KR950002857B1 - Inhibiting plant pathogens with an antagonistic microorganism - Google Patents

Abstract

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Description

Method of suppressing plant pathogens using antagonistic microorganisms

FIG. 1 is a bar graph showing percent decay rates of three groups of grapes and control grapes treated with NRRL Y-18314 showing Rhizopus decay inhibition.

2 shows the decay area diameter (mm) versus time (1) of apples infected with Botrytis cinerea for (1) a control sample treated with water only and (2) a sample treated with NRRL Y-18314. Line graph representing the day.

Figure 3 shows the area of decay (mm) versus time (in days) of apples infected with Penicillium expansum for (1) a control sample treated with water only and (2) a sample treated with NRRL Y-18314. A line graph representing).

FIG. 4 is a bar graph of percent infection showing the relative efficacy of yeast isolates in inhibiting penicillium digitatum rot of grapefruit.

Figure 5a is a photograph of peanuts treated with Aspergillus flavus and NRRL Y-18314 according to Example V.

FIG. 5B is a photograph of peanuts treated with Aspergillus plabus only in accordance with Example V.

FIG. 6a is a photograph of peanuts treated with Aspergillus niger and NRRL Y-18314 according to Example VI.

6b is a photograph of peanuts treated with Aspergillus nigerman according to the method described in Example VI.

Description of related application

This application is a partial consecutive application of US Patent Application No. 387,669, filed Jul. 31, 1989, which is part of US Patent Application No. 177,236, filed April 4,988.

Field of invention

The present invention relates to the biological suppression of plant diseases (eg pre- or post-harvest diseases) of agricultural products such as fruits. More specifically, the invention relates to (1) one or more microorganisms that are antagonists of (a) one or more calcium salts and plant pathogens, or (b) one or more antagonists of plant pathogens but not antibiotics. Methods of biologically inhibiting plant diseases of agricultural products (eg post-harvest rot) using microorganisms; (2) a composition useful for the above method; And (3) a product produced by the method. In addition, the present invention provides a method for harvesting agricultural products using Pichiaguilliermondii (anamorph Candida guilliermondii strain and Hansenia spora uvarum strain. It relates to a method for biologically inhibiting post-corruption.

Description of the Prior Art

Post-harvest disease of fruits results in 15-25% annual loss of fruit lessons worldwide. Fungicides, which are primary weapons in the control of these diseases, are often ineffective and harmful to humans and the environment. Therefore, new methods for suppressing post-harvest disease are urgently needed.

Recently, it has been found that treating post-harvest fruit with antagonistic microorganisms is an effective approach to the inhibition of post-harvest rot. A surprising effect has been shown in the inhibition of peach brown rot caused by Monilinia furcticola (Wint. Honey) and Bacillus subtilis (Pusey et al.) [Plant Dls. 86: 753-756 (1986). Matos was able to reduce the incidence of mold from 35% to 8% when inoculated with Trichoderma species with penicillium digitatum on lemon peels [De Matos, Ph.D. Dissertation, University of California , Riverdale, (1983). Singh and Deverall described bioregulation with bacterial antagonists against the citrus pathogen Alteraria Citri (Pierce) and Geotrichum [Trans.Br.Myco1.Soc.83: 487- 490 (1983). Scratched citrus fruit was immersed in a suspension of bacterial cells, in particular Bacillus subtilis (Ehrenber, Cohn) strains to delay rot by three rotative pathogens.

Summary of the Invention

The first aspect of the present invention relates to the use of one or more microorganisms in agricultural products (in this specification, "application" is limited to the deliberate administration of microorganisms to agricultural products as opposed to naturally occurring microorganisms in agricultural products) The present invention relates to a method for inhibiting the spread of plant pathogens of agricultural products, wherein the one or more microorganisms are antagonists to plant pathogens but are not antibiotics, and are used in an effective amount capable of inhibiting the spread of plant pathogens of agricultural products. The most striking and novel aspect of the present invention is the use of microorganisms that do not produce antibiotics to suppress disease of agricultural products. This method is very important for consumers because it avoids the potential adverse effects of antibiotics in food supply, such as causing antibiotic resistance of human pathogens.

The second aspect of the present invention relates to a method for inhibiting the spread of plant pathogens of agricultural products consisting of using one or more microorganisms which are antagonists (preferably not antibiotics) against one or more calcium salts and plant pathogens. At this time, one or more calcium salts and one or more microorganisms are used in the agricultural products in an effective amount capable of inhibiting the spread of plant pathogens of the agricultural products.

A third aspect of the invention relates to a composition that can be used to perform the methods. Such compositions include: (1) a group of one or more microorganisms that are antagonistic to plant pathogens but not antibiotics, and (2) gels, gums, waxes, oils, talc, starches and mixtures thereof. A composition consisting of a mixture of carriers for the one or more microorganisms selected from; A composition consisting of a mixture of one or more microorganisms and a carrier for the one or more microorganisms, wherein at least 99% of the number of the one or more microorganisms are antagonistic to plant pathogens but are not antibiotics; And / or a composition consisting of a mixture of one or more microorganisms which are antagonistic against one or more calcium salts and plant pathogens and which are preferably not antibiotics, preferably (a) essentially (B) greater than or equal to 99% of the microorganism consisting of calcium salt and one or more microorganisms, or (b) being antagonistic to plant pathogens, and (c) not exceeding 99 At least% is a composition).

A fourth aspect of the present invention relates to a product comprising the following: An agricultural product comprising at least about 10 ⁵ colony forming units per cm 2 of at least one microorganism antagonistic to plant pathogens but not antibiotics. product ; Products consisting of agricultural products containing one or more microorganisms, most of which are antagonistic to plant pathogens but are not antibiotics; A product comprising an agricultural product comprising at least one microorganism that is antagonistic (preferably not an antibiotic) to calcium salts and plant pathogens at concentrations of at least about 10 ⁵ colony forming units per cm 2; And / or an agricultural product comprising calcium salt and at least one microorganism, most of which is antagonistic to plant pathogens.

The fifth aspect of the present invention relates to a biologically pure culture of Hansenia spora ubarum isolate having the identification properties of isolate NRRL Y-18527.

The microorganism (s) described above can be selected from the group consisting of, for example, fungi (eg yeast), bacteria, viruses and mixtures thereof.

Regarding a preferred embodiment of the present invention, the inventors have discovered a novel yeast strain that is very effective in inhibiting various plant pathogens (eg, fruit-rot) that affect a wide range of agricultural products. Four isolates of the novel strain are described in Northern Regional Research Center [U.S. Department of Agriculture, Peoria, Illinois 61604] was assigned accession 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 Guillehermondi (Anamorph Candida Tuileriemondi), and NRRL Y-18527 has been identified as Hansenia sporabarum (Nichaus, Shehata, Mark et Phaff). The deposited materials have been recognized in particular under the Budapest Treaty, an international convention of microbial deposits for procedures. In addition, (1) the depository provides for the permanence of the deposit and is readily available to the public if patented. (2) The substance has been deposited in the course of pending patent applications under the conditions that access to this substance is available to persons designated by the chief officer of patents and trademarks qualified under 37 CFR 1.14 and 35 USC 122. All restrictions on the availability of this strain propagation to the public will be removed irrevocably when patented.

It is therefore an object of the present invention to provide novel biological inhibitors which are very effective in suppressing various plant pathogens causing pre- and post-harvest diseases in various agricultural products (eg fruits) and which are not harmful to consumers.

Another object of the present invention is to provide a method for biologically suppressing plant diseases (eg, post-harvest diseases) of agricultural products (eg, fruits) that do not require the use of fungicides.

In a preferred embodiment of the present invention, the agricultural product is a yeast isolate having the identification properties of an isolate selected from the group consisting of NRRL Y-18313, NRRL Y-18314, NRRL Y-18527, NRRl Y-18654 and mixtures thereof. Treated with an aqueous suspension. In fact, the microorganisms multiply and take over the surface of the scratched fruit to prevent infection by plant pathogens (eg fruit-rot).

Description of the Preferred Embodiments

Isolates NRRL Y-18313 and NRRL Y-18314 were washed repeatedly with water to obtain from the surface of citrus fruit. Isolate NRRL Y-18654 was obtained from the surface of the lemon by repeated washing. Isolator NRRL Y-18527 was isolated from the surface of the grapes. The microorganisms were then colonized and grown on any nutrient rich medium capable of growing the microorganisms sufficiently. Preferably, the medium is nutritive yeast dextrose agar (NYDA) or yeast-malt extract agar (YM).

Isolates NRRL Y-18313 and NRRL Y-18314 have the following identification characteristics. That is, the colonies are milky white, slightly elevated, glossy, rounded and smooth. Gastric mycelia were not observed.

After one week on Corn Meal agar, V-8 juice agar, YM or acetate no follicular spores were produced. On solid YM, cells were single cells in liquid culture after 1 day. Small globular cells were observed mainly in many chains or clusters with one bump. Isolate NRRL Y-18654 Colony is milky white, slightly elevated, glossy, flat and rounded at the edges.

The isolate NRRL Y-18527 was identified by the American Type Culture Collection as follows. That is, in liquid medium, the cells are in the form of lemons and have bipolar germination. In solid medium, the cells remain single cells or are free of filaments. Colonies are white, round with slightly elevated surfaces. No gastric mycelia were produced. One round follicle spore was produced per cell.

The microorganisms are grown under aerobic conditions at any temperature satisfactory for the growth of the microorganisms, ie from about 10 ° C to about 30 ° C. Preferred temperature ranges are about 20 ° C to 25 ° C. The pH of the nutrient medium is neutral, ie 6.7 to 7.2. Incubation time is the time required for the growth of microorganisms to reach a stationary phase. For NRRL Y-18314 and NRRL Y-18313, incubation times of about 40 to 60 hours are preferred. For NRRL Y-18654, incubation time of about 24 to 48 hours is preferred. Isolate NRRL Y-18527 is preferably grown for a culture time of 18 to 24 hours at a temperature of 25 to 28 ° C. such that the cells are in the growth log phase.

For small fermentation runs, isolates NRRL Y-18313, NRRL Y-18314, NRRL Y-18527, and NRRLY-18654 can be grown in any conventional shake flask. For large runs it is convenient to carry out the culture while shaking and venting the liquid medium inoculated in the fermentation tank. After incubation, the microorganisms were harvested by conventional sedimentation, ie centrifugation or filtration. Cultures are stored on silica gel and frozen until use.

The isolates NRRL Y-18313, NRRL Y-18314, NRRL Y-18527, and NRRL Y-18654 are useful for inhibiting various plant pathogens, particularly plant pathogens that cause post-harvest disease in fruits. Examples of plant pathogens include Penicillium italicum Wemer, Penicillium digitatum, Botrytis cinerea, Rhizopus stolonifer, Geotrichum candidum, Penny Silium xpansum and Alternaria alternata.

The microorganisms of the present invention are useful for inhibiting plant pathogens for a variety of agricultural products, including, but not limited to, fruits, vegetables (eg celery), grains, cereals, nuts, seeds and stored feed. Examples of fruits capable of carrying out the present invention include, but are not limited to, citrus fruits, grapes, apples, pears, tomatoes, persimmons, strawberries, peaches, apricots, cherries and papayas. Examples of citrus fruits include grapefruit, oranges, lemons, kumquats, limes and purmelos. Examples of such nuts are peanuts, almonds and pecans. Examples of such cereals are wheat, corn, sugar cane, soybeans and barley. The microorganisms of the invention can also be used in processed agricultural products, including, for example, raisins, dried plums, figs, dried apricots and date palm fruits.

Microorganisms of the present invention include (1) a gel or gum based carrier (eg, xanthan gum); (2) water based carriers (eg, microorganisms may be mixed with or suspended in water. Other water based carriers include water added wetting agents and / or diffusing agents); (3) Oil based carriers such as "Fresh Mark" or "Fresh Wax 58P" (this is a paste wax for peach, plum and nectarine, containing white oil paraffin wax, petroleum and oleic acid), all fresh marks ) (Orlando, Florida Chemicals) products; (4) Wax based carriers [e.g., wax coatings typically used in citrus fruit and apples, such as "Britex 551" or "Britex 559", kefar-Saba, Brosha (chemical) Pharmaceutical products) (including Broshar (Chemicals) Ltd.); (5) a powder carrier component which provides the composition in powder form and disperses the microorganism (s) therein to a desired concentration in the powder composition [examples of such powder carrier components include starch (eg corn starch) and / or talc In this]; And (6) mixed with various additives, including carriers such as mixtures of the above materials, for use in agricultural products. Antagonists typically survive well in oil based carriers, so it is preferable to use oil based carriers rather than water based carriers. When the microorganism is grown in the liquid medium, the microorganism is supplied to the suspension containing the liquid medium, but in order to improve the inhibition rate, it is preferable to supply in the presence of water or at least one carrier. The compositions of the present invention may also contain (1) insecticides, for example fungicides (eg, "TBZ", available from FMC); (2) one or more preservatives, i.e. environmental enhancers such as compositions that maintain moisture and / or help to keep microorganism (s) alive during storage and / or use, for example, (a) gums, eg Natural gums such as, for example, guar gum, carob pod gum, indian rubber, tragacanth gum or preferably xanthan gum; (b) methyl cellulose; (c) silica gel and (d) said preservative mixture; (3) surfactants and wetting agents, such as Tween and Triton 20 X-100 (manufactured by Rhom and Hass Company); (4) additives to enhance diffusion of the composition of the present invention; (5) additives for enhancing the adhesion of the composition of the present invention to agricultural products; (6) nutrients for microorganisms of the present invention; And (7) other additives including mixtures of the above additives. These additives must be used in amounts that do not interfere with the microbial efficacy of the present invention when used. Typically, a suitable composition preparation method requires only mixing of the microorganism (s) and additives. Typical methods of preparation consist of adding together the microorganism (s), preservatives and powder components and / or grinding the components together. Powder compositions can be used in agricultural products or can be used in combination with liquids (eg water) and subsequently in agricultural products. The compositions of the present invention have good storage properties, do not require refrigeration, do not face contamination problems and remain effective around representative fruit, vegetable and cereal storage.

Concentrations of suspensions useful in the present invention can be any concentration that inhibits the infestation of the target plant pathogens when added to the fruit. As will be apparent to a person skilled in the art, the effective concentration is (1) the type of agricultural product; (2) the degree of ripening of agricultural products; (3) the concentration of pathogens affecting agricultural products; (4) scratches of agricultural products; (5) temperature and humidity; And (6) factors such as age of plant pathogens. The concentration ranges from about 1 × 10 ⁴ to 1 × 10 ⁹ CFU / ml, with about 1 × 10 ⁷ to 1 × 10 ⁹ CFU / ml being most preferred. For the purposes of the present invention, the abbreviation "CFU" is used herein to refer to "Colony forming units".

The microorganisms of the present invention are used in agricultural products using conventional methods such as dipping, spraying and brushing. In addition, the microorganism of the present invention, wax, Or in other protective coatings used for processing agricultural products.

Agricultural products can be processed at any time before or after harvest. Typically, post harvest and storage or embarkation are preferred treatment times. For some grapes, pre-harvest is the preferred treatment time.

It is within the scope of the present invention to treat the fruit either alone or in combination with isolates NRRL Y-18313, NRRL Y-18313, NRRL Y-18527 or NRRL Y-18654. Other inhibitors may be used in combination with microorganisms that are useful for inhibiting the spread of plant pathogens in agricultural products. These reagents should be used in amounts that will not interfere with the efficacy of the microorganisms of the present invention, as readily apparent to those skilled in the art upon use.

The natural or defined concentrations of isolates NRRL Y-18318, NRRL Y-18314, NRRL Y-18654, and NRRL Y-18527 for fruits may typically vary from 0 to 100 CFU / cm 2. Hansenia spora uvarum or its amorphous Kloeckera apiculata is commonly found as a natural component of the flora of microorganisms that inhabit fruit surfaces [kamra N. and Madan, M. (l987) Microbios. Lett. 34: 79; Stollarova, V. (1982), Biologica (Bratsi 1) 37: 1115-1121]. However, in the past, these yeast species have not been reported to have biological control properties, so the ability of these yeasts to inhibit plant pathogens was not expected. One aspect of the invention provides a microorganism (s) of the invention at concentrations significantly higher than the natural / regulated concentrations described above, for example at least about 10 ⁶ CFU per cm 2, or preferably at least about 10 ⁶ CFU per cm 2. It's about things. In this regard, another aspect of the present invention relates to agricultural products comprising calcium salt and at least one antagonistic microorganism of the present invention at a concentration of at least about 10 ⁵ CFU / cm 2.

By using one or more calcium salts in combination with one or more microorganisms of the present invention, the inventors have found that plant pathogens (among them, particularly lysopus stolonifers of peaches, major rot pathogens of table grapes, penicillium and botrytis of apples) We found surprising and unexpected findings that it would be easier to improve the inhibitory effects of Botrytis rot and Penicillium rot of grapefruit. Enhancing the ability of the microorganisms of the present invention to inhibit plant pathogens in the presence of one or more calcium salts is a study by Conway, which shows that post-harvest rot does not decrease in fruits topically treated with calcium chloride [1981-Plant Disease 66: 402-403 and Conway et al., 1983 Plant Pathology 73: 1068-1011. From the facts, it was not particularly predicted. While not wishing to be bound by theory, the dramatic effect of calcium salt (s) on biological control may be due to the effects of or affecting metabolism of antagonistic microorganisms surviving the scratch site or by interaction with their metabolites. It is believed that this may be the result of the interaction of (s) with calcium cations. In a preferred embodiment of the invention relating to the use of calcium chloride, calcium chloride supplied as topical treatment is useful as a reagent to enhance the biological control of plant pathogens; Calcium chloride is more effective at improving biological control than other salts containing similar cations and anions; It is particularly surprising and unpredictable that the effects of salt calcium are exerted on a wide range of plant pathogens and proved to be various biological control agents. One or more calcium salts and one or more microorganisms are added separately to the agricultural product or as a mixture (eg containing one or more of the above additives) for ease of use. Representative examples of calcium salts include calcium chloride, calcium carbonate, calcium propionate, and mixtures thereof. For example, calcium chloride may be used at a concentration of about 1 g / 100 ml to about 10 g / 100 ml, preferably about 1 g / 100 ml to about 5 g / 100 ml, most preferably about 2 g / 100 ml.

The following examples are intended to further illustrate the invention and do not limit it to the scope defined by the claims.

The following seven citrus varieties were used to determine the efficacy of Pichia vulgarienmondi NRRL Y-18314: grapefruit (Citrus paradisi Macf cultivar 'Marsh Seedless'); 'Shamouti' and 'Valencia' orange (C.sinensis Osbeck); Lemon (C.lemon L. Burm 'Eureka'); Temple Orange (Tanger hybrid, C. reticulata XC. Sinensis); Kumquat (Fortunella margarita); And Fumelo (C.grandis). The decaying pathogens of the fruits tested include penicillium digitatum, penicillium italicum and geotricum candidum links, Link. Ex Pers., And green fungal disease, blue mold disease, and rancidity after harvest, respectively. Causes

The biologically pure culture of isolate NRRL Y-18314 was obtained according to the following method: Lemon was placed in a 600 ml beaker containing 200 ml of sterile water to wash the surface of the lemon. The beaker with fruits was placed in a 100 rpm rotating shaker for 10 minutes. 10 ml (once) of wash water were then sprayed onto NYDA plates and incubated for 24 hours before colonies were selected. The same fruit was separated and washed three times and followed the same procedure. Appeared colonies were isolated and purified using standard purification techniques. All cultures were stored on silica gel in the refrigerator until use. NRRL Y-18313 and NRRL Y-18654 can be obtained using similar methods.

Isolate NRRL Y-18314 was grown for 48 hours at 30 ° C. in a flask containing nutritive yeast dextrose broth (NYDB) on an inverting shaker. The culture was centrifuged at 7000 rpm for 10 minutes and the resulting pellets were suspended in water at various concentrations. The concentration of the aqueous suspension was adjusted on the spectrophotometer.

Freshly harvested fruits were wiped with 95% ethanol and placed 24 per tray on wet paper in a 50 × 100 × 15 cm plastic tray. 2 to 4 conical scratches were made on the skin 3 mm deep. The scratch was brushed with an aqueous suspension of NRRL Y-18314. The concentration range of the aqueous suspension is 1 × 10 ⁵ to 1 × 10 ¹⁰ CFU / ml. After 1-2 hours, 20 μl of an aqueous spore suspension (1 × 10 ⁴ spores / ml) of the target pathogen was pipetted into the scratch. Control fruits were inoculated with only an aqueous aqueous suspension of target fungal bacteria. After incubation the tray was covered with a high density polyethylene sleeve and kept at room temperature for several days.

The number of inoculation sites where corruption was prevalent was measured daily. In three or more replicate experiments with six fruits, each treatment of each experiment consisted of 24-75 inoculation sites per treatment. Each test was repeated two or more times.

The results are shown in Tables 1, 2 and 3.

Relative Efficacy of NRRL Y-18314 to Suppress Penicillium Digitatum Septicosis in Different Citrus Fruit Varieties

TABLE 1

a Number of contact areas per treatment in parentheses under the breed name.

b Use whole fruits without artificial inoculation. The fruit is briefly soaked in a liquid culture of 48 hours of NRRL Y-18314. NYDB is used as a control.

Inhibition of Penicillium Italicum Corruption of Grapefruit and Orange by NRRL Y-18314

TABLE 2

a Number of inoculation sites per treatment in parentheses under the breed name.

Inhibition of Geotricum Candid Decay in Grapefruit and Lemon by NRRL Y-18314

TABLE 3

a Number of inoculation sites per treatment is given in parentheses under the breed name.

As shown in Table 1, isolate NRRL Y-18314 was very effective in inhibiting penicillium digitatum rot on citrus fruit in all test varieties. The efficacy of NRRL Y-18314 varied with the cultivation of cultivar against rot. Isolator NRRL Y-18314 was more effective against fumelo fruit and less effective against temple, lemon, orange or kumquat fruit compared to its efficacy in grapefruit.

Table 2 shows that isolate NRRL Y-18314 is effective in inhibiting phenylsilium italicum rot of grapefruit, orange and other citrus fruit varieties. As in penicillium digitatum, NRRL Y-18314 controls penicillium italicum in grapefruit more effectively than in orange. NRRL Y-18314 is also effective in suppressing the geotricum candoom prevalence in citrus fruits. However, as shown in Table 3, Geotrikum Candidum was less controlled than the extent of penicillium rot, especially in lemons.

Example II

Pichia coli ermondyi NRRL Y-18314 has been shown to inhibit the spread of Lycopus rot in grapes.

Biologically pure cultures of NRRL Y-18314 were isolated and purified as described in Example I.

NRRL Y-18314 was incubated for 48 hours at 28 ° C. in 100 ml of NYDB in a 250 ml Ellenmeyer flask on a rotary shaker (100 rpm). Freshly harvested grapes of the Pellet and Thompson Seedless varieties were briefly immersed in a microbial suspension in NYDB. The berry was treated as a whole cluster of intact berry, as a damaged berry that was pulled out of the stem and scratched, or as each damaged berry that was scratched by punctured the undamaged berry. Control berry was soaked only in extinction NYDB.

After 1-2 hours of immersion of the berry in the suspension, the berry was dried and then inoculated by immersion in an aqueous suspension containing the target pathogen spores at a concentration of 1 × 10 ⁴ spores / ml. Alternatively, the berry was inoculated by placing one decayed berry in the center of the intact berry group (ie "nest"). Treated berries were placed in polyethylene coated cardboard and kept at room temperature for 5 days. The entire treated cluster was placed directly on a commercial shipping cardboard.

The incidence of rot was measured by counting the number of berry infected. Each treatment in each test consisted of at least three repetitions for 20 berries placed in shipping cardboard half or four repetitions for five complete clusters.

The results are shown in FIG. As shown in FIG. 1, Pichia juliermondidie is effective in reducing rot in both damaged and intact grapes. Decay was seen in uninjured berry inoculated before inoculation.

Example 3

The efficacy of the Pichia fierliermondi NRRL Y-18314 isolate, which inhibits Botrytis cinerea rot and Penicillium xpansum rot, was tested on apples.

Yellow apples were surface sterilized by washing with 2% sodium hypochlorite. After air drying, the apples were placed on a styrofoam tray in a plastic tray with a lid. 100 ml of water was added to each tray for wetting. The needle was used to scratch the apple. The scratch size was 4mm in width and 4mm in depth. Three-day shake cultures of NRRL Y-18314 without NYDB growth at a concentration of 1 × 10 ⁹ CFU / ml were added to the scratch at 50 μl / scratch. The apples were left to air dry. Thereafter, an aqueous suspension (1 × 10 ⁴ spores / ml) of Botrytis cinerea spores or penicillium expansum spores was added to the scratches at 20 μl / scratch. Control was inoculated with water only.

Infection area was measured 5, 7, 9 days after inoculation. The results are shown in FIG. Nine days after inoculation, small lesions developed. The degree of protection against penicillium xpansum was less than that of Botrytis Cinerea. Nevertheless, apples treated with NRRL Y-18314 are clearly shown in FIG. 3 as having significantly reduced penicillium xpansum prevalence compared to untreated controls.

Example IV

It is already coined the efficacy of Pichia Galliermondi NRRL Y-18314, which inhibits penicillium digitatum in grapefruit. The efficacy of the eight isolates of Hanseni was compared.

Eight isolates were obtained from the American Type Culture Collection (ATCC) at Rockville Parkrun Drive 12301, 20252, USA. The test isolates were identified as follows: ATCC 18538, ATCC20220, ATCC 36239, ATCC 34022, ATCC 9367, ATCC 36767 and ATCC 18107.

Each test isolate was incubated for 48 hours in NYDB liquid medium at 28 ° C. After centrifugation, the resulting pellet was washed twice with water and then suspended in water. The concentration of the aqueous suspension is in the range of 1.3 × 10 ⁷ to 1.3 × 10 ⁹ CFU / ml.

The surface of the grapefruit was sterilized with 95% ethanol and placed on a wet paper in a 50 × 100 × 15 cm plastic tray. Then, the surface of this fruit was scratched using a needle. 2 to 4 conical scratches in the rind were cut out to a depth of 3 mm. An aqueous suspension of the isolate was brushed on the scratch surface. Each isolate was tested for 48 inoculation sites. After 1-2 hours, an aqueous suspension of penicillium digatum (1 × 10 ⁵ spores / ml) was added to the scratch at 20 μl / scratch. Control was inoculated with water only.

After 7 days of inoculation the percentage of infection of the fruit was recorded. The data was analyzed to obtain mean and deviation by Duncan's multi-range test. Values leading to other characters are quite different at the 1% level. The results are shown in FIG.

NRRL Y-18314 is a conventional D. It has been clearly shown to be an excellent inhibitor of penicillium digitatum compared to the isolated isolate of Hanseni. Seven days after inoculation, grapefruit inoculated with the isolate from ATCC developed 25-65% infection, while grapefruit inoculated with NRRL Y-18314 was fully protected.

Example Ⅴ

The purpose of this example is to show the efficacy of isolate NRRL Y-18314 that inhibits Aspergillus plabus in peanuts. Peanuts were grown in the following manner. Each peanut surface is scratched. NRRL Y-18314 was treated as described in Example I. Similarly, Aspergillus plabus was treated as described for the pathogens in Example I. Treated peanuts were incubated at 26 ° C. for 14 days. FIG. 5A is a photograph of peanuts treated with Aspergillus Flavers and NRRL Y-18314, and FIG. 5B is a photograph of peanuts treated only with Aspergillus Flavers. As shown in these photographs, the suppression of pathogen growth by yeast is evident: FIG. 5a is only 33% growth, which is a (low or moderate) pathogen growth of the scratch compared to the 5b which indicates 100% extensive pathogen growth of the scratch. Indicates.

Example VI

The purpose of this example is to show the efficacy of isolate NRRL Y-18314 that inhibits Aspergillus niger in peanuts. Peanuts were grown in the following manner. Each peanut surface is scratched. Y-18314 was treated as described in Example I. Similarly, Aspergillus niger was treated as described for the pathogen in Example I. Treated peanuts were incubated at 26 ° C. for 14 days. FIG. 6a is a photograph of peanuts treated with Aspergillus niger and NRRL Y-18314, and FIG. 6b is a photograph of peanuts treated with Aspergillus niger only. As shown in these photographs, 100% infection of the untreated control (the yeast-treated peanut compared to degree 6b appears to completely inhibit pathogen growth (Figure 6a).

Example Ⅶ

25 ml of 48 hour NRRL Y-18314 culture was centrifuged. The obtained pellet is resuspended in 10 ml of each of the following (A), (B), (C) and (D): (A) obtained from Durant-Wayland Inc., La Grande, GA. Waxes containing paraffin mineral oil base; (B) fresh wax; (C) stayfresh water based waxes obtained from FMC Corporation (Woodstock, VA); (D) "Fresh Wax 58p" with Paraffin Mineral Oil Base For each wax, an initial dilution multiple (CFU / ml) was made (ie, initial, time 0). 1 ml of each mixture of (a) wax and culture was diluted at the time intervals listed in Table 4 (excluding those listed in the table above) by mixing with 9 ml of each wax. The resulting mixture was then plated onto yeast malt agar plates. The plate was maintained at about 20-25 ° C. The results are shown in Table 4. Table 4 lists all of the clony forming units per milliliter (CFU / ml).

TABLE 4

** Too many to count.

* Not tested (ie no dilution plate prepared).

In this example, RRL Y-18314 shows surprisingly high viability in commercially available waxes at room temperature even for extended periods of time.

Example Ⅷ

The purpose of this example is to show that lyophilized cells or whole cells of NRRL Y-18314 can survive in commercially available waxes (ie, fresh mark waxes) for extended periods of time. Lyophilized cells were frozen in liquid nitrogen and placed in a lyophilizer for 48 hours (4 times the volume of freeze-dried cells) and mixed with wax. The results are shown in Table 5. Table 5 lists all colony forming units per milliliter (ie CFU / ml).

TABLE 5

** Too many to count.

* Not tested (ie no dilution plate prepared).

In this example, NRRL Y-18314 exhibits surprisingly high viability in commercially available waxes at room temperature even for extended periods of time.

Example Ⅸ

5 ml of YM broth culture of NRRL Y-18314 (5.6 × 10 ⁸ CFU / ml) was mixed with 5 ml of gum. The gum concentration of the 5 ml solution is in the range of 1-20%, as shown in Table 6. Cultures and gum mixtures were added to either 40 cm 3 of corn starch (25.7 g) or silica gel (27.1 g). This formulation was mixed and dried at 54 ° C. for 4 days, then ground in mortar and ground with a pestle to give a fine powder. The powder was then stored at 4 ° C. 1 g of this powder was added to 10 ml of sterile water, mixed for 20 minutes using a stirring bar, and plated in dilution to determine the NRRL Y-18314 frequency. The results are shown in Table 6 as CFU / ml.

TABLE 6

These results clearly show that NRRL Y-18314 can survive for a long time in various gums mixed with corn starch or silica gel.

Example Ⅹ

The same procedure as in Example V is followed except that talc (28 g) is used instead of corn starch or silica gel. The results are shown in Table 7 as CFU / ml.

TABLE 7

These results clearly show that NRRL Y-18314 can survive for a long time in various mixtures of talc and gum.

Example XI

The purpose of this example is to show that CaCl ₂ and CaCO ₃ promote biosuppression more effectively than other inorganic salts and surprisingly unexpected synergism is obtained. A needle was used to deliberately scratch the yellow apple 3mm deep. Salts according to the first portion of the apples respectively in Table 8 2g / 100ml concentration 50μl portion of a solution of an aqueous solution consisting of NRRL Y-18314 in sterile distilled water (where, FeSO ₄ is yiyongdoem to 5mM concentration) and a concentration of 10 ⁷ CFU / ml Treated with. The second portion of apples was treated with 50 μl of an aqueous solution of each of the salts listed in Table 8 at a concentration of 2 g / 100 ml, with FeSO ₄ being used at a concentration of 5 mM. The control group was also treated with sterile distilled water. Two hours after the solution treatment, each apple was treated with 20 μl of 10 ⁵ spores / ml suspension of Botrytis cinerea. The average fruit infection percentage of four samples (8-10 runs per sample) was measured 10 days after inoculation with Botrytis cinerea. The results are shown in Table 8.

TABLE 8

The values in parentheses are the standard error. Asterisks indicate that the series of mean values differs significantly (P ≦ 0.05) from the deviation in the arcsin square root-modified data according to SAS GLM analysis. It can be observed that the salt alone did not statistically reduce the infection significantly (ie, the value of salt alone did not differ significantly from the case of saline alone, 100% infection). NRRL Y, as evidenced by the fact that CaCl ₂ and CaCO ₃ alone provide about 5% and about 18% infection reduction and NRRL Y-18314 alone provides about 40% infection reduction. The combination of -18314 and calcium salt clearly provides a synergistic reduction in infection. Therefore, the additive effect can be expected to be 45% or 58%, respectively. In practice, however, a combination of CaCl ₂ and NRRL Y-18314 provides a 96.7% reduction in infection, more than double the expected 45%, and a combination of CaCO ₃ and NRRL Y-18314 reduces infection by 72.5%, much higher than the expected 58%. To provide.

Example XII

The purpose of this example is to show that calcium salts provide enhanced biosuppression with various yeast strains of different species. The yellow apple was scratched according to the previous example. The apples were then treated with 50 μl of 10 ⁸ CFU / ml suspension of each yeast in sterile distilled water with or without ₂ g / 100 ml CaCl ₂ as described in Table 9.

After 2 hours the apples were treated with 20 μl of 10 ⁴ spores / ml suspension of Botrytis cinerea. Percent infection was observed 7 days after inoculation. The results are shown in Table 9.

TABLE 9

The values written above are the average of three runs in each treatment. The values in parentheses are the standard error of the mean. Asterisks indicate that the fruit rot when yeast treated with calcium chloride is significantly less (P ≦ 0.05) than when treated with yeast alone. It can be observed that either use of yeast results in significant reduction of infection and greater mixing of CaCl ₂ with each yeast results in greater reduction of infection.

Example XIII

The purpose of this example is to demonstrate the efficacy of NRRL Y-18314 and its CaCl ₂ incorporation to inhibit penicillium decay. The yellow apple was intentionally scratched according to Example XI. The scratched apples were then treated with 50 μl of a suspension of NRRL Y-18314 in sterile distilled water with or without CaCl ₂ (concentration 2 g / 100 ml) as described in Table X. After 2 hours, the apples were treated with 20 μl of a spore suspension of penicillium xpansum at the concentrations listed in Table X. 7 days after inoculation, lesion diameter was observed. The results are shown in Table X.

TABLE 10

The values listed in Table 10 are averages of five repetitions for each treatment. Table that is from, CaCl ₂ the single piece processing in the absence NRRL Y-18314 does not reduce significantly the corruption during most of the process compared to the water control and NRRL Y-18314 absence CaCl ₂ treatment group (over 50%) Can be observed. However, using NRRL Y-18314 with CaCl ₂ reduced rot by more than 50% at all yeast concentrations and penicillium spore concentrations tested.

Example XIV

The purpose of this example is to explain the synergistic effect of mixing the microorganism of the present invention with CaCl ₂ at various concentrations. The grapefruit was scratched in the same manner as in Example I. The flawed grapefruit was then treated with 50 μl of a partial solution of the strains identified in Tables XI and XII in sterile distilled water. After 2 hours the apples were treated with 20 μl of 10 ⁴ spores / ml suspension of Penicillium digitatum. Grapefruit was observed after incubation at 24 ° C. for 5 days. The average error decay percentage was obtained as follows (data is the average of two to three runs at each treatment).

TABLE 11

* Indicates NT "not tested".

TABLE 12

Example XV

The purpose of this example is to show that yeast cells that are not yeast culture broths provide biosuppression and washed yeast cells provide enhanced biosuppression than a combination of yeast cells and culture broths. After deliberately scratching the embryo, the cells were treated with 50 μl of washed yeast cells prepared by pelleting yeast cells from the culture broth by centrifugation at 5,000 RCF (relative centrifugal force), and resuspending the yeast cells in sterile distilled water. And repellet by centrifugation as before and resuspend with control of concentration in water or culture broth to reach the concentrations listed in Table XIII. Portions of embryos were treated only with culture broth (no yeast cells). Two hours later, the embryos were inoculated with 20 μl of 10 ⁴ spores / ml suspension of Reuspus Stolniper. After 4 days the average infection rate was observed. The results are as follows (each value is an average of two to five runs).

TABLE 13

Example XVI

A single seedless grape was pulled from the stem and scratched. The grapes were then immersed in a suspension of yeast specified in Table 14 at concentrations of 10 ⁸ to 10 ⁹ CFU / ml and incubated at 220 ° C. Percentage of fruit decay caused by naturally occurring microorganisms (e.g. Aspergillus niger and Rizopus stolonifer) on days 5 and 6 is as follows (data is 20 berries 3 times per fruit).

TABLE 14

1NYDB = Sterile Culture Broth, Nutritional Yeast Dextrose Broth

Fruit rot due to lyfupus was not observed in the yeast treated grapes.

Example XVII

"Perlett" whole clusters of grapes were immersed in a suspension of yeast antagonists as specified in Table 15 at concentrations of 10 ⁸ to 10 ⁹ CFU / ml. Twice in 6 fruit clusters at the time of processing. After 7 days of storage at 20 ° C., spontaneous fruit decay rates were observed. The result is as follows.

TABLE 15

n = number of errors in every treatment

The foregoing detailed description and examples have been described for purposes of illustration. Modifications and variations may be made without departing from the spirit and scope of the invention.

Claims (77)

A biologically pure culture of one or more isolates of Pichia guilliermondii having the identifying properties of isolates selected from the group consisting of NRRL Y-18313, NRRL Y-18314 and NRRL Y-18654. The biologically pure culture of claim 1, having the identification properties of NRRL Y-18313. The biologically pure culture of claim 1, having the identification properties of NRRL Y-18314. The biologically pure culture of claim 1, having the identification characteristics of NRRL Y-18654. A method for inhibiting the spread of plant pathogens of agricultural products, comprising using at least one microorganism that is antagonistic to plant pathogens but not antibiotics in an effective amount capable of inhibiting the spread of plant pathogens of agricultural products. A method for inhibiting the spread of plant pathogens of agricultural products, comprising using at least one microorganism that is antagonistic to at least one calcium salt and plant pathogens in an effective amount capable of inhibiting the spread of plant pathogens of agricultural products. 7. The method of claim 6, wherein said one or more microorganisms are not antibiotics. 6. The method of claim 5, wherein said at least one microorganism is selected from the group consisting of fungi, bacteria, viruses and mixtures thereof. 10. The method of claim 8, wherein said at least one microorganism is yeast. The method of claim 9, wherein the first microorganism is yeast having identification characteristics of accession number NRRL Y-18313, yeast having identification characteristics of accession number NRRL Y-18314, yeast having identification characteristics of accession number NRRL Y-18654 and And at least one yeast selected from the group consisting of yeast having the identification properties of accession number NRRL Y-18527. A method according to claim 10, wherein said at least one microorganism is a yeast having identification properties of accession number NRRL Y-18313. The method of claim 10, wherein said at least one microorganism is a yeast having identification properties of accession number NRRL Y-18314. The method of claim 10, wherein said at least one microorganism is a yeast having identification properties of accession number NRRL Y-18527. The method of claim 10, wherein said at least one microorganism is a yeast having identification properties of accession number NRRL Y-18654. 6. The method of claim 5, wherein said application step comprises using an additive selected from the group consisting of pesticides, preservatives, carriers, surfactants, wetting agents and mixtures thereof for said agricultural product. The agricultural product according to claim 11, wherein the application step is selected from the group consisting of gel-based carriers, gum-based carriers, water-based carriers, wax-based carriers, oil-based carriers, active-based carriers, starch-based carriers and mixtures thereof. A method comprising applying. 6. The method of claim 5, wherein said at least one microorganism is applied to said agricultural product as an agent essentially free of other microorganisms. 7. The method of claim 6, wherein said application step comprises applying a mixture of said at least one calcium salt and said at least one microorganism to said agricultural product. The method of claim 5, wherein the plant pathogen is Penicillium italicum Wemer, Penicillium digitatum, Botrytis cinerea, Rizopus stolonifer ), Geotrichum candidum, Penicillium expansum, Alternaria alternate, Alterna, Aspergillus flavus, Aspergillus niger Rhizopus arrhizus, Gilbertella persicovia, Mucor spp., Pezicula malicorticas, Monilinia spp. And bacterial pathogens. 19. The method of claim 18, wherein said plant pathogen is selected from the group consisting of Monilinia fructicola and Monilinia laxa. 7. The method 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. 6. The method of claim 5, wherein the agricultural product is selected from the group consisting of fruits, vegetables, grains, cereals, nuts, seeds and stored feed. 23. The fruit of claim 22, wherein the agricultural product is selected from the group consisting of citrus fruits, grapes, apples, pears, tomatoes, persimmons, strawberries, peaches, apricots, cherries, papayas, raisins, dried plums, figs, dried apricots and date palm fruits. Method characterized by that. 24. The method of claim 23, wherein the agricultural product is a citrus fruit selected from the group consisting of grapefruit, orange, lemon, kumquat, lime, and fumelo. 23. The method of claim 22, wherein said agricultural product is a nut selected from the group consisting of peanuts, almonds and pecans. 23. The method of claim 22, wherein said agricultural product is cereals selected from the group consisting of wheat, corn, sugar cane, soybeans and barley. 6. The method of claim 5, wherein said application step comprises spraying, injecting, rubbing, spraying or brushing said agricultural product containing said at least one microorganism. 6. The method of claim 5, wherein said application step comprises immersing or rolling said agricultural product in a composition containing said at least one microorganism. A composition comprising a mixture of carriers for one or more microorganisms selected from the group consisting of gels, gums, waxes, oils, talc, starches and mixtures thereof and one or more microorganisms which are antagonistic but not antibiotics against plant pathogens. A composition consisting of a mixture of one or more microorganisms and a carrier for the one or more microorganisms, wherein at least 99% of the number of the one or more microorganisms are antagonistic to plant pathogens but are not antibiotics. A composition consisting of a mixture of at least one calcium salt and at least one microorganism that is antagonistic to plant pathogens. 32. The composition of claim 31, wherein said at least one microorganism is not an antibiotic. 32. The composition of claim 31 consisting essentially of said at least one calcium salt and said at least one microorganism. 32. The composition of claim 31, wherein at least 99% of the number of microorganisms in the composition is antagonistic to plant pathogens. 32. The composition of claim 31, wherein at least 99% of the number of microorganisms in the composition are not antibiotics. 32. The composition of claim 31, wherein said at least one microorganism is selected from the group consisting of fungi, bacteria, viruses and mixtures thereof. 37. The composition of claim 36, wherein said at least one microorganism is yeast. 33. The yeast of claim 32, wherein the one or more microorganisms have identification properties of accession number NRRL Y-18313, yeasts having identification properties of accession number NRRL Y-18314, yeasts having identification properties of accession number NRRL Y-18654. And yeast having identification properties of Accession No. NRRL Y-18527. The composition according to claim 38, wherein said at least one microorganism is a yeast having identification properties of accession number NRRL Y-18314. The composition according to claim 38, wherein said at least one microorganism is a yeast having identification properties of accession number NRRL Y-18527. The composition according to claim 38, wherein said at least one microorganism is a yeast having identification properties of accession number NRRL Y-18313. 39. The composition of claim 38, wherein said at least one microorganism is a yeast having identification properties of accession number NRRL Y-18654. 25. The composition of claim 24, further comprising 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. 32. The composition of claim 31, further comprising 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 gels, gums, waxes, oils, talc, starches, water and mixtures thereof. 47. The composition of claim 46, wherein said carrier is xanthan gum. 46. The composition of claim 45, further comprising a preservative selected from the group consisting of gums, methylcellulose, silica gel, and mixtures thereof. 46. The composition of claim 45, wherein said preservative is xanthan gum. 30. The composition of claim 29, wherein said carrier is selected from the group consisting of starch, talc and mixtures thereof. 30. The composition of claim 29, further comprising at least one calcium salt. 49. The composition of claim 48, wherein said at least one calcium salt is selected from the group consisting of calcium chloride, calcium propionate, calcium carbonate and mixtures thereof. 29. The composition of claim 26 or 28, wherein said at least one calcium salt is selected from the group consisting of calcium chloride, calcium propionate, calcium carbonate and mixtures thereof. A product consisting of agricultural products containing at least 10 ⁵ colony-forming units per cm 2 of at least one microorganism that is antagonistic to plant pathogens but not antibiotics. 55. The product of claim 54, wherein the serf is at least about 106 colony forming units per cm 2. A product consisting of agricultural products containing at least one microorganism, most of which are antagonistic to plant pathogens but are not antibiotics. 57. The product of claim 54 or 56, wherein said agricultural product comprises calcium salt. 58. The product of claim 57, wherein the calcium salt is selected from the group consisting of calcium chloride, calcium pyroionate, calcium carbonate and mixtures thereof. A product comprising agricultural products containing calcium salts and microorganisms antagonistic to plant pathogens at concentrations of at least about 10 ⁵ colony forming units per cm 2. 60. The product of claim 59, wherein the one or more microorganisms are not antibiotics. Products consisting of agricultural products containing calcium salt in agricultural products and one or more microorganisms, most of which are antagonists of plant pathogens 62. The product according to claim 59 or 61, wherein the calcium salt is always selected from the group consisting of calcium chloride, calcium pyrionate, calcium carbonate and mixtures thereof. 62. The product of claim 54, 56, 59 or 61, wherein said at least one microorganism is selected from the group consisting of fungi, bacteria, viruses and mixtures thereof. 64. The product of claim 63, wherein the one or more microorganisms are yeasts. 65. The yeast having identification properties of accession number NRRL Y-18313, yeast having identification properties of accession number NRRL Y-18314, and yeast having identification properties of accession number NRRL Y-18654. And yeast having the identification characteristics of Accession No. NRRL Y-18527. 66. The product of claim 65, wherein the at least one microorganism is a yeast having identification properties of Accession No. NRRL Y-18313. 67. The product of claim 65, wherein said at least one microorganism is a yeast having identification properties of accession number NRRL Y-18314. 67. The product of claim 65, wherein said at least one microorganism is a yeast having identification properties of accession number NRRL Y-18527. 67. The product of claim 65, wherein the at least one microorganism is a yeast having identification properties of Accession No. NRRL Y-18654. 62. The product of claim 54, 56, 59 or 61, wherein said agricultural product is selected from the group consisting of fruit, vegetables, grains, grains, nut seeds and stored feed. 71. The fruit of claim 70, wherein the agricultural product is selected from the group consisting of citrus fruits, grapes, apples, pears, tomatoes, persimmons, strawberries, peaches, apricots, cherries, papayas, raisins, dried plums, figs, dried apricots and date palm fruits. Product characterized in that. 72. The product of claim 71, wherein the agricultural product is a citrus fruit selected from the group consisting of grapefruit, oranges, lemons, kumquats, limes and purmelos. 71. The product of claim 70, wherein said agricultural product is a nut selected from the group consisting of peanuts, almonds and pecans. 71. The product of claim 70, wherein the agricultural product is cereals selected from the group consisting of wheat, corn, sugar cane, beans and barley. 62. The product of claim 54, 56, 59 or 61, wherein said agricultural product further comprises an additive selected from the group consisting of pesticides, preservatives, carriers, surfactants, wetting agents and mixtures thereof. Biologically pure culture of the isolate of Hansenia spora uvarum with the identification properties of isolate NRRL Y-18527. 30. The composition of claim 29, wherein said carrier is xanthan gum.