of 25-28C 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 WO91/01~1 PCT~US90/04290 to the inoculated liauid 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 diaitatum, Botyrtis cinerea. Rhizo~us stolonifer, Geotrichum candidum. Penicillium expansum, 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. Examples of 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, kumauat, lime and pummelo.
Said nuts may for example include: peanuts, almonds and WO91/01~1 PCT/US90/04290 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 1~ water based carrier (e.g. the microorganisms may be mixed/suspended in water. Other water based carriers include wa~er plus wetting and/or spreading agents); (3) an oil based carrier (e.g. "Fresh Mark" or "Fresh Wax 58PI' ~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, Orlando, FL); (4) a wax based carrier (e.g.
including wax coatings typically used on citrus fruit and apples, for example "Britex 551" or "Britex 559", both from Broshar (Chemicals) Ltd., Kefar-Saba, Israel); (5) 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 WO91/01~1 PCT/US90/04290 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. When grown in a liquid medium, the microorganisms may be applied in suspension with the liquid medium, however it is preferred in order to improve control, to apply the microorganisms in the presence of water or one or more of the aforementioned carriers. Compositions of the present invention may also include other additives including: (1) pesticides, such as ~ungicides (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 andtor use, including e.g.: (a) 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 Rhom and Hass Company; (4) WO91/01~1 PCT/US90/~290 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. 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. Typically, 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.

WO91/01~1 PCT/US90/~290 Concentrations of suspensions useful in the invention are any concentrations which inhibits the development of the targeted plant pathogen when applied to the fruit.
As will be obvious to one skilled in the art, 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 thçlplant pathogen. Exemplary concentrations range from about 1 x 104 to 1 x 109 CFU/ml, most preferably, from about 1 x 107 to 1 x 109 CFU/ml. For purposes of the in~ention, the abbreviation' 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. In addition, 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. Typically, the preferred time W O 91/01641 PC~r/US90/04290 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.
It is within the scope of the invention to treat the S fruits with isolates 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.
The natural or normal concentration of isolates NRRL
Y-18313, NRRL Y-18314, NRRL Y-186S4 and NRRL Y-18S27 on lS fruit may typically vary from 0 to lOo CFU/cmZ.
Hanseniaspora uvarum. or its asexual form Kloeckera a~iculata, 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).
However, the ability of these yeasts to control plant pathogens was unexpected since these yeast species have not previously been reported to have biological control WO91/01641 PCT/~S90/04290 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 105 CFU per cm2, or preferably at least about lo6 CFU per cm2. 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 105 CFU/cm2 .
It has surprisingly and unexpectedly been discovered that use of at least one calcium salt with the at least one microorganism of the present invention facilitates improved control of plant pathogens (notably, Rhizo~us 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;

WO91/01~1 PCT/US90/04290 1981-Plant Disease 66:402-403 and, Conway et al 1983 Phytopathology 73:1068-1011. While not wishing to be bound by a theory, it was believed that the dramatic effect of the calcium salt(s) on biocontrol may be the S result of calcium cation interaction with the microorganism(s), perhaps by affecting the antagonistic microorganisms survival at the wound site or by affecting its metabolism or by interaction with its metabolic products. In regard to preferred embodiments of the present invention relating to use of calcium chloride, it is especially surprising and unexpected that: 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).

WO91/01~1 PCT/US90/04290 Typical examples of the calcium salt include: calcium chloride, calcium carbonate, calcium propionate, and mixtures thereof. For example, calcium chloride may be utilized in concentrations of about l gm/lO0 ml, to about l0 gm/l00 ml, preferably about l gm/lO0 ml to about 5 gm/lOO ml and most preferably about 2 gm/l00 ml.
The following examples are intended to further illustrate the the invention and not to limit the scope as defined by the claims.
Example I
The effectiveness o~ Pichia auilliermondii NRRL Y-18314 was evaluated using the following seven citrus cultivars: grapefruit (Citrus aradisi Macf. cv 'Marsh Seedless'); 'Shamouti' and 'Valencia' orange (C.,sinensis Osbeck); lemon (C. lemon L. Burm 'Eureka'): Temple orange (Tanger hybrid, C. reticulata X C. sinensis); Kumauat ~ tunella maraarita); and pummelos, (C. ,arandis~.
Fruit rot pathogens tested included Penicillium diaitatum, Penicillium italicum and Geotrichum candidum Link. ex Pers., fungi responsible for the postharvest diseases green-mold, blue-mold and sour-rot, respectively.
A biologically pure culture of isolate NRRL Y-18314 WO91/01~1 PCT/US90/04290 .

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.
~he 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 24 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 us~ng similar procedures.
Isolate NRR~ Y-18314 was grown in flasks containing nutrient yeast dextrose broth (NYDB) on a reciprocal shaker at 30C 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.
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, WO91/01~1 PCT/US90/04290 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 105 to 1 x 10~ CFU/ml. One to two hours later, 20 microliters of an aqueous spore suspension of the targeted pathogen, 1 x 104 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 o~ 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.
The results are given in Tables I, II, and III:

WO 91/01641 PCI`/US90/042gO

TAB~E I

Relative effectiveness of NF~ZL Y-18314 in inhibiting Penicillium diqitatum decay of different citrus cultivars.

.
Citrus Antagonist Incubation time (days) cl~ltivar 4 5 6 7.

. _ Percent Infectiona Grapefruit MRRL Y-18314 0 2 6 11 (72) Control 90 97 100 100 Ch~Lnge, 'Sh3mcuti' NgRL Y-18314 0 3 10 17 ~42) Control 93 100 100 100 Ch~u~ge, 'Valencia' MRR~ Y-18314 2 4 8 17 ~42) Cbntrol 90 94 97 100 Lemon N}WL Y-18314 0 2 10 15 (42) Control 98 100 100 100 Temple NRKL Y-18314 2 4 10 14 (48) Control 95 96 99 100 Pummelo NF~ZL Y-1~314 0 0 2 2 -(24~ Control 83 90 92 96 . Kumquatb NRgL Y-18314 4 8 12 ; (150) Conbrol 19 23 37 _ a Nhm~r of inoculation sites per treatment is indicated in parentheses under the cultivar's name.
b Whole fruits were used without artificial inoculation. The fruit was dipped nx~xentarily in a 48 hr-old liquid culture o~ the N~RL Y-18314.
NYDB was used as control.

W O 9t/01641 P ~ /US90/04290 TABI~ II

Inhibition of Penicillium italicum decay of grapefruit and orange by NEWL Y-18314 Citrus Antagonist Incukation time (days) var 3 4 5 6 Percent Infectiona Grapefruit N~WL Y-18314 3 3 4 6 (72) Control 97 lOO 100 lOO
Orange 'V~lencia' NE~ZL Y-18314 3 8 lO lg (72) Control 84 95 97 lOO
Orange 'Shz~uti' NEEZL Y-18314 3 6 8 15 (72) Control 90 95 lOO lOO

a Nhm~Er of incculation sites.per treatment is indicated in parentheses uux~r the culti~nLr's name.

W O 91/01641 PC~r/US90/04290 TABLE III
Inhibition of Geotrichum candidum decay of grapefruit and lemon by NRRL Y-18314 . . .
Citrus Angatonist Incubation time ~days) ~11tivar 3 4 5 6 . . .
Percent infectiona Grapefruit NRRL Y-18314 33 8 9 (72) Co.. k ul 30 56 78 86 Lemon NRRL Y-18314 12 17 18 18 (30) Control 75 77 77 77 a Number of incculatlon sites per treatment is indicated in parentheses under the cultivar;s,name.

WO91/01~1 PCT/US90/04290 As shown in Table I, isolate NRRL Y-18314, was highly effective in inhibiting Penicillium diqitatum 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.
Table II shown that isolate NRRL Y-18314 was effective in inhibiting Penicillium italicum decay on grapefruit, oranges and other citrus fruit cultivars. As in the case of Penicillium diaitatum, 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 ~able III, Geotrichum candidum was controlled to a lesser extent that the Penicillia decays, particularly in lemons.

WO 91/01641 PCr/US90/042g Example II
The ability of Pichia auilliermondii NRRL Y-18314 to inhibit Rhizopus rot development in grapes was demonstrated.
A biologi~cally 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 28c 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 c~using a wound, or as injured single berries wounded by piercing non-injured berries with a needle. Control berries were dipped in sterile NYDB only.
one to two hours after the berries had been dipped in the suspension, 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 1 o4 spores/ml.

WO91/01~1 PCT/US90/04290 Alternatively, 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.
The results are shown in Figure 1. As shown in Figure 1, Pichia auilliermondii was effective in reducing ~h~QpUS rot in both injured and non-injured grape berries. Reduction of decay was most pronounced in berries that were not injured prior to inoculation and inoculated by nesting.

WO91/01~1 PCT~US90/~290 Exam~le 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 109 CFU/ml concentra'cion were added to the wounds, 50 microliters/wound. Apples were allowed to air dry.
Thereafter, an aqueous suspension of Botrvtis cinerea or Penicillium expansum spores, 1 x 104 spores/ml, were added to the wounds, 20 microliters/wound. Controls were inoculated with water only.
Measurements of infected areas were taken 5, 7, 9 days after inoculation. Results are shown in Figures 2 WO91/01~1 PCT/US90/042 after inoculation, with only small lesion development after nine days. Protection against Penicillium expansum was to a lesser extent than against Botrytis cinerea.
Nevertheless, Figure 3 clearly shows that applies treated with NRRL Y-18314 had a significant decrease in the development of Penicillium ex~ansum when compared to the untreated controls.

Example IV
The effectiveness of Pichia auilliermondii NRRL Y-18314, to inhibit Penicillium diaitatum on grapefruit was compared to the effectiveness of eight previously identified isolates of D.hansenii.
The eight isolates were obtained from the American Type Culture Collection, hereinafter referred to as "ATCC", located at 12301 Parklawn Drive, Rockville, Maryland 20252, USA. Identification of the isolates tested were as follows: ATCC 18538, ATCC 20220, ATCC
36239, ATCC 34022, ATCC 36239, ATCC 9367, ATCC 36767, and ATCC 18107.
Each isolate tested was incubated in NYDB liquid medium at 28C for 48 hours. Following centrifugation, the resulting pellets were washed twice with water and .

W09t/01~1 PCT/US90tO4290 thereafter suspended in water. Concentrations of the aqueous suspensions ranged from 1.3 x 107 to 1.3 x 109 CFU/ml.
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 aaueous 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 aqùeous suspension of Penicillium diaitatum, 1 x 105 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 diaitatum when compared to prior identified isolates of D. hansenii. After seven days of WO91/01~1 PCT/US90/04290 3s 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 Asperaillus 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 Asperaillus flavus was applied as described for the pathogen in Example 1. The treated nuts were incubated 14 days at 26' C. Figure 5A
15 i5 photograph of the peanuts treated with both Asperaillus fla~us and NRRL Y-18314, and Figure 5B is a photograph of peanuts treated only with As~eraillus flavus. As shown in these photographs, the results clearly show the inhibition by the yeast of the pathogen growth: Figure SA 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.

WO91/0!~1 PCT/US90/04290 EXAMPLE VI
The purpose of this example is to show the effectiveness of isolate NRRL-Y-18314 at inhibiting Aspergillus niaer 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. Similarly, the As~eraillus niaer 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 Asperaillus niger and NRRL-Y-18314, and Figure 6B is a photograph of peanuts treated only with Asperaillus ~i~Ç~- 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 VII
Twenty-five milliliters of a 48-hour-old NRRL-Y-18314 culture was centrifuged. The resulting pellet was resuspended in 10 ml. of each of the following: (A) a wax including a paraffin mineral oil base obtained from Durant-Wayland Inc., La Grange, GA; (B) Fresh Wax; (C) WO91/01~1 PCT/US90/~290 Stayfresh water based wax from FMC Corporation, Woodstock, VA; (D) "Fresh Wax 58P" including a paraffin mineral oil base, referred to herein above. Initial dilution counts (of CFU/ml) were made in each wax (i.e.
initial, time zero counts). Dilutions were carried out at the time intervals indicated in Table IV (except as noted in said table), by mixing: (a) .l milliliter of each mixture of wax and culture, with: (b) .9 milliliter of the respective wax. The resultant mixtures were then plated on yeast malt agar plates. The plates were maintained at about 20 to 25 C. Results are shown in Table IV. Entries in Table IV are all in colony forming units per milliliter.

W O 91/01641 PC~r/US90/04290 -.TABLE IV

Durand~Wavland Fresh Mark Sta~fresh Fresh Wax 58P
Initially 1 X 105 l.0 X 107 8.9 X lO8 l.0 X 195 (Time Zero) less ~ -19 Days 2.5 X 106 2.0 X 105 2.9 X 106 l.0 X lO
35 Days 2.7 X 106 1.4 X lo6 9.0 X 104 l.9 X 105 46 Days 5.9 X 106 8.4 X 105 3.0 X 104 l.5 X 105 less than 60 Days l.l X 107 2.3 X 106 l.0 X 104 4.9 X 106 76 Days 3.0 X 106 4.9 X lO6 less ~ 1.5 X 106 258 Days NT* tnNTc** NT tnNrc 342 Days NT TNTC NT TNTC
**TNIC stands for too nLm~nous to count.
*NT stands for not tested, i.e. dilution plates were not made.

This example clearly indicates the surprisingly and unexFY<~b3~ly highviability of NRRLrY-18314 `in cc~nY~rcially available ~axes at room temperature, even for extended p~riods of time.

WO91/01~1 PCT/US90/W2 EXAMPLE VIII
The purpose of this example is to show that either freeze dried cells or whole cells of NRRL Y-18314 can remain viable in a commercially available wax (i.e. Fresh S Mark Wax) for long periods of time. Freeze dried cells were frozen in liquid nitrogen and placed on a lyophilizer for 48 hours and mixed with the wax, (4 volumes of wax to one volume of freeze dried cells).
Whole cells were centrifuged into a pellet at 5000 RCF
and resuspended in the wax. (4 volumes of wax to one volume of whole cell pellet). The results are shown in Table V. Entries in Table V are all in colony forming units (i.e. CFU1 per milliliter.

.~ , W O 91/0164t P ~ /US90/04290 , TABLE IV

Ourand-Wavland Fresh Mark StavfreshFresh Wax 58P
Initially 1 X }05 1.0 X 107 8.9 X 108 1.0 X 105 (Time Zero) less tha~-19 Days 2.5 X 106 2.0 X 10S 2.9 X 106 1.0 X 10~
35 Days 2.7 X 106 1.4 X 106 9.0 X 104 1.9 X 105 46 Days 5.9 X 106 8.4 X 105 3.0 X 104 1.5 X 105 less than 60 Oays 1.1 X 107 2.3 X 106 1.0 X 104 4.9 X 106 less than 76 Days 3.0 X 106 4,9 X 106 1.0 X 104 1.5 X 106 258 Days NT* TNTC** NT INTC
342 Days NT TNIC NT TNIC
**l~nrC stands for too numercus to count.
*NT stands for Fot t~sted, i.e. dilution plates were not made.

miS example clearly indicates the surprisingly and unexpectedly high viability of NRRLrY-18314 'in cr~m~Ercially available waxes at room temperature, even for extended peri~C of time.

WO91/01~1 PCT/US90/04290 EXAMPLE IX
Five milliliters of a YM broth culture of NRRL-Y-18314 (5.6 X 108 CFU/ml) were mixed with 5 milliliters of gum. Gum concentration of the s milliliter solutions ranged from 1-20% as indicated in Table VI. The culture and gum mixture was added to 40 cm3 of either corn starch (25.7 g) or silica gel ~27.1 g). This preparation was mixed and dried at 54C for 4 days and then ground in a mortar and pestle to a fine powder. The powder was then stored at 4-C. One gram of this powder was added to 10 milliliters of sterile water and mixed with a stirring bar for 20 minutes and dilution plating was done to determine NRRL-Y-18314 populations. The results are shown in Tàble VI in units of colony forming units per milliliter.

TAB~ Vl CO~N,STP~K~ 9 DAYS 24 DAYS 37 DAYS 56 DAYS
Tragacanth less ~ l~CC than 1% 1.0 X 10 l.O'X 104 Karaya 10~ 3.0 X 105 5.0 X 104 2.0 X 104 5.0 X 104 Locust Bean less ~
15% 1.0 X 10 2.0 X 104 3.0 X 104 1.8 X lO5 Xanthan 20% 8.0 x 105 7.3 x 105 3.0 x 105 5 9 x 105 Iragacanth less tha~ less than 1% 1.0 X 10~ 1.0 X 104 less than 10% 2.0~X,105 1.0 X 104 1.0 X 104 Locust Bean les5 t ~ less than 15~ ` 1.0 X 10 1.0 X 104 Xanth3n 20% 1.0 X ~05 1.1 X 105 1.0 X 104 , m ese re~.~lts clearly show that the NRRLrY-18314 remained viable for an extended period of time in any of a variety of gums ccmbined with either corn starch or silica qel.

WOgl/01~1 PCT/US90/04290 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.

WO 91/01641 PCl`/US90/04290 ._ K ~ K ~ K ~ K ~ x ~ K

1¦ K K illl K ~ K

n ~ K ¦X ¦ K ¦ K

~ ~; il K X il K K 1¦1 K K ~ K K

~ I 'Oo.Do I e~o~o~Do ~ ol~or~ ~ oro l`~l` ~o'Do~Oo~Oo o~ XX XXX XXXX XXXX XXXX XXXX
_N O ~D _ O N _ O N ~ ~ _ O N ~ ~ ~ O ~<
m v~ ~ o _ N ~-- ~ N O ~ 'O ~ _i _ _ N _ _ ~ I` 'O

~Do~Oo e~O~O~oO 'o'`ol`ol`o 'o~`o'o~`o t~ t-o .Do~.Oo~Oo~Oo XX XXX XXXX XXXX XXXX XXXX
__ _o_ ~ 1`~ _-oo_ ~m-l O~o~--V~ ~1 ~ _ ; _ N - ~ _ In _ _ _ lil _ 0 It _ _ O ~ _ _ _ _ ~ _ _ ~ _ ~ _ ~

WO91/01~1 PCT/US90/~290 These results clearly show that the NRRL Y-18314 remained viable for extended periods of time in various combinations of talc and gum.

EXAMPLE XI
The purpose of this example is to show that CaCl2 and CaC03 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 a~ueous solution consisting o~ NRRL Y-18314 in sterile distilled water at a concentration of 107 CFU/ml with each of the salts listed in Table VIII at a concentration of 2 grams/lO0 ml (with the exception of FeS04 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 - 20 VIII at a concentration of 2 grams/lO0 ml (with the exception of FeS04 which was utilized in a concentration of 5 millimolar). Also a control was run using only sterile distilled water. Two hours after application of W O 91/01641 PC~r/US90/04290 the above solutions, each of the apples was challenged with 20 microliters of a 105 spore/ml suspension of Botrytis cinerea. The average percent fruit infection of four trials (8 to 10 replicates per trial) was measured 5 10 days after inocula~ion with the Botrytis cinerea. The results are shown in Table VIII.

TABT~ VIII

Percent Infection Inorgan~c Salt Alone Salt and Salt (No NRN Y-18314~ N~ Y-18314 CaC12 95.0 (+ 5.0~ 3,3 (+ 3,3)*
cac03 71.9 (+ 13.5) 27.5 (+ 24.3)*
FeS04 87.5 (+ 12.5) 57.5 (+ 21.0) KCl 100.0 (+ O.o) 47.5 (+ 20.6)*
~C12 100.0 (+ 0.0) 61.3 (+ 17.4) MnC12 100.O (~ O.O) 97.5 (+ 2.5) NaCl loO.0 (+ o.o) 71.6 (+ lS.7) C~ONI~IS
~2 100.0 (+ O.o) 59.5 (+ 14.4) WO 91/01~1 PCT/US90/04290 Values in parentheses are standard errors of the mean.
Asterisk indicates that means within a row are significantly (P ~ 0.05) different according to SAS GLM
analysis of variance on arcsin square root-transformed data. It may be observed that none of the salts used alone provided statistically significant reduction of infection (i.e. none of the values for use of salt alone differ significantly from use of salt water alone i.e.
100% infection). Further the combination of NRRL Y-18314 and the calcium salts clearly provide synergistic infection reduction, as evidenced by the fact that CaC12 and CaC03 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.
However, in actuality, the combination of CaCl2 with NRRL
Y-18314 provided more than twice the expected value of 45% i.e. about 96.7%; and the combination of CaCo3 with NRRL Y-18314 provided 72.5% which is significantly higher than the expected value of 58%.

W O 91/01641 PC~r/US90/04290 EX~MPLE XII

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 108 CFU/ml suspension of the respective yeasts in sterile distilled water, with or without 2 grams/100 ml of CaC12 as referred to in Table IX. Two hours later the apples were challenged with 20 microliters of a suspension of 104 spores/milliliters of Botrvtis cinerea. Seven days after inoculation percent infection was observed. Results are shown in Table IX.

W O 91/01641 P ~ /US90/04290 T~IE IX

-Y~Ct Average Strain CaC12 Percent Infection NRRLrY-18527 YES 6.7 (+ 6.7)*
" " NO 41.7 (+ 18.8) NRRLrY-18314 YES 0.0 (+ 0-0)*
" " NO 26.7 (+ 6.7) NCNE YES 100.0 (+ 0.0) " " NO 100. 0 (+ O. O) m e above entries are the average of 3 trials per treatment. Values in paren~Keses are standar1 errors of the mean. Asterisk indicates that fruit rot in yeast treatments with calcium chloride is significantly tP ~ 0.05) less than that of fruit treated with yeast alone. It may be observed that significant infection reduction wa,s achiev~d using either of the y~ct, and that further ~nfection reduction w~s achieved using the ccTbination of CaC12 with each yeast.

EXAMPLE XIII
The purpose of this example is to demonstrate the effectiveness of NRRL Y-18314 and combinations thereof with CaCl2 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 CaCl2 (concentration of 2 gram/lO0 ml) as noted in Table X.
Two hours later the apples were challenged with 20 microliters of a spore suspension of Penicillium ex~ansum at the concentrations referred to in Table X. Seven days after inoculation lesion diameter was observed. Results are shown in Table X.

P ~ /US90/04290 TABLE X

__ NRRLrY-18314Penicillium sporeAverage Presence of Concentration Concentration TPcion Diameter Standard C~C12 (CFU/nl)(spores/ml~ (m~) Error NO 107 103 32.4 8.4 " " " 104 .49.8 2.1 " " " I 105 40.8 6.1 NO lo8 . 103 18.4 7~9 " " " 104 35.2 2.1 " " " 105 36.2 9.2 YES 107 103 14.4 7.4 104 9.0 6.3 ~ 105 15.2 9.3 YES 108 103 7.6 5.3 " " " 104 17.4 5.8 " " " 105 19.2 6.7 YES ~ 103 40.4 1.9 " " 104 45.2 1.6 " " 105 47.0 1.6 NO 0 103 47.2 2.3 . :

WO91/01~1 PCT/US90/04290 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 CaCl2, did not facilitate significant reduction of decay (greater than 50%) in most treatments, as compared to the water control and CaCl2 treatments without NRRL Y-18314.
However, when NRRL Y-18314 was applied with CaCl2 decay was reduced greater than 50% at all yeast concentrations and at all Penicillium spore concentrations tested.
EXAMPLE XIV
The purpose of this example is to illustrate the synergistic effects of combinations of various concentrations of CaCl2 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 104 spore/milliliter suspension of Penicillium diaitatum.
The grapefruit was incubated for 5 days at 24C before observations were taken. Results of average percent fruit rot were as follows (data is the average of 2-3 trials per treatment):

WO 91101641 Pcr/usso/o429o ~r~u3r~

FOR STRAIN NRRLrY-18314 Averaqe Percent Fruit Rot NRRLrY~18314 Concentration (cfu/ml) Concen~ration 0 106 107 1o8 % 59.5 31.5 10.5 o 1 % 39.0 22.8 1.5 Nl~
2 % 22.8 7.5 1.5 NT

.. .. .. . _ .
* Nr stands for ~not ~ed".

~B~: XII
FOR STRAIN NRRLrY-18313 Averaae Percent Fruit Rot NRRLrY-18313 Concentration (cfu/ml) Concentration 0 106 107 108 , 0 % 91.7 69.0 23.3 6.5 1 % 63.0 3.0 6.0 1.5 2% 33.0 7.3 7.3 14.3 WO91~01~1 PCT/US90/04290 EXAMPLE XV
The purpose of this example is to show that yeast cells rather than the yeast cultu're 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). Two hours later the peaches were inoculated with 20 microliters of a 104 spores/ml suspension of Rhizo~us stolonifer.
Average percent infection was observed 4 days later. The results are as follows (each value is the average of two to five trials):

4~ PCl`/US90/042gO

~; ~, o o .
oOoOoO oO

~ o~ 5 5 1 N N N

+1 l ¦ ¦ ~ ~ + ~
U~

. : l t_ N ~" N
+l +~ +l +l +

~ ~ ~v~ z~ ~

WO 91/01641 Pcr/usgo/042gO

EXAMPLE XVI
Single Thompson seedless grapes were wounded by pulling from stems. The grapes were then dipped in a suspension of the yeasts specified in Table XIV at concentration of 108 bo 109 cfu/ml and incubated at 220C.
At 5 and 6 days the percent fruit rot by naturally occurring organisms (e.g. As~eraillus niaer and Rhizopus stolonifer! was as follows (data is for 3 replicates of 20 berries per fruit treatment);

TABLE XIV

Percent Fruit Rot ~ it Treatment 5 OAYS 6 O~YS
N~rY-18527 5.0 % 13.0 %
N~rY-18314 18.0 % 31.0 %
Water 90.0 ~ 100.0 %
NyDBl 50.0 % 92.0 %
-NYDB = sterile ~ ture broth, nutrient y~ct dexbxse broth.

Fruit rot due to Rhizopus ~Jas not observed in gray~ treated with any of the yeast tredtments.

WO91/01~1 PCT/US90/04290 EXAMPLE XVII
Whole clusters of "Perlett~ grapes were dipped in suspensions of the yeast antagonists specified in Table XV at concentrations of lO8 to 109 cfu/ml. Two replicates of 6 fruit clusters' were used for each treatment.
Percent of naturally occurring fruit rot was observed after 7 days of storage at 20~C. The results were as follows:

TABT~ XV

-Percent Fruit Rot Yeast Treatment n As~erqillus Botrvtis Rhizo ~ Total NR~L~Y-18527 825 1.8 %2,0 % 3.2 % 7.~ ~
NRRLrY-18314 825 0.7 %0.6 % 8.2 % 9.5 %
15 Water 890 1.4 %3.0 % 16.2 % 20.6 %
n = nu~xr o~ fruit per treatment.

The foregoing detailed descriptions and examples are given merely for purposes of illustration. Modifications and variations may be made therein without departing from the spirit and scope of the invention.

Claims (77)

WHAT IS CLAIM:

1. A biologically pure culture of at least one isolate of Pichia guilliermondii having the identifying characteristics of an isolate selected from the group consisting of NRRL Y-18313, NRRL Y-18314, and NRRL Y-18654.

2. The biologically pure culture of claim 1, having the identifying characteristics of NRRL Y-18313.

3. The biologically pure culture of claim 1, having the identifying characteristics of NRRL Y-18314.

4. The biologically pure culture of claim 1, having the identifying characteristics of NRRL Y-18654.

5. A process for inhibiting plant pathogen development on an agricultural commodity comprising :
applying to an agricultural commodity at least one microorganism, which is an antagonist against plant pathogens but is not antibiotic, in an amount effective to inhibit plant pathogen development on said agricultural commodity.

6. A process for inhibiting plant pathogen development on an agricultural commodity comprising:
applying to an agricultural commodity at least one calcium salt and at least one microorganism, said at least one microorganism being an antagonist against plant pathogens, wherein said at least one calcium salt and said at least one microorganism are applied to said agricultural commodity in an amount effective to inhibit plant pathogen development on said agricultural commodity.

7. The process of claim 6, wherein said at least one microorganism is not antibiotic.

8. The process of claim 5, wherein said at least one microorganism is selected from the group consisting of fungi, bacteria, viruses and mixtures thereof.

9. The process of claim 8, wherein said at least one microorganism is a yeast.

10. The process of claim 9, wherein said at least one microorganism is at least one yeast selected from the group consisting of: a yeast having the identifying characteristics of deposit NRRL Y-18313, a yeast having the identifying characteristics of deposit NRRL Y-18314, a yeast having the identifying characteristics of deposit NRRL Y-18654 and a yeast having the identifying characteristics of deposit NRRL Y-18527.

11. The process of claim 10, wherein said at least one microorganism is a yeast having the identifying characteristics of deposits NRRL Y-18313.

12. The process of claim 10, wherein said at least one microorganism is a yeast having the identifying characteristics of deposit NRRL Y-18314.

13. The process of claim 10, wherein said at least one microorganism is a yeast having the identifying characteristics of deposit NRRL Y-18527.

14. The process of claim 10, wherein said at least one microorganism is a yeast having the identifying characteristics of deposit NRRL Y-18654.

15. The process of claim 5, wherein said step of applying includes applying to said agricultural commodity an additive selected from the group consisting of pesticides, preservatives, carriers, surfactants, wetting agents and mixtures thereof.

16. The process of claim 11, wherein said step of applying includes applying to said agricultural commodity a carrier, wherein said carrier is selected from the group consisting of: a gel based carrier, a gum based carrier, a water based carrier, a wax based carrier, an oil based carrier, a talc based carrier, a starch based carrier and mixtures thereof.

17. The process of claim 5, wherein said at least one microorganism is applied to said agricultural commodity in a preparation which is essentially free of other microorganisms.

18. The process of claim 6, wherein said step of applying includes applying to said agricultural commodity a mixture of said at least one calcium salt and said at least one microorganism.

19. The process of claim 5, wherein said plant pathogen is selected from the group consisting of Penicillium italicum Wehmer, Penicillium digitatum, Botrytis cinerea, Rhizopus stolonifer, Geotrichum candidum, Penicillium expansum, Alternaria alternate, Asperaillus flavus, Asperaillus niger Rhizopus arrhizus, Gilbertella persicovia, Mucor spp., Pezicula malicorticas, Monilinia spp. and bacterial pathogens.

20. The process of claim 18, wherein said plant pathogen is selected from the group consisting of Monilinia fructicola and Monilinia laxa.

21. The process of claim 6, wherein said at least one calcium salt is selected from the group consisting of calcium chloride, calcium propionate, calcium carbonate and mixtures thereof.

22. The process of claim 5, wherein said agricultural commodity is selected from the group consisting of fruits, vegetables, cereals, grains, nuts, seeds and silage.

23. The process of claim 22, wherein said agricultural commodity is a fruit selected from the group consisting of a citrus fruit, grape, apple, pear, tomato, persimmon, strawberry, peach, apricot, cherry, papaya, raisin, prune, fig, dried apricot and date.

24. The process of claim 23, wherein said agricultural commodity is citrus fruit selected from the group consisting of grapefruit, orange, lemon, kumquat, lime and pummelo.

25. The process of claim 22 wherein said agricultural commodity is a nut selected from the group consisting of peanuts, almonds and pecans.

26. The process of claim 22 wherein said agricultural commodity is a grain selected from the group consisting of wheat, corn, sorghum, soybeans and barley.

27. The process of claim 5 wherein said step of applying includes dusting, injecting, rubbing, spraying or brushing said agricultural commodity with a composition containing said at least one microorganism.

28. The process of claim 5 wherein said step of applying includes dipping or rolling said agricultural commodity in a composition containing said at least one microorganism.

29. (Amended) A composition comprising a mixture of:
at least one yeast selected from the group consisting of: a yeast having the identifying characteristics of deposit NRRL Y-18313, a yeast having the identifying characteristics of deposit NRRL Y-18654 and a yeast having the identifying characteristics of deposit NRRL Y-18527 and mixtures thereof, wherein the at least one yeast is an antagonist against plant pathogens but is not antibiotic; and a carrier for said at least one yeast which is selected from the group consisting of:
a gel, gum, wax, oil, talc, starch and mixtures thereof.

30. (Amended) A composition comprising a mixture of:
at least one yeast selected from the group consisting of: a yeast having the identifying characteristics of deposit NRRL Y-18313, a yeast having the identifying characteristics of deposit NRRL Y-18314, a yeast having the identifying characteristics of deposit NRRL Y-18654 and a yeast having the identifying characteristics of deposit NRRL Y-18527 and mixtures thereof; and a carrier for said at least one yeast, wherein at least 99% by count of said at least one yeast is antagonistics against plant pathogens but is not antibiotic.

31. (Amended) A composition comprising a mixture of:
at least one calcium salt and at least one yeast selected from the group consisting of: a yeast having the identifying characteristics of deposit NRRL Y-18313, a yeast having the identifying characteristics of deposit NRRL Y-18314, a yeast having the identifying characteristics of deposit NRRL Y-18654 and a yeast having the identifying characteristics of deposit NRRL Y-18527 and mixtures thereof, wherein the at least one yeast is an antagonist against plant pathogens.

32. (Amended) The composition of claim 31 wherein said at least one yeast is not antibiotic.

33. (Amended) The composition of claim 31 consisting essentially of said at least one calcium salt and said at least one yeast.

34. (Amended) The composition of claim 31 wherein at least 99% by count of yeasts in said composition are antagonistic to plant pathogens.

35. (Amended) The composition of claim 31 wherein at least 99% by count of yeasts in said composition are not antibiotic.

36. Cancelled

37. Cancelled

38. Cancelled

39. Cancelled

40. Cancelled

41. Cancelled

42. Cancelled

43. (Amended) The composition of claim 29 further including an additive selected from the group consisting of pesticides, preservatives, surfactants, wetting agents and mixtures thereof.

44. The composition of claim 43 wherein said additive is a preservative selected from the group consisting of methylcellulose, silica gel and mixtures thereof.

45. The composition of claim 31 further including an additive selected from the group consisting of pesticides, preservatives, carriers, surfactants, wetting agents and mixtures thereof.

46. The composition of claim 45 wherein said additive is a carrier selected from the group consisting of a gel, gum, wax, oil, talc, starch, water and mixtures thereof.

47. The composition of claim 46 wherein said carrier is xanthan gum.

48. The composition of claim 45 further including a preservative selected from the group consisting of a gum, methylcellulose, silica gel and mixtures thereof.

49. The composition of claim 45 wherein said preservative is xanthan gum.

50. The composition of claim 29 wherein said carrier Is selected from the group consisting of starch, talc and mixtures thereof.

51. The composition of either claim 29 further including at least one calcium salt.

52. (Amended) The composition of claim 51 wherein said at least one calcium salt is selected from the group consisting of calcium chloride, calcium propionate, calcium carbonate and mixtures thereof.

53. (Amended) The composition of either claim 30 or 31 wherein said at least one calcium salt is selected from the group consisting of calcium chloride, calcium propionate, calcium carbonate and mixtures thereof.

54. (Amended) A manufacture comprising an agricultural commodity having thereon a concentration of at least about 105 colony forming units per square centimeter of at least one yeast selected from the group consisting of: a yeast having the identifying characteristics of deposit NRRL Y-18313, a yeast having the identifying characteristics of deposit NRRL Y-18314, a yeast having the identifying characteristics of deposit NRRL Y-18654 and a yeast having the identifying characteristics of deposit NRRL Y-18527 and mixtures thereof, wherein the at least one yeast is an antagonist against plant pathogens but is not antibiotic.

55. The manufacture of claim 54 wherein said concentration is at least about 106 colony forming units per square centimeter.

56. (Amended) A manufacture comprising an agriculture commodity having thereon at least one yeast selected from the group consisting of: a yeast having the identifying characteristics of deposit NRRL Y-18313, a yeast having the identifying characteristics of deposit NRRL Y-18314, a yeast having the identifying characteristics of deposit NRRL Y-18654 and a yeast having the identifying characteristics of deposit NRRL Y-18527 and mixtures thereof, wherein the at least one yeast is an antagonist against plant pathogens but is not antibiotic.

57. The manufacture of either claim 54 or 56 wherein said agricultural commodity has a calcium salt thereon.

58. The manufacture of claim 57 wherein said calcium salt is selected from the group consisting of calcium chloride, calcium propionate, calcium carbonate and mixtures thereon.

59. (Amended) A manufacture comprising an agricultural commodity having thereon a calcium salt and at least one yeast selected from the group consisting of;
a yeast having the identifying characteristics of deposit NRRL Y-18313, a yeast having the identifying characteristics of deposit NRRL Y-18314, a yeast having the identifying characteristics of deposit NRRL Y-18654 and a yeast having the identifying characteristics of deposit NRRL Y-18527 and mixtures thereof, wherein the at least one yeast is an antagonist against plant pathogens in a concentration of at least about 105 colony forming units per square centimeter.

60. (Amended) The manufacture of claim 59 wherein said at least one yeast is not antibiotic.

61. (Amended) A manufacture comprising an agricultural commodity having thereon a calcium salt and at least one yeast selected from the group consisting of:
a yeast having the identifying characteristics of deposit NRRL Y-18313, a yeast having the identifying characteristics of deposit NRRL Y-18314, a yeast having the identifying characteristics of deposit NRRL Y-18527 and mixtures thereof, wherein the at least one yeast is an antagonist against plant pathogens.

62. The manufacture of either claim 59 or 61 wherein said calcium salt is selected from the group consisting of calcium chloride, calcium propionate, calcium carbonate and mixtures thereof.

63. Cancelled

64. Cancelled

65. Cancelled

66. Cancelled

67. Cancelled

68. Cancelled

69. Cancelled

70. The manufacture of either claim 54, 56, 59 or 61 wherein said agricultural commodity is selected from the group consisting of fruits, vegetables, cereals, grains, nuts, seeds and silage.

71. The manufacture of claim 70 wherein said agricultural commodity is a fruit selected from the group consisting of a citrus fruit, grape, apple , pear, tomato, persimmon, strawberry, peach, apricot, cherry, papaya, raisin, prune, fig, fried apricot and date.

72. The manufacture of claim 71 wherein said agricultural commodity is a citrus fruit selected from the group consisting of grapefruit, orange, lemon, kumquat, lime and pummelo.

73. The manufacture of claim 70 wherein said agricultural commodity is a nut selected from the group consisting of peanuts, almonds and pecans.

74. The manufacture of claim 70 wherein said agricultural commodity is a grain selected from the group consisting of wheat, corn, sorghum, soybeans and barley.

75. The manufacture of either claim 54, 56, 59 or 61 wherein said agricultural commodity also has thereon an additive selected from the group consisting of pesticides, preservatives, carriers, surfactants, wetting agents and mixtures thereof.

76. A biologically pure culture of an isolate of Hanseniaspora uvarum having the identifying characteristics of isolate NRRL Y-18527.

77. The composition of claim 29 wherein said carrier is xanthan gum.