BACKGROUND OF THE INVENTION

Field of the Invention The present invention relates to the biological control of postharvest diseases in fruits. More par¬ ticularly, this invention relates to the use of Acremon- ium breve, "A. breve", to biologically control the post- harvest disease, grey-mold, in pome fruits. Description of the Prior Art

Grey-mold, a major postharvest disease of fruit, is caused by the fungus, Botrytis cinerea Pers. ex Fril, "B. cinerea". Primiarly, a storage disease, grey-mold rot, as well as other fungi-induced postharvest dis- eases, causes considerable economical losses to the fruit industry each year.

Traditional attempts to control post-harvest dis¬ eases have involved the treatment of the fruit, after harvest and before storage, with a fungicide. However, increasing numbers of fungicide-tolerant strains of pathogens associated with postharvest diseases, as well as the need for fungicides which are safe to the en¬ vironment and humans, have created the necessity for alternative methods to control these diseases. One alternative has been the use of biological agents to control postharvest diseases in fruit. Brown

rot in peaches caused by Monilinia fructicola (Wint. ) Honey was successfully controlled with Bacillus sub- tilis. Pusey, et al.. Plant Pis. , 6:753-756 (1984); Wilson, et al.. Plant Pis., 9:375-378 (1985). Tricho- derma viride Pers. & S.F. Gray applied to strawberry plants in the field partially controlled grey-mold on stawberry fruits after harvest. Tronsmo, et al., Neth. J. Plant Pathol., 83(suppl. l):449-455 (1977). Also, partial control of rot in apples caused by B. cinerea flower infection was obtained by applying conidia T. harzianium Rifai to apple trees during bloom. Control was comparable to prior known fungicidal treatments. Tronsmo, et al.. Plant. Pis., 4:1009 (1980).

SUMMARY OF THE INVENTION I have discovered a strain of A. breve which is highly effective to control grey-mold rot in pome fruits. Viable cultures of the strain have been deposi¬ ted with the culture collection at the North Regional Research Center, U.S. Pepartment of Agriculture, Peoria, Illinois 61604, under the accession number NRRL 18307. Progenies of the strain 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 the progenies of the strain to the public will be irrevocably removed upon the granting of the patent of which the strain is the subject.

Accordingly, it is an object of the invention to provide a biological control agent which is highly ef¬ fective to control grey-mold in pome fruits.

It is also an object of the invention to provide a method for biologically controlling grey-mold in pome fruits, before or after harvest. Another object of the invention is to provide a method for biologically controlling grey-mold in these

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fruits which eliminates the use of fungicidal treat¬ ments.

Still another object of the invention is to provide a method for simultaneously controlling two or more pathogens causing postharvest diseases in pome fruits. In accordance with my invention, pome fruits are subjected to an aqueous suspension of strain NRRL 18307 in an amount effective to inhibit the development of the pathogen, B. cinerea.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 indicates the effect of A. breve, strain NRRL 18307, on grey-mold development on wounded Golden Delicious apples.

FIG. 2 outlines lesion development on wounded Gol¬ den Delicious apples which was intially inoculated with A. breve, strain NRRL 18307, and subsequently in¬ oculated with B. cinerea spores at different time inter¬ vals following treatment with strain NRRL 18307.

FIG. 3 outlines the effect of antagonists mixtures of A. breve, strain NRRL 18307, and Pseudomonas sp. on lesion development resulting from inoculation of wounded Golden Delicious apples with B. cinerea and

P. expansum spore mixtures.

DETAILED PESCRIPTION OF THE INVENTION Strain NRRL 18307 is isolated from apple leaves by repeatedly washing the leaves with a suitable aqueous buffer, i.e. a phosphate buffer and the like. There¬ after, the organisms are plated and grown on a nutri¬ tionally rich medium sufficient to support growth of the organism. For optimum growth, the preferred medium is either nutrient yeast dextrose broth (NYDB) or nutri¬ ent yeast dextrose agar (NYDA).

Strain NRRL 18307 has the following characteristic description: Colonies were 32 mm in diameter after 6 days on potato dextrose agar at 24°C, mostly moist

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with synnematogen especially towards the center. Colony color is dull yellow to cream under fluorescent light, becoming orange under black light. Phialides, often indistinguishable from hypha, are unbranched, smooth, 15 to 20 μlong with no collarette. Conidia are globose to obovate and are formed into heads.

Growth of strain NRRL 18307 is effected under aero¬ bic conditions, at any temperature satisfactory for growth of the organism, i.e. from about 4°C to about 28°C; the preferred temperature range is about 22°C to 26°C. The pH of the nutrient media is about pH 6.5 to 6.9. The incubation time is that time necessary for the organism to reach a stationary phase of growth, preferably, from about 40 to 60 hours. Strain NRRL 18307 may be grown in any conventional shake flask for small fermentation runs. For larger 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 organism is harvested by conventional sedimentary methodology, i.e. centrifugation or filter¬ ing. The resulting pellets are stored until use.

For purpose of the invention, the term "pome fruit" is used herein to designate fruits having a fleshy outer layer and a central core with seeds enclosed in a capsule. Suitable pome fruits include, but are not limited to, apples, pears and the like.

In accordance with the invention, strain NRRL 18307 is applied to the fruits in suspension with water. The suspension may optionally contain conventional addi¬ tives such as surfactants and antioxidants. Aqueous suspensions of strain NRRL 18307 may be applied to the fruit using any conventional techniques , such as spray¬ ing, dipping or brushing. The treatments may be applied to the fruits before or after harvest. Preferably, the treatments are applied after harvest and prior to storage.

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Concentrations of aqueous suspensions of strain NRRL 18307 useful in the process of the invention are any concentrations which inhibit the development of B. cinerea when applied to pome fruits. As will be obvious to the skilled artisan, effective concentrations will vary depending upon such factors as the ripeness of the fruits and the concentration of the pathogen affecting the fruit. Exemplary concentrations range from about 2.5 x 10 ⁵ cells/ml to about 9.5 x 10 cell/ ml; the most preferred concentration ranges from 3.4 x 10 ⁶ to 9.5 x 10 ⁶ cells/ml.

It is within the compass of the invention to treat the fruits with strain NRRL 18307 alone or in combina¬ tion with other biological control agents which are effective to control other pathogens inciting posthar¬ vest diseases in pome fruits. When used, these agents should be used in an amount, as readily determined by one skilled in the arts, which will not interfere with the effectiveness of an organism of the invention. 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 I The effectiveness of strain NRRL 18307 to inhibit the development of B. cinerea spores in wounded Golden Delicious apples was demonstrated.

Strain NRRL 18307 was isolated using the following procedure: Leaves were picked throughout the 1985 grow¬ ing season from Golden Delicious apple trees in 4 blocks of 5 trees in each block, scattered over approximately a 4 acre area. 10 leaves were collected from different parts of trees in each block. The samples were washed in phosphate buffer by shaking in a beaker on a rotary shaker for 10 minutes at 100 rpm. The buffer from the first washing was discarded and the leaves were washed a second time with 30 seconds of sonication

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in a Bransonic 521 sonicator at the beginning of the wash. The washing was plated on NYPA medium and incu¬ bated for 48 hours. Appearing colonies were isolated and purified using standard purification techniques. Strain NRRL 18307 was grown in NYPB liquid medium for 48 hours on a shaker at 150 rpm. 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 spectrophotometer.

Apple wounds 3 mm deep and 3 mm wide were inocu¬ lated with 20 μl of an aqueous suspension of strain NRRL 18307 having a concentration of 2.5 x 10 cells/ ml, 3.4 x 10 ⁶ cells/ml or 9.5 x 10 ⁶ cells/ml. Shortly thereafter, the wounds were inoculated with 20 μl of 4 1 x 10 spore/ml of an aqueous suspension of B. cinerea spores. Control apples were inoculated with 20 μl of the aqueous suspension of B. cinerea spores only.

Lesion diameter was measured after 7 days incuba- tion at 24 ^e C. Each apple constituted a single replicate and each treatment was replicated 3 times.

At each concentration tested, strain NRRL 18307 inhibited the development of the pathogen, B. cinerea.

As shown in Fig. 1, total protection occurred on all treated apples. Further, protection was ongoing for a prolonged period of time. After the apples were left standing for 10 weeks, no new lesions developed.

Example II The ability of strain NRRL 18307 to inhibit B_ _; _ cinerea spore germination in vitro was demonstrated. The abbreviation "CFU" is used herein to desigante col¬ ony forming units.

An aqueous suspension of strain NRRL 18307 was prepared as described in Example I at a concentration of 9.5 x 10 cells/ml. In 16 mm diameter wells of plas¬ tic culture plates, three 100 μl portions of an aqueous

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suspension of strain NRRL 18307 was mixed respectively with 100 μl of an aqueous suspension containing either

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1 x 10 spores/ml, 1 x 10 spores/ml or 1 x 10 spores/ ml of the pathogen B. cinerea. The plates were incubated at 24°C. The plates were first examined at 72 hours of incubation and at this point, equal volumes of freshly squeezed apple juice were added to the plates. After an additional 48 hours of incubation (120 hrs from the beginning of the experiment), the plates were examined a second time. Each well constituted a single replicate and each treat¬ ment was replicated three times. Germination was deter¬ mined according to arbitrary scales by examination under an inverted microscope. Results were analyzed and are recorded in Table I.

As shown in Table I, strain NRRL 18307 strongly inhibited spore germination xn_ vitro after 72 hours. The addition of apple juice did not increase germina- tion. Thus, while the mode of action of strain NRRL 18307 to inhibit B. cinerea is not known, these results suggest that strain NRRL 18307 may, at least partially, operate to inhibit spore germination of B. cinerea in the fruit.

Example III

Combination treatments containing various concen¬ trations of A. breve, strain NRRL 18307, and Pseudomanas sp. were tested on Golden Delicious apples using spore mixtures of B. cinerea and Penicillium expansu , "P. expansum" , the pathogen which causes the postharvest disease, blue-mold.

20 μl of a water suspension containing a mixture of strain NRRL 18307 and Pseudomanas sp. was applied to apple wounds 3 mm wide and 3 mm deep. Within about 15 r.inutes, 20 μl of an aqueous suspension of B. cin- erea/P. expansum spore mixture were added to the wounds .

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The spore mixtures consisted of a constant concentration of 1 x 10 ⁴ B. cinerea spores/ml and a concentration from 0 to 1 x 10 in ten-fold increases of P. expansum spores . Concentrations of mixtures of strain NRRL 18307 and Pseudomanas sp. in the aqueous suspension varied as shown in Table II.

Lesions diameter was measured 7 days after inocu¬ lation at 24 ^β C. Each apple consisted of a single repli¬ cate and each treatment was replicated 3 times. The results were as presented in Fig. 3.

Both pathogens, P. expansum and B. cinerea, were effectively controlled simultaneously when the apples were inoculated with mixtures containing A. breve, NRRL 18307 and Pseudomanas sp. There was a strong tendency toward reduction in lesion size in all spore mixtures as concentrations of the control agents increased.

As shown in Fig. 3, the two highest concentrations of

NRRL 18307/Pseudomonas sp. mixtures prevented lesion development on fruit inoculated with spore mixtures

3 containing from 0 to 1 x 10 spores/ml of P. expansum. This was also true for the third highest concentration of antagonists mixtures, except for spore mixtures contain¬ ing P. expansum at 1 x 10 ^ spores/ml where small lesions occurred. Also, no lesions developed at concentrations of 95% and 80% transmittance with 1 x 10 and 1 x 10 ⁴ spores/ml of P. expansum, respectively, in the mixtures.

Advantageously, strain NRRL 18307 acts very rapid¬ ly on pome fruits to control grey-mold in commercially acceptable concentrations. Further, the treatments may be used in combination with other biological agents to simultaneously control two or more pathogens causing postharvest diseases in pome fruits.

It is understood that modifications and variations may be made to the foregoing disclosure without depart- ing from the spirit and scope of the invention.

SϋBSΪfflilϊ SHEET

TABLE I

Germination of B. cinerea spores _in vitro in water suspensions of Acremonium breve, strain NRRL 18307. ^a

Pathogen spore concentration (spore/milliliter)

Pathogen Antagonist hr 10' 10 10"

- = No germination; + = Less than 1/3 germinated; ++ = 1/3 - 2/3 germinated; and +++ = More than 2/3 germinated.

TABLE II

Conversion of Concentrations of Aqueous Suspensions of Antagonist Mixtures from % Transmittance to CFU/ml.

CFU/ml in mixture

% Transmittance Pseudomonas sp, Acremonium breve NRRL at 420 nm 18307

100 98 8 x 10 1.5 x 10 95 9 x 10 2.5 x 10 ^" 90 1 x 10 1.2 x 10 80 3.6 x 10 3.4 x 10

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