Inhibiting plant pathogens with an antagonistic microorganism(s)

The present invention is drawn to biological control of plant pathogens (e.g. either preharvest or postharvest diseases) on agricultural commodities (such as fruits, vegetables, cereals, grains, nuts, seeds and silage) by use of at least one microorganism which is an antagonist against plant pathogens.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to the biological control of plant diseases 
(e.g. either pre-harvest or postharvest diseases) in agricultural 
commodities such as fruit. More particularly, this invention relates to: 
(1) methods for biologically controlling plant diseases (such as post 
harvest 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) manufactures produced by such methods. 
2. 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 without posing such hazards to humans or the 
environment. 
Recently, it has been shown that the postharvest treatment of fruit with 
antagonistic microorganisms is an effective 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 Matoswas able to 
reduce mold incidence from 35% to 8% when a species of Trichoderma was 
inoculated with Penicillium digitatum 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 candidum link. ex Pers., and 
P. digitatum. Singh et al. [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 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 may 
be 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.sup.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.sup.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 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. 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 (e.g. fruit-rot) pathogens which affect a wide variety of 
agricultural commodities. Three isolates of the new strains have been 
deposited with the culture collection at The Northern Regional Research 
Center, U.S. Department of Agriculture, Peoria, Ill. 61604, under the 
acquisition numbers NRRL Y-18313, NRRL Y-18314 and NRRL Y-18527. NRRL 
Y-18314 has been identified as Pichia quilliermondii and NRRL Y-18527 has 
been identified as Hanseniaspora uvarum (Nichaus) Shehata, Mrak 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. 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 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 and mixtures 
thereof. In effect, the organisms multiply and occupy the surfaces of 
wounded fruit, thereby preventing infection by plant (e.g. fruit-rot) 
pathogens.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Aspects of the present invention my be practiced with a variety of 
microorganisms which are antagonistic against plant pathogens (such 
microorganisms my for example exert their antagonism by either out 
competing the pathogen for available nutrients or rendering the infection 
site unfavorable for the pathogen) but which are not antibiotic, 
including: fungi (e.g. yeasts, for example, at least one yeast selected 
from the group consisting of yeasts having the identifying characteristics 
of deposite NRRL Y-18313, NRRL Y-18314, and NRRL Y-18527) bacteria, 
viruses and mixtures thereof. 
Microorganisms useable in the present invention my for example be 
identified by the following procedure: 
(1) screening agricultural commodities (e.g. the surface(s) of said 
agricultural commodities) for the presence of a microorganism(s); 
(2) recovering (e.g. by washing or rinsing from the agricultural commodity) 
and isolating said microorganism(s); 
(3) testing said microorganism(s) for antagonistic activity against plant 
pathogens, and; 
(4) in regard to those aspects of the instant invention relating to 
nonantibiotic microorganisms, testing for absence of antibiotic activity. 
However, it should be understood that said microorganism(s) may be 
obtained from sources other than said agricultural commodities. 
In regard to a preferred embodiment of the present invention, isolates of 
NRRL-Y-18313 and NRRL-Y-18314 were obtained from the surface of citrus 
fruits by repeatedly washing the fruit with water, and NRRL Y-18527 was 
isolated from the surface of a grape. NRRL Y-18527 has been identified as 
Hanseniaspora uvarum (Nichaus) Shehata, Mrak et Phaff. The organisms are 
thereafter plated and grown on any nutritionally rich medium sufficient to 
support growth of the organisms. 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 as determined by the American Type Culture Collection: 
colonies are 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-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. 
Biochemical and physiological tests of the isolates were as follows: 
______________________________________ 
Carbon 
Assimilation: 
NRRL-Y-18314 
NRRL-Y-18313 
NRRL-Y-18527 
______________________________________ 
Glucose + + + 
Galactose 
+ + - 
L-sorbose 
+ + - 
Maltose + + - 
Sucrose + + - 
Cellobiose 
+ + + 
Trehalose 
+ + - 
Lactose - - - 
Melibiose 
+ - - 
Raffinose 
+ + - 
Melezitose 
+ + - 
Inulin + + - 
Soluble w w NT* 
Starch 
D-xylose + + - 
L-arabinose 
+ + - 
D-arabinose 
+ + - 
D-ribose + + NT 
L-rhamnose 
+ w - 
D-glucos- 
+ w NT 
amine 
Ethanol w w - 
Erythritol 
w - NT 
Glycerol + + - 
Adonitol + + - 
(Ribitol) 
Dulcitol + + - 
(Galactitol) 
D-mannitol 
+ + NT 
D-sorbitol 
+ + - 
(glucitol) 
a-methly- 
+ + - 
D-glucoside 
Salicin + + + 
Inositol - - NT 
Lactic acid 
w + NT 
Citric acid 
+ + NT 
Succinic acid 
+ + NT 
______________________________________ 
Nitrogen 
assimilation: 
NRRL-Y-18314 
NRRL-Y-18313 
NRRL-Y-18527 
______________________________________ 
NH-NO.sub.3 
+ + + 
KNO.sub.3 
+ + + 
NO.sub.2 w w NT 
Ethylamine 
+ + + 
______________________________________ 
Fermentation: 
NRRL-Y-18314 
NRRL-Y-18313 
______________________________________ 
Glucose + + + 
Galactose 
w + - 
Maltose - - - 
Sucrose + + - 
Lactose - - - 
Raffinose 
- - - 
Melibiose 
- - - 
Inulin w - - 
Cellobiose 
- - + 
Melezitose 
- - - 
Starch - - - 
Trehalose 
- - + 
______________________________________ 
w = weak 
NT = not tested 
Growth of isolates NRRL Y-18313 and NRRL Y-18314 may be effected under 
aerobic conditions at any temperature satisfactory for growth of the 
organisms, i.e. from about 10.degree. C. to about 30.degree. C. The 
preferred temperature range is about 20.degree. C. to 25.degree. C. 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 isolates to reach a stationary phase 
of growth, preferably, from about 40 to 60 hours. Growth of isolate NRRL 
Y-18527 is preferably achieved at a temperature range of 
25.degree.-28.degree. 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 and NRRL Y-18527 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 isolates are harvested by conventional 
sedimentary methodology (e.g. centrifugation) or filtering. Cultures are 
stored on silica gel and frozen at -20.degree. C. until use. 
The microorganisms of the present invention (including isolates 
NRRL-Y-18313, NRRL-Y-18314, and/or NRRL Y-18527) are useful to control a 
variety of plant pathogens. Exemplary species of plant pathogens include, 
but are not limited to, Penicillium italicum Wehmer, Penicillium 
digitatum, Botrytis cinerea, Rhizopus stolonifer, Geotrichum candidum, 
Penicillium expand, Alternaria alternata, Aspergillus flavus, Aspergillus 
niger, Rhizopus arrhizus, Gilbertella persicovia, Mucov spp., Pezicula 
malicorticas, Monilinia spp. (e.g. Monilinia fructicola or Monilinia 
laxa), and bacterial pathogens. 
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, 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, 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 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 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.: (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) additives which promote spreading of the compositions oft he 
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 dusted on an agricultural commodity, or the powdered 
composition may be mixed 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. 
When the compositions of the present invention are in the form of a liquid 
mixture or suspension, any concentration of constituents may be used which 
inhibits plant pathogen development of the targeted plant pathogen when 
applied to an agricultural commodity. As will be apparent to one skilled 
in the art, effective concentrations may vary depending upon such factors 
as: (1) the type of agricultural commodity; (2) the physiological 
condition of the agricultural commodity (e.g. ripeness ); (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.times.10.sup.4 to 1.times.10.sup.9 CFU/ml, most preferably, from 
about 1.times.10.sup.7 to 1.times.10.sup.9 CFU/ml. For purposes of this 
invention, the abbreviation "CFU" is used herein to designate "colony 
forming units." According to one aspect of the present invention the 
microorganism(s) are applied to the agricultural commodity in a 
preparation which is essentially free of other microorganisms. 
The microorganisms of the invention may be applied to agricultural 
commodities using conventional methods such as dusting, injecting, 
rubbing, rolling, dipping, spraying or brushing. In addition, the 
microorganisms of the invention may be incorporated into a variety of 
compositions suitable for application to agricultural commodities, 
including waxes, wraps or other protective coatings used in processing the 
agricultural commodity. 
The natural or normal concentration of isolates NRRLY-18313, NRRLY-18314, 
and NRRLY-18527 on fruit may typically vary from 0 to 100 CFU/cm.sup.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). 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 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.sup.5 CFU per cm.sup.2, or 
preferably at least about 10.sup.6 CFU per cm.sup.2. It should be 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.sup.5 CFU/cm.sup.2. 
The agricultural commodities maybe treated any time before or after 
harvest. Typically, 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. 
It is within the scope of the present invention to treat the agricultural 
commodity with isolates NRRL-Y-18313, NRRL-Y-18314 or NRRLY-18527 alone, 
or in combination. 
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, 
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(s) 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. While not wishing to be bound by a theory, 
Applicants believe that the dramatic effect of the calcium salt(s) on 
biocontrol may be the 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 Fore 
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. For example, 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 gin/100 ml, and most 
preferably about 2 gm/100 ml. 
The following examples are intended to further illustrate the invention and 
not to limit the scope of the invention as defined by the claims. 
EXAMPLE 1 
The effectiveness of yeast 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. reticulata X 
C. sinensis); Kumquat (Fortunella margarita); and pummelos, (C. grandis). 
Fruit rot pathogens tested included Penicillium digitatum, Penicillium 
italicum and Geotrichum candidurn 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 was obtained using the 
following procedures: The surface of lemons was washed by placing the 
fruit in a 600 ml beaker containing 200 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 24 hours before colonies were selected. The 
same fruit received three separate washings and the same procedures were 
followed. Appearing colonies were isolated and purified using standard 
purification techniques. All cultures were stored on silica gel in a 
freezer until use. 
Isolate NRRL-Y-18314 was grown in flasks containing nutrient yeast dextrose 
broth (NYDB) on a reciprocal shaker at 30.degree. 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. 
Freshly harvested fruit was wiped with 95% ethanol and placed on moist 
paper in 50.times.100.times.15 cm plastic trays, 24 fruits per tray. Two 
to four conical wounds, 3 mm deep, were cut in the fruit peel. The wounds 
were brushed with an aqueous suspension of NRRL Y-18314. The concentration 
of the aqueous suspension was 1.times.10.sup.9 CFU/ml. One to two hours 
later, 20 microliters of an aqueous spore suspension of the targeted 
pathogen, 1.times.10.sup.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. 
Results were analyzed and are recorded in Tables I, II, and III below. 
TABLE I 
______________________________________ 
Relative effectiveness of NRRL Y-18314 in inhibiting 
Penicillium digitatum decay of different citrus cultivars. 
Citrus Incubation time (days) 
cuitivar Antagonist 4 5 6 7 
______________________________________ 
Percent Infection.sup.a 
Grapefruit NRRL Y-18314 0 2 6 11 
(72) Control 90 97 100 100 
Orange, `Shamouti` 
NRRL Y-18314 0 3 10 17 
(42) Control 93 100 100 100 
Orange, `Valencia` 
NRRL Y-18314 2 4 8 17 
(42) Control 90 94 97 100 
Lemon NRRL Y-18314 0 2 10 15 
(42) Control 98 100 100 100 
Temple NRRL Y-18314 2 4 10 14 
(48) Control 95 96 99 100 
Pummelo NRRL Y-18314 0 0 2 2 
(24) Control 83 90 92 96 
Kumquat.sup.b 
NRRL Y-18314 4 8 12 -- 
(150) Control 19 23 37 -- 
______________________________________ 
.sup.a Number of inoculation sites per treatment is indicated in 
parentheses under the cultivar's name. 
.sup.b Whole fruits were used without artificial inoculation. The fruit 
was dipped momentarily in a 48 hrold liquid culture of the NRRL Y18314. 
NYDB was used as control. 
TABLE II 
______________________________________ 
Inhibition of Penicillium italicum decay of 
grapefruit and orange by NRRL Y-18314 
Citrus Incubation time (days) 
cultivar Antagonist 3 4 5 6 
______________________________________ 
Percent Infection.sup.a 
Grapefruit NRRL Y-18314 3 3 4 6 
(72) Control 97 100 100 100 
Orange `Valencia` 
NRRL Y-18314 3 8 10 19 
(72) Control 84 95 97 100 
Orange `Shamouti` 
NRRL Y-18314 3 6 8 15 
(72) Control 90 95 100 100 
______________________________________ 
.sup.a Number of inoculation sites per treatment is indicated in 
parentheses under the cultivar's name. 
TABLE III 
______________________________________ 
Inhibition of Geotrichum candidum decay of 
grapefruit and lemon by NRRL Y-18314 
Incubation time (days) 
Citrus cultivar 
Antagonist 3 4 5 6 
______________________________________ 
Percent infection.sup.a 
Grapefruit NRRL Y-18314 3 3 8 9 
(72) Control 30 56 78 86 
Lemon NRRL Y-18314 12 17 18 18 
(30) Control 75 77 77 77 
______________________________________ 
.sup.a Number of inoculation sites per treatment is indicated in 
parentheses under the cultivar's name. 
As shown in Table I, 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 cultivar 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 shows 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 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 than the Penicillia 
decays, particularly in lemons. 
EXAMPLE II 
The ability of NRRLY-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. 
NRRLY-18314 was incubated in 100 ml of NYDB in 250 ml Erlenmeyer flasks on 
a rotary shaker (100 rpm) at 28.degree. 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. 
One to two hours after t he 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.times.10.sup.4 spores/ml. 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 were analyzed and are shown in FIG. 1. 
As shown in FIG. 1, NRRLY-18314 was effective in reducing Rhizopus rot in 
both injured and non-injured grape berries. Reduction of decay was most 
pronounced in those berries that were not injured prior to inoculation and 
in those inoculated by nesting. 
EXAMPLE III 
The effectiveness of isolate NRRLY-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 the bottom 
of each plastic tray in order to maintain high humidity. The apples were 
wounded using a needle. Wound size was 4 mm wide by 5 mm deep. Three-day 
old shake cultures of NRRL Y-18314 growing on NYDB at a 1.times.10.sup.9 
CFU/ml concentration was added to the wounds, 50 microliters/wound. 
Treated apple wounds were allowed to air dry. Controls were inoculated 
with water only. There were 10 replicates per treatment each consisting of 
a single wound per fruit. Thereafter, an aqueous suspension of Botrytis 
cinerea or Penicillium expansum spores, 1.times.10.sup.4 spores/ml, were 
added to the wounds, 20 microliters/wound. 
Measurements of infected areas were taken 5, 7, and 9 days after 
inoculation. Results were analyzed and are shown in FIGS. 2 and 3. 
NRRL Y-18314 effectively controlled both Borytis cinerea and Penicillium 
expansum rots in apples. As shown in FIG. 2, total protection against 
Botrytis cinerea occurred in treated apples up to about 7 days after 
inoculation, with only small lesion development after nine days. 
Protection against Penicillium expansum was to a lesser extent than 
against Botrytis cinerea. Nevertheless, FIG. 3 clearly shows that apples 
treated with NRRLY-18314 had a significant decrease in the development of 
Penicillium expansum when compared to the untreated controls. 
EXAMPLE IV 
The effectiveness of NRRL Y-18314, to inhibit Penicillium digitatum 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, Md. 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 28.degree. C. 
for 48 hours. Following centrifugation, the resulting pellets were washed 
twice with water and thereafter suspended in water. Concentrations of the 
aqueous suspensions ranged from 1.3.times.10.sup.7 to 1.3.times.10.sup.9 
CFU/ml. 
The surface of the grapefruit was sterilized with 95% ethanol and placed on 
moist paper in 50.times.100.times.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 each isolate was brushed onto the surface of a wound. Each 
isolate was tested on 48 sites of inoculations. One to two hours later, an 
aqueous suspension of Penicillium digitatum, 1.times.10.sup.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 New Multiple Range Test. Different letters are 
significant at a 1% level. The results are recorded in FIG. 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 
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 Aspergillus flavus was applied as described for the pathogen in 
Example 1. The treated nuts were incubated 14 days at 26.degree. C. FIG. 
5A is photograph of the peanuts treated with both Aspergillus flavus and 
NRRL-Y-18314, and FIG. 5B is a photograph of peanuts treated only with 
.Aspergillus flavus. As shown in these photographs, the results clearly 
show the inhibition by the yeast of the pathogen growth: FIG. 5A shows 
only 11 (33%) Of the wounds on which the pathogen grew (low to medium 
growth) compared with FIG. 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 NRRLY-18314 was applied as described in Example 1. Similarly, the 
Aspergillus niger was applied as described for the pathogen in Example 1. 
The treated nuts were incubated 14 days at 26.degree. C. FIG. 6A is a 
photograph of the peanuts treated with both Aspergillus niger and 
NRRL-Y-18314, and FIG. 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 (FIG. 
6A) compared with 100% infection in the non-treated control (FIG. 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) 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) 0.1 milliliter of 
each mixture of wax and culture, with: (b) 0.9 milliliter of the 
respective wax. The resultant mixtures were then plated on yeast malt agar 
plates. The plates were maintained at about 20.degree. to 25.degree. C. 
Results are shown in Table IV. Entries in Table IV are all in colony 
forming units per milliliter. 
TABLE IV 
______________________________________ 
Durand- 
Fresh Fresh 
Wayland 
Mark Stayfresh 
Wax 58P 
______________________________________ 
Initially 1 .times. 10.sup.5 
1.0 .times. 10.sup.7 
8.9 .times. 10.sup.8 
1.0 .times. 10.sup.5 
(Time Zero) 
19 Days 2.5 .times. 10.sup.6 
2.0 .times. 10.sup.5 
2.9 .times. 10.sup.6 
less than 
1.0 .times. 10.sup.5 
35 Days 2.7 .times. 10.sup.6 
1.4 .times. 10.sup.6 
9.0 .times. 10.sup.4 
1.9 .times. 10.sup.5 
46 Days 5.9 .times. 10.sup.6 
8.4 .times. 10.sup.5 
3.0 .times. 10.sup.4 
1.5 .times. 10.sup.5 
60 Days 1.1 .times. 10.sup.7 
2.3 .times. 10.sup.6 
less than 
4.9 .times. 10.sup.6 
1.0 .times. 10.sup.4 
76 Days 3.0 .times. 10.sup.6 
4.9 .times. 10.sup.6 
less than 
1.5 .times. 10.sup.6 
1.0 .times. 10.sup.4 
258 Days NT* TNTC** NT TNTC 
342 Days NT TNTC NT TNTC 
______________________________________ 
**TNTC stands for too numerous to count. 
*NT stands for not tested, i.e. dilution plates were not made. 
This example clearly indicates the surprisingly and unexpectedly high 
viability of NRRL-Y-18314 in commercially available waxes at room 
temperature, even for extended periods of time. 
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 Mark Wax) for long periods of time. Freeze dried cells 
were frozen in liquid nitrogen and placed on a lyphilyzer 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. CFU) per milliliter. 
TABLE V 
______________________________________ 
Freeze Dried Cells Whole Cells 
of NRRL-Y-18314 of NRRL-Y-18314 
Stored at Stored at 
Room Stored Under 
Room Stored Under 
Week Temp. Refrigeration 
Temp. Refrigeration 
______________________________________ 
1 3.52 .times. 10.sup.3 
TNTC* TNTC TNTC 
2 350 TNTC TNTC TNTC 
3 40 TNTC TNTC TNTC 
5 0 TNTC TNTC TNTC 
7 0 TNTC TNTC TNTC 
9 0 TNTC TNTC TNTC 
11 0 TNTC TNTC TNTC 
13 20 9.88 .times. 10.sup.3 
TNTC TNTC 
16 0 5.8 .times. 10.sup.3 
TNTC TNTC 
19 20 6.28 .times. 10.sup.3 
TNTC TNTC 
24 10 5.54 .times. 10.sup.3 
TNTC TNTC 
37 0 1.83 .times. 10.sup.3 
TNTC TNTC 
57 TNTC TNTC 
______________________________________ 
*TNTC = too numerous to count, no count greater than 1 .times. 10.sup.4 
CFU/ml was made. 
EXAMPLE IX 
Five milliliters of a YM broth culture of NRRL-Y-18314 (5.6.times.10.sup.8 
CFU/ml) were mixed with 5 milliliters of gum. Gum concentration of the 5 
milliliter solutions ranged from 1-20% as indicated in Table VI. The 
culture and gum mixture was added to 40 cm.sup.3 of either corn starch 
(25.7 g) or silica gel (27.1 g) . This preparation was mixed and dried at 
54.degree. C. for 4 days and then ground in a mortar and pestle to a fine 
powder. The powder was then stored at 4.degree. 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 Table VI in units of colony forming 
units per milliliter. 
TABLE VI 
______________________________________ 
9 DAYS 24 DAYS 37 DAYS 56 DAYS 
______________________________________ 
CORN STARCH 
Tragacanth 
less than 
less than 
1% 1.0 .times. 10.sup.5 
1.0 .times. 10.sup.4 
Karaya 3.0 .times. 10.sup.5 
5.0 .times. 10.sup.4 
2.0 .times. 10.sup.4 
5.0 .times. 10.sup.4 
10% 
Locust Bean 
less than 
2.0 .times. 10.sup.4 
3.0 .times. 10.sup.4 
1.8 .times. 10.sup.5 
15% 1.0 .times. 10.sup.5 
Xanthan 8.0 .times. 10.sup.5 
7.3 .times. 10.sup.5 
3.0 .times. 10.sup.5 
5.9 .times. 10.sup.5 
20% 
SILICA GEL 
Tragacanth 
less than 
less than 
1% 1.0 .times. 10.sup.5 
1.0 .times. 10.sup.4 
Karaya 2.0 .times. 10.sup.5 
1.0 .times. 10.sup.4 
less than 
10% 1.0 .times. 10.sup.4 
Locust Bean 
less than 
less than 
15% 1.0 .times. 10.sup.5 
1.0 .times. 10.sup.4 
Xanthan 1.0 .times. 10.sup.5 
1.1 .times. 10.sup.5 
1.0 .times. 10.sup.4 
20% 
______________________________________ 
These results clearly show that the NRRL-Y-18314 remained viable for an 
extended period of time in any of a variety of gums combined with either 
corn starch or silica gel. 
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. 
TABLE VII 
__________________________________________________________________________ 
5 DAYS 
19 DAYS 
33 DAYS 
48 DAYS 
61 DAYS 
80 DAYS 
88 DAY 
__________________________________________________________________________ 
Methylcellulose 
1% 6.4 .times. 10.sup.6 
5.2 .times. 10.sup.6 
3.0 .times. 10.sup.5 
8.6 .times. 10.sup.5 
6.8 .times. 10.sup.5 
4.3 .times. 10.sup.5 
5% 6.1 .times. 10.sup.6 
6.8 .times. 10.sup.6 
Guar 
1% 3.1 .times. 10.sup.6 
3.8 .times. 10.sup.6 
less than 
8.0 .times. 10.sup.4 
2.3 .times. 10.sup.5 
8.0 .times. 10.sup.4 
1.0 .times. 10.sup.5 
5% 6.0 .times. 10.sup.5 
1.1 .times. 10.sup.6 
10% 1.3 .times. 10.sup.6 
2.0 .times. 10.sup.6 
Xanthan 
1% 4.3 .times. 10.sup.6 
7.2 .times. 10.sup.6 
5% 1.3 .times. 10.sup.7 
2.1 .times. 10.sup.7 
10% 2.7 .times. 10.sup.7 
2.8 .times. 10.sup.7 
1.8 .times. 10.sup.6 
20% 3.6 .times. 10.sup.7 
3.2 .times. 10.sup.7 
3.6 .times. 10.sup.6 
4.1 .times. 10.sup.6 
6.7 .times. 10.sup.6 
2.6 .times. 10.sup.6 
Locust Bean 
1% 4.1 .times. 10.sup.6 
3.8 .times. 10.sup.6 
5% 1.0 .times. 10.sup.7 
6.2 .times. 10.sup.6 
10% 5.8 .times. 10.sup.6 
7.4 .times. 10.sup.6 
5.0 .times. 10.sup.5 
15% 1.1 .times. 10.sup.7 
1.2 .times. 10.sup.7 
8.0 .times. 10.sup.5 
4.9 .times. 10.sup.5 
6.3 .times. 10.sup.5 
1.3 .times. 10.sup.6 
Karaya 
1% 1.6 .times. 10.sup.7 
1.1 .times. 10.sup.7 
5% 1.2 .times. 10.sup.7 
1.0 .times. 10.sup.7 
10% 5.5 .times. 10.sup.7 
2.2 .times. 10.sup.7 
1.7 .times. 10.sup.6 
1.2 .times. 10.sup.6 
2.9 .times. 10.sup.6 
1.8 .times. 10.sup.6 
25% 1.3 .times. 10.sup.7 
1.9 .times. 10.sup.7 
2.2 .times. 10.sup.6 1.8 .times. 10.sup.6 
Tragacanth 
1% 8.0 .times. 10.sup.6 
1.3 .times. 10.sup.7 
1.4 .times. 10.sup.6 
5.4 .times. 10.sup.5 
9.1 .times. 10.sup.5 
7.6 .times. 10.sup.5 
5% 5.1 .times. 10.sup.6 
9.6 .times. 10.sup.6 
8.0 .times. 10.sup.5 
10% 1.9 .times. 10.sup.6 
7.8 .times. 10.sup.6 
25% 2.4 .times. 10.sup.6 
6.2 .times. 10.sup.6 
__________________________________________________________________________ 
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 CaCl.sub.2 and CaCO.sub.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.sup.7 CFU/ml with each 
of the salts listed in Table VIII at a concentration of 2 grams/100 ml 
(with the exception of FeSO.sub.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 FeSO.sub.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 10.sup.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. 
TABLE VIII 
______________________________________ 
Percent Infection 
Inorganic Salt Alone Salt and 
Salt (No NRRL Y-18314) 
NRRL Y-18314 
______________________________________ 
CaCl.sub.2 95.0 (.+-.5.0) 
3.3 (.+-.3.3)* 
CaCO.sub.3 71.9 (.+-.13.5) 
27.5 (.+-.24.3)* 
FeSO.sub.4 87.5 (.+-.12.5) 
57.5 (.+-.21.0) 
KC1 100.0 (.+-.0.0) 
47.5 (.+-.20.6)* 
MgCl.sub.2 100.0 (.+-.0.0) 
61.3 (.+-.17.4) 
MnCl.sub.2 100.0 (.+-.0.0) 
97.5 (.+-.2.5) 
NaCl 100.0 (.+-.0.0) 
71.6 (.+-.15.7) 
CONTROLS 100.0 (.+-.0.0) 
59.5 (.+-.14.4) 
H.sub.2 O 
______________________________________ 
Values in parentheses are standard errors of the mean. Asterisk indicates 
that means within a row are significantly (P&lt;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 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 CaCl.sub.2 and CaCO.sub.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. However, in actuality, the combination of CaCl.sub.2 with 
NRRL Y-18314 provided more than twice the expected value of 45% i.e. about 
96.7%; and the combination of CaCO.sub.3 with NRRL Y-18314 provided 72.5% 
which is significantly higher than the expected value of 58%. 
EXAMPLE 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 10.sup.8 CFU/ml 
suspension of the respective yeasts in sterile distilled water, with or 
without 2 grams/100 ml of CaCl.sub.2 as referred to in Table IX. Two hours 
later the apples were challenged with 20 microliters of a suspension of 
10.sup.4 spores/milliliters of Botrytis cinerea. Seven days after 
inoculation percent infection was observed. Results are shown in Table IX. 
TABLE IX 
______________________________________ 
Yeast Average 
Strain CaCl.sub.2 
Percent Infection 
______________________________________ 
NRRL-Y-18527 YES 6.7 (.+-.6.7)* 
NRRL-Y-18527 NO 41.7 (.+-.18.8) 
NRRL-Y-18314 YES 0.0 (.+-.0.0)* 
NRRL-Y-18314 NO 26.7 (.+-.6.7) 
NONE YES 100.0 (.+-.0.0) 
NONE NO 100.0 (.+-.0.0) 
______________________________________ 
The above entries are the average of 3 trials per treatment. Values in 
parentheses are standard errors of the mean. Asterisk indicates that fruit 
rot in yeast treatments with calcium chloride is significantly 
(P.ltoreq.0.05) less than that of fruit treated with yeast alone. It my be 
observed that significant infection reduction was achieved using either of 
the yeast, and that further infection reduction was achieved using the 
combination of CaCl.sub.2 with each yeast. 
EXAMPLE XIII 
The purpose of this example is to demonstrate the effectiveness of 
NRRLY-18314 and combinations thereof with CaCl.sub.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 NRRLY-18314 in sterile distilled water, with or 
without CaCl.sub.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 
______________________________________ 
Penicillium Average 
Presence 
NRRL-Y-18314 
spore Lesion 
of Concentration 
Concentration 
Diameter 
Standard 
CaCl.sub.2 
(CFU/ml) (spores/ml) (mm) Error 
______________________________________ 
NO 10.sup.7 10.sup.3 32.4 8.4 
NO 10.sup.7 10.sup.4 49.8 2.1 
NO 10.sup.7 10.sup.5 40.8 6.1 
NO 10.sup.8 10.sup.3 18.4 7.9 
NO 10.sup.8 10.sup.4 35.2 2.1 
NO 10.sup.8 10.sup.5 36.2 9.2 
YES 10.sup.7 10.sup.3 14.4 7.4 
YES 10.sup.7 10.sup.4 9.0 6.3 
YES 10.sup.7 10.sup.5 15.2 9.3 
YES 10.sup.8 10.sup.3 7.6 5.3 
YES 10.sup.8 10.sup.4 17.4 5.8 
YES 10.sup.8 10.sup.5 19.2 6.7 
YES 0 10.sup.3 40.4 1.9 
YES 0 10.sup.4 45.2 1.6 
YES 0 10.sup.5 47.0 1.6 
NO 0 10.sup.3 47.2 2.3 
______________________________________ 
The entries of Table X are the average of 5 replicates per treatment. It 
maybe observed from the table that the isolate NRRLY-18314, applied in the 
absence of CaCl.sub.2, did not facilitate significant reduction of decay 
(greater than 50%) inmost treatments, as compared to the water control and 
CaCl.sub.2 treatments without NRRLY-18314. However, when NRRLY-18314 was 
applied with CaCl.sub.2 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 CaCl.sub.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.sup.4 spore/milliliter suspension of Penicillium digitatum. The 
grapefruit were incubated for 5 days at 24.degree. C. before observations 
were taken. Results of average percent fruit rot were as follows (data is 
the average of 2-3 trials per treatment): 
TABLE XI 
______________________________________ 
FOR STRAIN NRRL-Y-18314 
Average Percent Fruit Rot 
CaCl.sub.2 NRRL-Y-18314 Concentration (cfu/ml) 
Concentration 
0 10.sup.6 10.sup.7 
10.sup.8 
______________________________________ 
0% 59.5 31.5 10.5 0 
1% 39.0 22.8 1.5 NT* 
2% 22.8 7.5 1.5 NT 
______________________________________ 
*NT stands for "not tested". 
TABLE XII 
______________________________________ 
FOR STRAIN NRRL-Y-18313 
Average Percent Fruit Rot 
CaCl.sub.2 NRRL-Y-18313 Concentration (cfu/ml) 
Concentration 
0 10.sup.6 10.sup.7 
10.sup.8 
______________________________________ 
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 
______________________________________ 
EXAMPLE XV 
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 13. 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 10.sup.4 spores/ml suspension of 
Rhizopus stolonifer. Average percent infection was observed 4 days later. 
The results are as follows (each value is the average of two to five 
trials): 
TABLE XIII 
__________________________________________________________________________ 
AVERAGE PERCENT INFECTION 
Washed Yeast Cells 
Resuspended 
Yeast Washed Yeast Cells (cfu/ml) 
in Culture Broth 
Culture Broth 
Strain 10.sup.9 
10.sup.8 
10.sup.7 
10.sup.9 
10.sup.8 (without Yeast 
__________________________________________________________________________ 
Cells) 
NRRL-Y-18527 10.0 (.+-.7.1) 
15.0 (.+-.8.2) 
57.0 (.+-.21.6) 
32.5 (.+-.7.5) 
75.0 (.+-.25.0) 
100.0 (.+-.0.0) 
NRRL-Y-18314 2.5 (.+-.2.5) 
35.0 (.+-.11.5) 
90.0 (.+-.10.0) 
30.0 (.+-.20.0) 
87.5 (.+-.12.5) 
100.0 (.+-.0.0) 
NRRL-Y-18313 3.3 (.+-.3.3) 
34.4 (.+-.14.1) 
87.5 (.+-.12.5) 
52.5 (.+-.22.5) 
100.0 (.+-.0.0) 
100.0 (.+-.0.0) 
Zygosaccharomyces rouxii 
40.0 (.+-.20.0) 
63.3 (.+-.27.3) 
76.7 (.+-.23.3) 
75.0 (.+-.25.0) 
100.0 (.+-.0.0) 
100.0 (.+-.0.0) 
(ATTC #10682) 
Zygosaccharomyces rouxii 
49.7 (.+-.20.2) 
76.3 (.+-.13.2) 
96.7 (.+-.3.3) 
80.0 (.+-.0.0) 
100.0 (.+-.0.0) 
100.0 (.+-.0.0) 
(ATTC #34517) 
__________________________________________________________________________ 
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 10.sup.8 to 10.sup.9 cfu/ml and incubated at 
22.degree. 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); 
TABLE XIV 
______________________________________ 
Percent Fruit Rot 
Fruit Treatment 5 DAYS 6 DAYS 
______________________________________ 
NRRL-Y-18527 5.0% 13.0% 
Y-18314 18.0% 31.0% 
Water 90.0% 100.0% 
NYDB.sup.1 50.0% 92.0% 
______________________________________ 
.sup.1 NYDB = sterile culture broth, nutrient yeast dextrose broth. 
Fruit rot due to Rhizopus was not observed in grapes treated with any of 
the yeast treatments. 
EXAMPLE XVII 
Whole clusters of "Perlett" grapes were dipped in suspensions of the yeast 
antagonists specified in Table XV at concentrations of 10.sup.8 to 
10.sup.9 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.degree. C. The results were as follows: 
TABLE XV 
______________________________________ 
Yeast Percent Fruit Rot 
Treatment n Aspergillus 
Botrytis 
Rhizopus 
Total 
______________________________________ 
NRRL-Y-18527 
825 1.8% 2.0% 3.2% 7.0% 
NRRL-Y-18314 
825 0.7% 0.6% 8.2% 9.5% 
Water 890 1.4% 3.0% 16.2% 20.6% 
______________________________________ 
n = number of fruit per treatment. 
It is understood that modifications and variations may be made to the 
foregoing disclosure without departing from the spirit and scope of the 
invention.