CA2118267C - Bioencapsulated biopesticides and process for the manufacture thereof - Google Patents
Bioencapsulated biopesticides and process for the manufacture thereofInfo
- Publication number
- CA2118267C CA2118267C CA002118267A CA2118267A CA2118267C CA 2118267 C CA2118267 C CA 2118267C CA 002118267 A CA002118267 A CA 002118267A CA 2118267 A CA2118267 A CA 2118267A CA 2118267 C CA2118267 C CA 2118267C
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- Prior art keywords
- thuringiensis
- gel
- paste
- bacillus thuringiensis
- biopolymeric
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/26—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests in coated particulate form
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
- A01N63/23—B. thuringiensis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Environmental Sciences (AREA)
- Dentistry (AREA)
- Agronomy & Crop Science (AREA)
- Biomedical Technology (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Dispersion Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
A bioencapsulated biopesticide comprises cells or spores of Bacillus thuringiensis and natural biopolymers extracted from natural sources.
Description
BIOENCAPSULATED BIOPESTICIDES AND
PROCESS FOR THE MANUFACTURE THEREOF
Field of the Invention The present invention relates to a bioencapsulated biopesticide and a process for the preparation thereof; and, more particularly, it pertains to a biodegradable, environmentally safe biopesticide and a simple and economical process for the preparation thereof which comprises encapsulating Bacillus thuringiensis strains or their spores by using a biopolymer.
Background of the Invention In recent years, biopesticides have been in the limelight partly due to the stiffening regulations on the use of toxic chemicals as pesticides.
Biopesticides are attractive as an alternative or supplement to the existing chemical pesticides owing to their environmental safety and their specificity to target pathogens or insects.
Such biopesticides may include bioinsecticides, biofungicides, bioherbicides and plant growth regulators; and biological control agents commonly used for the preparation of biopesticides may include bacteria, yeasts, fungi, viruses and toxins such as B.T. toxin produced by Bacillus thurinqiensis.
Major problems associated with the biopesticides are high production cost and their instability or sensitivity to various environmental factors such as sunlight, desiccation, heat, ultraviolet light and rainwash when they are applied in the crop field, which limit the use of most biopesticides. For example, B.T. toxin produced by Bacillus thurinqiensis loses its insecticidal activity in a few days in the field under sunlight. Accordingly, the instability of B.T. toxin under the field conditions limits its utility despite its pesticidal effectiveness.
In this connection, there have been employed encapsulation technologies to protect chemical pesticides from environmental damages and provide controlled release of active ingredients(U.S. Patent No. 2,876,160). However, most of these encapsulation techniques developed for the chemical pesticides are n t suitable for biopesticides since microorganisms or biologically active agents contained in the biopesticides are much more sensitive than the chemical components. Therefore, needs for an effective technique for the stable encapsulation of biopesticide has continued to exist, which satisfy various requirements for its practical utility.
First of all, the materials to be used in carrying out the encapsulation should be natural biopolymers rather than synthetic polymers to provide an inexpensive and environmentally safe biopesticide.
Secondly, the biopolymer gel matrix prepared from the polymeric materials for the encapsulation of biological control agents should be able to protect the encapsulated biological control agents from various environmental damages such as sunlight, heat, W light and desiccation by supplying them with a protective layer and should have a high moisture holding capacity for their survival.
Thirdly, the biopolymer matrix should adhere to the crop and absorb moisture when it is applied in the field, which property is important for providing an even distribution of the biopesticide, efficient delivery to targets such as plant pathogens, harmful insects and weeds, and resistance against rainwash.
Fourthly, the bioencapsulating matrix layer should provide and maintain optimum physical and biological conditions for the activities of the biological control agents. To attain best results, when the bioencapsulated products are applied to the target, the microorganisms in the biomatrix layer should germinate, grow and proliferate efficiently to produce bioactive agents such as antibiotics, enzymes, toxins or other bioactive compounds which can serve as bioherbicide, bioinsecticide, biofungicide, nematocide, bactericide, molluscicide, acaricide, algicide, plant growth regulator, biofertilizer or fruit and vegetable preserving agents in postharvest storage.
Fifthly, the bioencapsulation technique should be cost effective to allow the biopesticides to compete against 211~267 chemical pesticides.
Various techniques for the encapsulation of enzymes and chemical pesticides in a synthetic polymer matrix have been studied extensively. However, none of these techniques have been satisfactory due to the lack of ability to fulfill the above requirements for biopesticides. For example, European Patent No. 0,320,483 discloses the use of chemical polymers such as polyvinylalcohol and polyvinyl pyrrolidone to encapsulate microorganisms such as Bacillus thuringiensis, Alternaria cassiae and Pseudomonas fluorescens. However, this 'echnique is not practical due to the high production cost and risk of environmental pollution which may be caused by the use of chemical polymers.
There have been also proposed bioencapsulation techniques which employ various biopolymer matrices to encapsulate biological control agents. For instance, H. Shigemitsu has disclosed in U.S. Pat. No. 4,647,537 a process for the bioencapsulation of microorganisms inhibiting plant pathogens in a carrageenan polymer matrix; however, this technique may have a limited utility due to the high cost of carrageenan.
U.S. Pat. No. 4,724,147(J.J. Marois) and U.S. Pat. No.
4,668,512(J.A. Lewis) have reported the use of alginate pellets to encapsulate fungi to be used as bioherbicide.
Again, the efficacy of this technique is rather limited due to the high price of alginate, low moisture holding capacity, and failure to provide sufficient adhesivity in case of a foliar application.
H. K. Kaya et al. have also disclosed a similar technique wherein calcium alginate is used to encapsulate nematodes (Environ. Entomol., 14, 572-574(1985)); however, this technique is not practical due to the high cost of calcium alginate.
Jung et al. have shown in French Patent No. 2,501,229 the inclusion of mycorrhizas in a biopolymer gel for viable storage, wherein the biopolymer gel is made of high molecular weight heteropolysaccharides produced by Xanthomonas. However, this technique is also handicapped by the high production cost of said biopolymer.
In European Patent No. 0,192,319, A.C. Barnes and S.G.
Cummings offer a novel bioencapsulation technique wherein B.T.
toxin molecules are immobilized inside of a whole cell of B.T.
toxin-producing Pseudomonas killed by the treatment with a chemical reagent. This product is called Cell-Cap*and shows an improved stability in the field due to the prevention of UV
damage under sunlight. However, this technique is also too expensive to be used commercially.
Among the bioencapsulation techniques so far disclosed, a most economical one is the stàrch encapsulation technique for entomopathogens disclosed in U.S. Patent No. 4,859,377 (B.S. Shasha and R.L. Dunkle). In this technique, a biogel is prepared by mixing pregelatinized corn starch powder, corn oil, cold water and biological control agents such as spores of Bacillus thuringiensis and B.T. toxin at a room temperature for 5 to 60 seconds. Reassociation of the amylose components ; *Trademark 211~267 in the pregelatinized starch results in a substantially homogeneous mass of starch matrix; and the biological agents are dispersed and entrapped uniformly throughout the continuous starch matrix. Their preferred starch concentration is about 25 to 40% for a granular form of the product. The gelled starch/biological agent mixture is placed on a tray at room temperature for about 30 minutes. The resulting nonsticky matrix is then ground by a suitable means into nonagglomerative particles. These gel particles are coated with dry pearl corn starch powder, air-dried at room temperature, and then sieved into various mesh sizes to obtain the final product which contains the starch, biological control agent and some water.
While this bioencapsulation technique is more economical than other prior art methods, a close scrutiny of the technique reveals a number of deficiencies oncluding the following:
firstly, the gel material and the final product show insufficient stickiness which is required for the efficient delivery of biopesticide to targets;
secondly, the gel matrix contains the only carbon source, which makes it difficult for the encapsulated microbes to grow, multiply and produce bioactive compounds efficiently in the crop field;
thirdly, it is still somewhat expensive to use processed pregelatinized starch as the encapsulation material; and fourthly, non-sterilized materials are employed during the encapsulation process, which makes the final product liable to be contaminated by undesirable microbes.
Therefore, there still exists a demand for the development of a simple, inexpensive and practical bioencapsulation process, which is capable of delivering bioinsecticides to the control subjects or targets with high efficiency.
Summary of the Invention It is an object of the present invention to provide a simple and economical process for the bioencapsulation of various Bacillus thurinqiensis strains.
It is another object of the present invention to provide biodegradable and environmentally safe biopesticides prepared in accordance with the inventive process.
It is a further object of the present invention to provide novel Bacillus thuringiensis strains exhibiting high insecticidal activities.
Detailed Description of the Invention As used herein, the term "biocapsulation" refers to entrapping a biological control agent inside a biopolymeric gel matrix prepared from a biopolymeric material.
_:' .
In accordance with the present invention, there is provided a simple and economical process for the preparation of a bioencapsulated biopesticide containing a Bacillus thurinqiensis strain, comprising the steps of:
heating one or more carbohydrate-rich biopolymers and/or one or more protein-rich biopolymers in the presence of water at an elevated temperature to prepare a biopolymeric gel or paste;
heat-sterilizing the biopolymeric gel or paste and then cooling it to a lower temperature;
mixing the biopolymeric gel or paste uniformly with cells and/or spores of said Bacillus thuringiensis strain; and drying(and formulating the mixture of the biopolymeric gel and the Bacillus thuringiensis cells and/or spores into a desired type of the bioencapsulated biopesticide.
Specifically, said carbohydrate-rich biopolymers and/or protein-rich biopolymers are added with a suitable amount of water to have a solid concentration ranging from 5 to 40 %, preferably 10 to 30 % on the basis of total weight of the resulting mixture. The mixture is boiled at a temperature ranging from 90~C to 130~C, preferably 100~C, for 0.5 to 2 hours, and, if necessary, the boiled mixture is ground or macerated to make a biopolymeric gel. To control the gelation, density and viscosity of the biopolymeric gel, gelatin and/or agar may be added to the mixture at a concentration of 0.1 %-
PROCESS FOR THE MANUFACTURE THEREOF
Field of the Invention The present invention relates to a bioencapsulated biopesticide and a process for the preparation thereof; and, more particularly, it pertains to a biodegradable, environmentally safe biopesticide and a simple and economical process for the preparation thereof which comprises encapsulating Bacillus thuringiensis strains or their spores by using a biopolymer.
Background of the Invention In recent years, biopesticides have been in the limelight partly due to the stiffening regulations on the use of toxic chemicals as pesticides.
Biopesticides are attractive as an alternative or supplement to the existing chemical pesticides owing to their environmental safety and their specificity to target pathogens or insects.
Such biopesticides may include bioinsecticides, biofungicides, bioherbicides and plant growth regulators; and biological control agents commonly used for the preparation of biopesticides may include bacteria, yeasts, fungi, viruses and toxins such as B.T. toxin produced by Bacillus thurinqiensis.
Major problems associated with the biopesticides are high production cost and their instability or sensitivity to various environmental factors such as sunlight, desiccation, heat, ultraviolet light and rainwash when they are applied in the crop field, which limit the use of most biopesticides. For example, B.T. toxin produced by Bacillus thurinqiensis loses its insecticidal activity in a few days in the field under sunlight. Accordingly, the instability of B.T. toxin under the field conditions limits its utility despite its pesticidal effectiveness.
In this connection, there have been employed encapsulation technologies to protect chemical pesticides from environmental damages and provide controlled release of active ingredients(U.S. Patent No. 2,876,160). However, most of these encapsulation techniques developed for the chemical pesticides are n t suitable for biopesticides since microorganisms or biologically active agents contained in the biopesticides are much more sensitive than the chemical components. Therefore, needs for an effective technique for the stable encapsulation of biopesticide has continued to exist, which satisfy various requirements for its practical utility.
First of all, the materials to be used in carrying out the encapsulation should be natural biopolymers rather than synthetic polymers to provide an inexpensive and environmentally safe biopesticide.
Secondly, the biopolymer gel matrix prepared from the polymeric materials for the encapsulation of biological control agents should be able to protect the encapsulated biological control agents from various environmental damages such as sunlight, heat, W light and desiccation by supplying them with a protective layer and should have a high moisture holding capacity for their survival.
Thirdly, the biopolymer matrix should adhere to the crop and absorb moisture when it is applied in the field, which property is important for providing an even distribution of the biopesticide, efficient delivery to targets such as plant pathogens, harmful insects and weeds, and resistance against rainwash.
Fourthly, the bioencapsulating matrix layer should provide and maintain optimum physical and biological conditions for the activities of the biological control agents. To attain best results, when the bioencapsulated products are applied to the target, the microorganisms in the biomatrix layer should germinate, grow and proliferate efficiently to produce bioactive agents such as antibiotics, enzymes, toxins or other bioactive compounds which can serve as bioherbicide, bioinsecticide, biofungicide, nematocide, bactericide, molluscicide, acaricide, algicide, plant growth regulator, biofertilizer or fruit and vegetable preserving agents in postharvest storage.
Fifthly, the bioencapsulation technique should be cost effective to allow the biopesticides to compete against 211~267 chemical pesticides.
Various techniques for the encapsulation of enzymes and chemical pesticides in a synthetic polymer matrix have been studied extensively. However, none of these techniques have been satisfactory due to the lack of ability to fulfill the above requirements for biopesticides. For example, European Patent No. 0,320,483 discloses the use of chemical polymers such as polyvinylalcohol and polyvinyl pyrrolidone to encapsulate microorganisms such as Bacillus thuringiensis, Alternaria cassiae and Pseudomonas fluorescens. However, this 'echnique is not practical due to the high production cost and risk of environmental pollution which may be caused by the use of chemical polymers.
There have been also proposed bioencapsulation techniques which employ various biopolymer matrices to encapsulate biological control agents. For instance, H. Shigemitsu has disclosed in U.S. Pat. No. 4,647,537 a process for the bioencapsulation of microorganisms inhibiting plant pathogens in a carrageenan polymer matrix; however, this technique may have a limited utility due to the high cost of carrageenan.
U.S. Pat. No. 4,724,147(J.J. Marois) and U.S. Pat. No.
4,668,512(J.A. Lewis) have reported the use of alginate pellets to encapsulate fungi to be used as bioherbicide.
Again, the efficacy of this technique is rather limited due to the high price of alginate, low moisture holding capacity, and failure to provide sufficient adhesivity in case of a foliar application.
H. K. Kaya et al. have also disclosed a similar technique wherein calcium alginate is used to encapsulate nematodes (Environ. Entomol., 14, 572-574(1985)); however, this technique is not practical due to the high cost of calcium alginate.
Jung et al. have shown in French Patent No. 2,501,229 the inclusion of mycorrhizas in a biopolymer gel for viable storage, wherein the biopolymer gel is made of high molecular weight heteropolysaccharides produced by Xanthomonas. However, this technique is also handicapped by the high production cost of said biopolymer.
In European Patent No. 0,192,319, A.C. Barnes and S.G.
Cummings offer a novel bioencapsulation technique wherein B.T.
toxin molecules are immobilized inside of a whole cell of B.T.
toxin-producing Pseudomonas killed by the treatment with a chemical reagent. This product is called Cell-Cap*and shows an improved stability in the field due to the prevention of UV
damage under sunlight. However, this technique is also too expensive to be used commercially.
Among the bioencapsulation techniques so far disclosed, a most economical one is the stàrch encapsulation technique for entomopathogens disclosed in U.S. Patent No. 4,859,377 (B.S. Shasha and R.L. Dunkle). In this technique, a biogel is prepared by mixing pregelatinized corn starch powder, corn oil, cold water and biological control agents such as spores of Bacillus thuringiensis and B.T. toxin at a room temperature for 5 to 60 seconds. Reassociation of the amylose components ; *Trademark 211~267 in the pregelatinized starch results in a substantially homogeneous mass of starch matrix; and the biological agents are dispersed and entrapped uniformly throughout the continuous starch matrix. Their preferred starch concentration is about 25 to 40% for a granular form of the product. The gelled starch/biological agent mixture is placed on a tray at room temperature for about 30 minutes. The resulting nonsticky matrix is then ground by a suitable means into nonagglomerative particles. These gel particles are coated with dry pearl corn starch powder, air-dried at room temperature, and then sieved into various mesh sizes to obtain the final product which contains the starch, biological control agent and some water.
While this bioencapsulation technique is more economical than other prior art methods, a close scrutiny of the technique reveals a number of deficiencies oncluding the following:
firstly, the gel material and the final product show insufficient stickiness which is required for the efficient delivery of biopesticide to targets;
secondly, the gel matrix contains the only carbon source, which makes it difficult for the encapsulated microbes to grow, multiply and produce bioactive compounds efficiently in the crop field;
thirdly, it is still somewhat expensive to use processed pregelatinized starch as the encapsulation material; and fourthly, non-sterilized materials are employed during the encapsulation process, which makes the final product liable to be contaminated by undesirable microbes.
Therefore, there still exists a demand for the development of a simple, inexpensive and practical bioencapsulation process, which is capable of delivering bioinsecticides to the control subjects or targets with high efficiency.
Summary of the Invention It is an object of the present invention to provide a simple and economical process for the bioencapsulation of various Bacillus thurinqiensis strains.
It is another object of the present invention to provide biodegradable and environmentally safe biopesticides prepared in accordance with the inventive process.
It is a further object of the present invention to provide novel Bacillus thuringiensis strains exhibiting high insecticidal activities.
Detailed Description of the Invention As used herein, the term "biocapsulation" refers to entrapping a biological control agent inside a biopolymeric gel matrix prepared from a biopolymeric material.
_:' .
In accordance with the present invention, there is provided a simple and economical process for the preparation of a bioencapsulated biopesticide containing a Bacillus thurinqiensis strain, comprising the steps of:
heating one or more carbohydrate-rich biopolymers and/or one or more protein-rich biopolymers in the presence of water at an elevated temperature to prepare a biopolymeric gel or paste;
heat-sterilizing the biopolymeric gel or paste and then cooling it to a lower temperature;
mixing the biopolymeric gel or paste uniformly with cells and/or spores of said Bacillus thuringiensis strain; and drying(and formulating the mixture of the biopolymeric gel and the Bacillus thuringiensis cells and/or spores into a desired type of the bioencapsulated biopesticide.
Specifically, said carbohydrate-rich biopolymers and/or protein-rich biopolymers are added with a suitable amount of water to have a solid concentration ranging from 5 to 40 %, preferably 10 to 30 % on the basis of total weight of the resulting mixture. The mixture is boiled at a temperature ranging from 90~C to 130~C, preferably 100~C, for 0.5 to 2 hours, and, if necessary, the boiled mixture is ground or macerated to make a biopolymeric gel. To control the gelation, density and viscosity of the biopolymeric gel, gelatin and/or agar may be added to the mixture at a concentration of 0.1 %-
2.0 ~.
The resulting biopolymeric gel is autoclaved at e.g., 121~C for 30 to 60 minutes to remove contaminating microorganisms from the biopolymeric gel matrix. After cooling the biopolymeric gel matrix to a temperature ranging from room temperature to 50~C, a sufficient amount, e.g., 106 to 10 cells per lg of biopolyperic gel, of B. thurinqiensis cells, spores or B.T. toxins is mixed with the sterile biopolymeric ge~ matrix for the bioencapsulation of the biological control agent.
Carbohydrate or protein components are denatured by the heating process and are made to form a uniform biopolymeric gel matrix after cooling. Thus, said cells, spores or B.T.
toxins of Bacillus thuringiensis are uniformly dispersed and entrapped inside the biopolymeric gel matrix.
About 104 to 1013 cells, preferably 107 to 101~ cells per lg of the biopolymeric gel matrix may be employed to attain a good biopesticidal activity.
The above mixture of biopolymeric gel matrix and biological control agent is dried at a temperature from 20 to 50~C, preferably from 20 to 40~C, dependyng on the stability of the biological control agent, and formulated into the form of powder, particles or pellets of a desired size ranging from 20 to 400 meshes to prepare the bioencapsulated biopesticide.
Representative of the carbohydrate-rich biopolymers which may be employ d in the present invention include grains such as rice, wheat, barley, corn, Italian millet, Indian millet, Chinese millet and buck wheat; tubers such as potato; tuberous roots such as sweet potato and casaba; powders and by-products ~118267 of these products such as corn meal, wheat bran and potato peel; and biopolymers extracted from these products.
Exemplary protein-rich biopolymers of the invention include many different types of beans, nuts, peanuts, cotton seeds and oil seeds; powders and by-products of these products such as soy flour, soy proteins, peanut meal, oil seed meal and cotton seed meal; proteins extracted from these products;
and fish or animal protein extracts and milk proteins.
Said carbohydrate-rich biopolymeric gels or protein-rich biopolymeric gels can be used either alone or in combination for the bioencapsulation of desired biological control agent. However, a hybrid biopolymeric gel containing both of the carbohydrate-rich biopolymers and protein-rich biopolymers is more preferred.
This hybrid biopolymeric gel may be better suited for the bioencapsulation of many different types of microorganisms as this type of hybrid biopolymeric gel matrix can protect the biological control agent effectively from adverse environmental conditions and also supply both carbon and nitrogen sources necessary for the encapsulated microorganisms to grow optimally and produce txe bioactive compounds.
Exemplary strains of the Bacillus thuringiensis which may be used in the present invention include B. thuringiensis ATCC
10792, B. thuringiensis KCTC 0107BP, B. thurinqiensis KCTC
0108BP and B. thurinqiensis subsp. israelensis ATCC 35646.
Further, novel B. thuringiensis strains exhibiting high insecticidal activities which have been isolated from soil 211~2G7 samples and screened for their ability to kill the larvae of Lepidopteran insects by the present inventors also may be employed. Two novel strains showing high B.T. toxin production were designated as Bacillus thuringiensis FM-BT-285 and B.
thurinqiensis FM-BT-14, and deposited at Korean Collection for Type Cultures(KCTC) on April 11, 1994 with the accession numbers of KCTC 0107BP and KCTC 0108BP, respectively, under the terms of Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure.
The biological control agents to be encapsulated in the biopolymeric gel matrix can be prepared by culturing a strain of said B. thuringiensis in a liquid medium or a semi-solid medium, e.g., 2% soytone-0.5% starch. Upon the completion of the culture preparation, the cells or spores are separated by centrifugation or filtration. Separated cells or spores as well as culture broth containing the microbial cells can be also subjected directly to the bioencapsulation into a biopolymeric gel matrix. Further, bioencapsulated biopesticides comprising B.T. toxin may be prepared by mixing dried powder of B. thurinqiensis with biopolymeric gel matrix.
In addition to the active biological control agent, other additives, e.g., U.V protectant, moisture preservant and/or nutritional supplements, may be added into the biopolymeric gel matrix.
Further, sterile chitin powders, cellulose, wheat bran, clay or soil may be added to the biopolymeric gel before 211~2G 7 encapsulation to provide the biological control agent with enough surface to stick to, and to protect them from the damages from U.V or sunlight.
Because of the nutritional requirements of B.
thurinqiensis strains, various nutritional components may be supplemented into the biopolymeric gel matrix for producing an optimal microbial activity. For instance, various carbohydrate sources, e.g., starch, glucose, sucrose, dextrin and corn syrup; nitrogen sources, e.g., soybean meal, peptone, yeast extract, casamino acids and inorganic nitrogen sources; and trace elements of, e.g., iron, manganese, zinc and cobalt may be added into the biopolymeric gel matrix.
The bioencapsulated biopesticides may be applied directly to target insects and plants in an amount of 0.4 Kg/acre.
On the other hand, one of the very important aspects of the present invention lies in its ability to allow the biopesticide to have more than one biocidal activity. Through a proper combination of microorganisms having antifungal, insecticidal or herbicidal activities, biopesticides having a number of different pesticidal activities may be prepared. In addition, synergism ln the pesticidal activity may be achieved by coencapsulating a properly selected number of microorganisms together.
As can be seen from the above description, the present invention employs inexpensive biopolymers for the bioencapsulation of microorganisms and, therefore, can produce bioinsecticides with a low production cost, rendering it 21182~7 competitive against existing agrochemicals. Further, the biopolymers used in this invention are mostly edible, biodegradable and environmentally safe.
The following Examples are intended to further illustrate the present invention without limiting its scope.
Percentages given below for solids in solid mixtures, liquids in liquids and solids in liquids are on a wt/wt, vol/vol and wt/vol basis, respectively, unless specifically indicated otherwise.
Example 1: Isolation of B. thuringiensis strains Soil samples were collected from various crop fields in Korea by scraping off the surface material with a sterile spatula and then obtaining a small portion of the soil located 5 to 10 cm below the surface. These samples were stored in sterile plastic bags at 4~C.
Grain dust samples were collected from rice brans and fodder at various crop field in Korea. A commercial compost(Bio-Meca, Dae-Sung Nong-San Co., Korea) was also used as a sample for the isolation of B. thuringiensis.
To isolate B. thuringiensis from various samples, 0.5g of each of the samples was added into a 125ml quadruple-baffled flask charged with lOml of L broth(tryptone 2g, yeast extract 5g, NaCl 5g per liter) bufferred with 0.25M sodium acetate and then cultured at 30~C for 4 hours with shaking at 250 rpm by using a rotary shaker. After 4 hours, lml of the culture ~118267 solution was taken, heated at 85~C for 10 minutes, spread on L agar plate, and then incubated at 30~C for 24 hours. Upon the completion of incubation, all of the colonies showing the growth characteristics of B. thuringiensis were selected, transferred to T3 agar medium(tryptone 3g, tryptose 2g, yeast extract 1.5g, MnCl2 5mg, agar 15g, pH 6.8, H2O lOOOml) by streaking and then incubated at 30~C for 24 to 48 hours for sporulation.
The cultures so obtained were examined by phase contrast microscopy for the presence of spores and B.T. toxin crystals.
About 300 Bacillus thurinqiensis colonies were isolated.
Example 2: Bioassay for insecticidal activity of Bacillus thuringiensisstrainsagainst diamondbackmoth(DBM) Diamondback moth(DBM), i.e., Plutella xylostella L. which has been raised for several years in a laboratory without exposure to any insecticides was used as a test subject for determining insecticidal activity of Bacillus thuringiensis.
The laboratory condition was maintained at a temperature of 25+1~C, a photoperiod of 16L:8D and relative humidity of 50 to 60~. Leaves of Chinese cabbage, i.e., Brassica oleracea var.
capitata L., was provided as a DBM diet.
300 Bacillus thuringiensis colonies obtained in Example 1 were inoculated respectively in soytone-yeast extract-glucose medium(Bacto peptone 20g, Bacto yeast extract 2g, soluble starch 5g, glucose lOg, MgSO4 7H2O 0.5g, FeSO4 7H2O
0.02g, MnSO4 0.02, ZnSO4 7H2O 0.02g) and then cultured for 5 days at 28~C with shaking at 250 rpm by using a shaker.
The resulting culture broths with bacterial mass were used in a bioassay for B.T. toxin activity as described 5 hereinbelow.
A leaf-dipping method was employed to determine the larvicidal activity of candidate Bacillus thurinqiensis strains. The culture broth was diluted with Triton X-lO0(100 ppm) at each dilution rate of 1/50,000, 1/10,000, 1/5,000 and 1/1,000. Excised cabbage leaf disks(5cm in diameter) were dipped into the above solution for 30 seconds. After drying it more than 30 minutes, leaf disks were placed in the petri dishes and 10 individuals of 2nd instar DBM larvae were put into each petri dish. All petri dishes were covered and held 15 in an incubator at 25+1~C. Insect mortalities were investigated at 24, 48 and 72 hours after the treatment.
Out of 300 strains tested, only two strains showed 100%
mortality against Diamondback moth when the 50,000-fold diluted culture broth was used. All other strains showed 20 activity at the concentration of less than 10,000-fold dilutions.
The two strains showed 2 times higher B.T. toxin production than a commercial Thuricide-producing Bacillus thurinqiensis strain isolated from Thuricide(Sandoz Co., 25 Germany).
The above two strains showing high B.T. toxin production were designated as Bacillus thuringiensis FM-sT-285 and B.
*Trademark 21182~7 thuringiensis FM-BT-14, and deposited at Korean Collection for Type Cultures(KCTC) on April 11, 1994 with the accession numbers of KCTC 0107BP and KCTC 0108BP, respectively, under the terms of Budapest Treaty on the International Recognition of the Deposit of Microorganism for the purpose of Patent Procedure.
B. thuringiensis FM-BT-285(KCTC 0107BP) and B.
thurinqiensis FM-BT-14(KCTC 0108BP) are motile rods with dimensions of 1.3 to 1.4 X 3.7 to 4.1 ~m. Their crystal formation was confirmed by phase contrast microscopy in which the form of crystals was observed to be bipyramidal.
Further, the molecular weights of the crystals formed by B.
thuringiensis FM-BT-285(KCTC 0107BP) and FM-BT-14(KCTC 0108BP) were determined to be 135 and 62 kilodaltons(kDa) and 135 kDa, respectively, by using polyacrylamide gel electrophoresis.
B. thuringiensis FM-BT-285(KCTC 0107BP) and FM-BT-14(KCTC
0108BP) exhibited negative reactions in the utilization of sucrose and Voges-Proskauer reaction, respectively, contrary to other conventional B. thuringiensis strains which exhibits positive reactions in both of the reactions.
The biochemical properties of B. thuringiensis FM-BT-285(KCTC 0107BP) and FM-BT-14(KCTC 0108BP) are shown in Table 1.
211~ 2 ~ r~
Table 1. Biochemical cheracteristics of B. thurinqiensis KCTC 0107BP and KCTC 0108BP
Biochemical Responcses of isolates characteristics Gram stain + +
Anaerobic growth + +
Mortility + +
Methyl-red reaction + +
Nitrate reduction + +
Hemolysis + +
Voges-Proskauer reaction +
Lysozyme resistance + +
Productions of indole H2S _ _ ~-galactosidase catalase + +
phenylalanine deaminase tryptophan deaminase lysine decarboxylase arginine dihydrolase + +
ornithine decarboxylase oxidase + +
urease + +
gelatinase + +
Gas from glucose Utilization of adonitol arabinose casein + +
citrate dulcitol esculine + +
glucose + +
inositol lactose maltose + +
mannitol raffinose rhamnose salicine sorbitol starch + +
sucrose - +
xylose * Note: (+) Positive reaction (-) Negative reaction 2 G ~
Example 3: Bioencapsulation of Bacillus thuringiensis (Step 1) Culture of Bacillus thuringiensis Bacillus thuringiensis FM-BT-285(KCTC 0107BP) was cultured in soytone-yeast extract-glucose medium at 28~C for 5 days and the resulting culture was centrifuged to collect the cells and spores.
(Step 2) Preparation of biopesticide using protein or carbohydrate biopolymeric gel matrix To make a protein biopolymeric gel, soybean powder 200g, CaCO3 lg, gelatin 2g, yeast extract lg, FeSO4 7H2O 50mg, MnCl2-4H2O 20mg and soil 20g were added into lOOOml of H2O and the mixture was boiled at 100~C for 1 hour with stirring to make a uniform biopolymeric gel matrix. The biopolymeric gel matrix was sterilized by using an autoclave at 121~C for 30 minutes.
After cooling the gel matrix to room temperature, about 2 X 1012 B. thuringiensis spores obtained in (Step 1) were mixed with the above biopolymeric gel matrix and the B.
thurinqiensis spore-biopolymeric gel matrix complexes were spread thinly on a plate and dried at room temperature for 2 days.
Dried spore-matrix complex was ground by using a crusher to obtain powder having 20 to 300 mesh particles. The dried matrix contained bioencapsulated B.t cells about 5 X 109 cells/g.
On the other hand, a biopesticide comprising carbohydrate biogel matrix was prepared by reapeating the same procedures as described in the above except that 200g of rice powder was used in place of soybean powder. All other components and their amounts employed were the same as above.
(step 3) Preparation of biopesticide using hybrid biopolymeric gel The same procedures as described in (Step 2) above were repeated except that a hybrid biopolymeric gel matrix made from rice powder 100g, soybean powder 100g, glucose 2g, pharmamedia 10g, CaCO3 lg, yeast extract lg, FeSO4 7H2O 50mg, MnCl2 4H2O 10mg, soil 2g and 1000ml H2O was used in place of the protein biopolymeric gel to obtain the biopesticide powder.
Example 4: Field Evaluation of bioencapsulated biopesticides Insecticidal activity of bioencapsulated B. thurinqiensis cells or spores was compared with those of naked B.
thurinqiensis cells and commerciàl B. thurinqiensis pesticide, Thuricide(Sandoz Co., Germany), under the field conditions as follows.
During early July in Korea under humid and warm conditions(20-32~C, high relative humidity with frequent rains during rainy monsoon season), about 200g of bioencapsulated B.
thurinqiensis powder obtained in (Step 3) of Example 4 was 211~ 2 fj r~
applied to 0.25 acre of kale field infested with diamondback moth. Sg of naked B. thuringiensis cells and lOOg of Thuricide were also applied under the same condition as above as a control and comparative pesticide, respectively.
After 2, 7 and 15 days from the application, mortality of diamondback moth for each test pesticide was determined on the basis of the initial number of moth before the application and the results are shown in Table 2.
Table 2. Insecticidal Activity of Biopesticides Mortality of DBM larvae Pesticides after Application(%) 2 days 7 days 15 days Naked B. thurinqiensis cells 20 0 0 Thuricide 100 30 0 Biopesticide of the present invention comprising -Hybrid biogel 100 60 50 -protein biogel 100 50 40 -carbohydrate biogel 100 40 20 As shown in Table 2, bioencapsulated Bacillus thuringiensis of the present invention showed stronger insecticidal activity for a long time under field conditions than other tested pesticides.
While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made and also fall 2 ~ 7 within the scope of the invention as defined by the claims that follow.
The resulting biopolymeric gel is autoclaved at e.g., 121~C for 30 to 60 minutes to remove contaminating microorganisms from the biopolymeric gel matrix. After cooling the biopolymeric gel matrix to a temperature ranging from room temperature to 50~C, a sufficient amount, e.g., 106 to 10 cells per lg of biopolyperic gel, of B. thurinqiensis cells, spores or B.T. toxins is mixed with the sterile biopolymeric ge~ matrix for the bioencapsulation of the biological control agent.
Carbohydrate or protein components are denatured by the heating process and are made to form a uniform biopolymeric gel matrix after cooling. Thus, said cells, spores or B.T.
toxins of Bacillus thuringiensis are uniformly dispersed and entrapped inside the biopolymeric gel matrix.
About 104 to 1013 cells, preferably 107 to 101~ cells per lg of the biopolymeric gel matrix may be employed to attain a good biopesticidal activity.
The above mixture of biopolymeric gel matrix and biological control agent is dried at a temperature from 20 to 50~C, preferably from 20 to 40~C, dependyng on the stability of the biological control agent, and formulated into the form of powder, particles or pellets of a desired size ranging from 20 to 400 meshes to prepare the bioencapsulated biopesticide.
Representative of the carbohydrate-rich biopolymers which may be employ d in the present invention include grains such as rice, wheat, barley, corn, Italian millet, Indian millet, Chinese millet and buck wheat; tubers such as potato; tuberous roots such as sweet potato and casaba; powders and by-products ~118267 of these products such as corn meal, wheat bran and potato peel; and biopolymers extracted from these products.
Exemplary protein-rich biopolymers of the invention include many different types of beans, nuts, peanuts, cotton seeds and oil seeds; powders and by-products of these products such as soy flour, soy proteins, peanut meal, oil seed meal and cotton seed meal; proteins extracted from these products;
and fish or animal protein extracts and milk proteins.
Said carbohydrate-rich biopolymeric gels or protein-rich biopolymeric gels can be used either alone or in combination for the bioencapsulation of desired biological control agent. However, a hybrid biopolymeric gel containing both of the carbohydrate-rich biopolymers and protein-rich biopolymers is more preferred.
This hybrid biopolymeric gel may be better suited for the bioencapsulation of many different types of microorganisms as this type of hybrid biopolymeric gel matrix can protect the biological control agent effectively from adverse environmental conditions and also supply both carbon and nitrogen sources necessary for the encapsulated microorganisms to grow optimally and produce txe bioactive compounds.
Exemplary strains of the Bacillus thuringiensis which may be used in the present invention include B. thuringiensis ATCC
10792, B. thuringiensis KCTC 0107BP, B. thurinqiensis KCTC
0108BP and B. thurinqiensis subsp. israelensis ATCC 35646.
Further, novel B. thuringiensis strains exhibiting high insecticidal activities which have been isolated from soil 211~2G7 samples and screened for their ability to kill the larvae of Lepidopteran insects by the present inventors also may be employed. Two novel strains showing high B.T. toxin production were designated as Bacillus thuringiensis FM-BT-285 and B.
thurinqiensis FM-BT-14, and deposited at Korean Collection for Type Cultures(KCTC) on April 11, 1994 with the accession numbers of KCTC 0107BP and KCTC 0108BP, respectively, under the terms of Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure.
The biological control agents to be encapsulated in the biopolymeric gel matrix can be prepared by culturing a strain of said B. thuringiensis in a liquid medium or a semi-solid medium, e.g., 2% soytone-0.5% starch. Upon the completion of the culture preparation, the cells or spores are separated by centrifugation or filtration. Separated cells or spores as well as culture broth containing the microbial cells can be also subjected directly to the bioencapsulation into a biopolymeric gel matrix. Further, bioencapsulated biopesticides comprising B.T. toxin may be prepared by mixing dried powder of B. thurinqiensis with biopolymeric gel matrix.
In addition to the active biological control agent, other additives, e.g., U.V protectant, moisture preservant and/or nutritional supplements, may be added into the biopolymeric gel matrix.
Further, sterile chitin powders, cellulose, wheat bran, clay or soil may be added to the biopolymeric gel before 211~2G 7 encapsulation to provide the biological control agent with enough surface to stick to, and to protect them from the damages from U.V or sunlight.
Because of the nutritional requirements of B.
thurinqiensis strains, various nutritional components may be supplemented into the biopolymeric gel matrix for producing an optimal microbial activity. For instance, various carbohydrate sources, e.g., starch, glucose, sucrose, dextrin and corn syrup; nitrogen sources, e.g., soybean meal, peptone, yeast extract, casamino acids and inorganic nitrogen sources; and trace elements of, e.g., iron, manganese, zinc and cobalt may be added into the biopolymeric gel matrix.
The bioencapsulated biopesticides may be applied directly to target insects and plants in an amount of 0.4 Kg/acre.
On the other hand, one of the very important aspects of the present invention lies in its ability to allow the biopesticide to have more than one biocidal activity. Through a proper combination of microorganisms having antifungal, insecticidal or herbicidal activities, biopesticides having a number of different pesticidal activities may be prepared. In addition, synergism ln the pesticidal activity may be achieved by coencapsulating a properly selected number of microorganisms together.
As can be seen from the above description, the present invention employs inexpensive biopolymers for the bioencapsulation of microorganisms and, therefore, can produce bioinsecticides with a low production cost, rendering it 21182~7 competitive against existing agrochemicals. Further, the biopolymers used in this invention are mostly edible, biodegradable and environmentally safe.
The following Examples are intended to further illustrate the present invention without limiting its scope.
Percentages given below for solids in solid mixtures, liquids in liquids and solids in liquids are on a wt/wt, vol/vol and wt/vol basis, respectively, unless specifically indicated otherwise.
Example 1: Isolation of B. thuringiensis strains Soil samples were collected from various crop fields in Korea by scraping off the surface material with a sterile spatula and then obtaining a small portion of the soil located 5 to 10 cm below the surface. These samples were stored in sterile plastic bags at 4~C.
Grain dust samples were collected from rice brans and fodder at various crop field in Korea. A commercial compost(Bio-Meca, Dae-Sung Nong-San Co., Korea) was also used as a sample for the isolation of B. thuringiensis.
To isolate B. thuringiensis from various samples, 0.5g of each of the samples was added into a 125ml quadruple-baffled flask charged with lOml of L broth(tryptone 2g, yeast extract 5g, NaCl 5g per liter) bufferred with 0.25M sodium acetate and then cultured at 30~C for 4 hours with shaking at 250 rpm by using a rotary shaker. After 4 hours, lml of the culture ~118267 solution was taken, heated at 85~C for 10 minutes, spread on L agar plate, and then incubated at 30~C for 24 hours. Upon the completion of incubation, all of the colonies showing the growth characteristics of B. thuringiensis were selected, transferred to T3 agar medium(tryptone 3g, tryptose 2g, yeast extract 1.5g, MnCl2 5mg, agar 15g, pH 6.8, H2O lOOOml) by streaking and then incubated at 30~C for 24 to 48 hours for sporulation.
The cultures so obtained were examined by phase contrast microscopy for the presence of spores and B.T. toxin crystals.
About 300 Bacillus thurinqiensis colonies were isolated.
Example 2: Bioassay for insecticidal activity of Bacillus thuringiensisstrainsagainst diamondbackmoth(DBM) Diamondback moth(DBM), i.e., Plutella xylostella L. which has been raised for several years in a laboratory without exposure to any insecticides was used as a test subject for determining insecticidal activity of Bacillus thuringiensis.
The laboratory condition was maintained at a temperature of 25+1~C, a photoperiod of 16L:8D and relative humidity of 50 to 60~. Leaves of Chinese cabbage, i.e., Brassica oleracea var.
capitata L., was provided as a DBM diet.
300 Bacillus thuringiensis colonies obtained in Example 1 were inoculated respectively in soytone-yeast extract-glucose medium(Bacto peptone 20g, Bacto yeast extract 2g, soluble starch 5g, glucose lOg, MgSO4 7H2O 0.5g, FeSO4 7H2O
0.02g, MnSO4 0.02, ZnSO4 7H2O 0.02g) and then cultured for 5 days at 28~C with shaking at 250 rpm by using a shaker.
The resulting culture broths with bacterial mass were used in a bioassay for B.T. toxin activity as described 5 hereinbelow.
A leaf-dipping method was employed to determine the larvicidal activity of candidate Bacillus thurinqiensis strains. The culture broth was diluted with Triton X-lO0(100 ppm) at each dilution rate of 1/50,000, 1/10,000, 1/5,000 and 1/1,000. Excised cabbage leaf disks(5cm in diameter) were dipped into the above solution for 30 seconds. After drying it more than 30 minutes, leaf disks were placed in the petri dishes and 10 individuals of 2nd instar DBM larvae were put into each petri dish. All petri dishes were covered and held 15 in an incubator at 25+1~C. Insect mortalities were investigated at 24, 48 and 72 hours after the treatment.
Out of 300 strains tested, only two strains showed 100%
mortality against Diamondback moth when the 50,000-fold diluted culture broth was used. All other strains showed 20 activity at the concentration of less than 10,000-fold dilutions.
The two strains showed 2 times higher B.T. toxin production than a commercial Thuricide-producing Bacillus thurinqiensis strain isolated from Thuricide(Sandoz Co., 25 Germany).
The above two strains showing high B.T. toxin production were designated as Bacillus thuringiensis FM-sT-285 and B.
*Trademark 21182~7 thuringiensis FM-BT-14, and deposited at Korean Collection for Type Cultures(KCTC) on April 11, 1994 with the accession numbers of KCTC 0107BP and KCTC 0108BP, respectively, under the terms of Budapest Treaty on the International Recognition of the Deposit of Microorganism for the purpose of Patent Procedure.
B. thuringiensis FM-BT-285(KCTC 0107BP) and B.
thurinqiensis FM-BT-14(KCTC 0108BP) are motile rods with dimensions of 1.3 to 1.4 X 3.7 to 4.1 ~m. Their crystal formation was confirmed by phase contrast microscopy in which the form of crystals was observed to be bipyramidal.
Further, the molecular weights of the crystals formed by B.
thuringiensis FM-BT-285(KCTC 0107BP) and FM-BT-14(KCTC 0108BP) were determined to be 135 and 62 kilodaltons(kDa) and 135 kDa, respectively, by using polyacrylamide gel electrophoresis.
B. thuringiensis FM-BT-285(KCTC 0107BP) and FM-BT-14(KCTC
0108BP) exhibited negative reactions in the utilization of sucrose and Voges-Proskauer reaction, respectively, contrary to other conventional B. thuringiensis strains which exhibits positive reactions in both of the reactions.
The biochemical properties of B. thuringiensis FM-BT-285(KCTC 0107BP) and FM-BT-14(KCTC 0108BP) are shown in Table 1.
211~ 2 ~ r~
Table 1. Biochemical cheracteristics of B. thurinqiensis KCTC 0107BP and KCTC 0108BP
Biochemical Responcses of isolates characteristics Gram stain + +
Anaerobic growth + +
Mortility + +
Methyl-red reaction + +
Nitrate reduction + +
Hemolysis + +
Voges-Proskauer reaction +
Lysozyme resistance + +
Productions of indole H2S _ _ ~-galactosidase catalase + +
phenylalanine deaminase tryptophan deaminase lysine decarboxylase arginine dihydrolase + +
ornithine decarboxylase oxidase + +
urease + +
gelatinase + +
Gas from glucose Utilization of adonitol arabinose casein + +
citrate dulcitol esculine + +
glucose + +
inositol lactose maltose + +
mannitol raffinose rhamnose salicine sorbitol starch + +
sucrose - +
xylose * Note: (+) Positive reaction (-) Negative reaction 2 G ~
Example 3: Bioencapsulation of Bacillus thuringiensis (Step 1) Culture of Bacillus thuringiensis Bacillus thuringiensis FM-BT-285(KCTC 0107BP) was cultured in soytone-yeast extract-glucose medium at 28~C for 5 days and the resulting culture was centrifuged to collect the cells and spores.
(Step 2) Preparation of biopesticide using protein or carbohydrate biopolymeric gel matrix To make a protein biopolymeric gel, soybean powder 200g, CaCO3 lg, gelatin 2g, yeast extract lg, FeSO4 7H2O 50mg, MnCl2-4H2O 20mg and soil 20g were added into lOOOml of H2O and the mixture was boiled at 100~C for 1 hour with stirring to make a uniform biopolymeric gel matrix. The biopolymeric gel matrix was sterilized by using an autoclave at 121~C for 30 minutes.
After cooling the gel matrix to room temperature, about 2 X 1012 B. thuringiensis spores obtained in (Step 1) were mixed with the above biopolymeric gel matrix and the B.
thurinqiensis spore-biopolymeric gel matrix complexes were spread thinly on a plate and dried at room temperature for 2 days.
Dried spore-matrix complex was ground by using a crusher to obtain powder having 20 to 300 mesh particles. The dried matrix contained bioencapsulated B.t cells about 5 X 109 cells/g.
On the other hand, a biopesticide comprising carbohydrate biogel matrix was prepared by reapeating the same procedures as described in the above except that 200g of rice powder was used in place of soybean powder. All other components and their amounts employed were the same as above.
(step 3) Preparation of biopesticide using hybrid biopolymeric gel The same procedures as described in (Step 2) above were repeated except that a hybrid biopolymeric gel matrix made from rice powder 100g, soybean powder 100g, glucose 2g, pharmamedia 10g, CaCO3 lg, yeast extract lg, FeSO4 7H2O 50mg, MnCl2 4H2O 10mg, soil 2g and 1000ml H2O was used in place of the protein biopolymeric gel to obtain the biopesticide powder.
Example 4: Field Evaluation of bioencapsulated biopesticides Insecticidal activity of bioencapsulated B. thurinqiensis cells or spores was compared with those of naked B.
thurinqiensis cells and commerciàl B. thurinqiensis pesticide, Thuricide(Sandoz Co., Germany), under the field conditions as follows.
During early July in Korea under humid and warm conditions(20-32~C, high relative humidity with frequent rains during rainy monsoon season), about 200g of bioencapsulated B.
thurinqiensis powder obtained in (Step 3) of Example 4 was 211~ 2 fj r~
applied to 0.25 acre of kale field infested with diamondback moth. Sg of naked B. thuringiensis cells and lOOg of Thuricide were also applied under the same condition as above as a control and comparative pesticide, respectively.
After 2, 7 and 15 days from the application, mortality of diamondback moth for each test pesticide was determined on the basis of the initial number of moth before the application and the results are shown in Table 2.
Table 2. Insecticidal Activity of Biopesticides Mortality of DBM larvae Pesticides after Application(%) 2 days 7 days 15 days Naked B. thurinqiensis cells 20 0 0 Thuricide 100 30 0 Biopesticide of the present invention comprising -Hybrid biogel 100 60 50 -protein biogel 100 50 40 -carbohydrate biogel 100 40 20 As shown in Table 2, bioencapsulated Bacillus thuringiensis of the present invention showed stronger insecticidal activity for a long time under field conditions than other tested pesticides.
While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made and also fall 2 ~ 7 within the scope of the invention as defined by the claims that follow.
Claims (9)
1. A process for the preparation of a bioencapsulated biopesticide containing a strain of Bacillus thuringiensis, comprising the steps of:
(a) heating one or more carbohydrate-rich biopolymers and/or one or more protein-rich biopolymers in the presence of water at an elevated temperature to prepare a biopolymeric gel or paste;
(b) sterilizing and then cooling the biopolymeric gel or paste to a lower temperature;
(c) mixing the biopolymeric gel or paste uniformly with cells and/or spores of the strain of Bacillus thurinqiensis; and, thereafter, (d) drying and formulating the mixture of the biopolymeric gel or paste and the Bacillus thuringiensis cells and/or spores into a desired form of the bioencapsulated biopesticide.
(a) heating one or more carbohydrate-rich biopolymers and/or one or more protein-rich biopolymers in the presence of water at an elevated temperature to prepare a biopolymeric gel or paste;
(b) sterilizing and then cooling the biopolymeric gel or paste to a lower temperature;
(c) mixing the biopolymeric gel or paste uniformly with cells and/or spores of the strain of Bacillus thurinqiensis; and, thereafter, (d) drying and formulating the mixture of the biopolymeric gel or paste and the Bacillus thuringiensis cells and/or spores into a desired form of the bioencapsulated biopesticide.
2. The process of claim 1, wherein said carbohydrate-rich biopolymer is selected from the group consisting of rice, wheat, barley, corn, Italian millet, Indian millet, Chinese millet, buck wheat, potato, sweet potato, casaba, corn meal, wheat bran, potato peel, and biopolymers extracted from these products.
3. The process of claim 1, wherein said protein-rich biopolymer is selected from the group consisting of beans, nuts, cotton seeds and oil seeds, proteins extracted therefrom, milk, fish and animal protein extracts.
4. The process of claim 1, wherein the solid content in the biopolymeric gel or paste in step (a) ranges from 5 to 40 % on the basis of the total weight of the gel or paste.
5. The process of claim 1, wherein said heating in step (a) is carried out at a temperature ranging from 90 to 130°C.
6. The process of claim 1, wherein the cells and/or spores of Bacillus thuringiensis are added in step (c) in an amount ranging from 104 to 1013 cells and/or spores per lg of the gel or paste.
7. The process of claim 1, wherein said strain of Bacillus thuringiensis is selected from the group consisting of B. thuringiensis ATCC 10792, B. thuringiensis FM-BT-285(KCTC 0107BP), B. thuringiensis FM-BT-14(KCTC 0108BP) and B. thuringiensis subsp. israelensis ATCC 35646.
8. A novel strain of Bacillus thuringiensis FM-BT-285(KCTC 0107BP) or Bacillus thuringiensis FM-BT-14(KCTC
0108BP).
0108BP).
9. A bioencapsulated Bacillus thuringiensis prepared in accordance with the process recited in claims 1, 2, 3, 4, 5, 6, or 7.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR93-22037 | 1993-10-22 | ||
| KR1019930022037A KR970007083B1 (en) | 1993-10-22 | 1993-10-22 | Method for manufacturing of biocides using bioencapsulation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2118267A1 CA2118267A1 (en) | 1995-04-23 |
| CA2118267C true CA2118267C (en) | 1997-12-30 |
Family
ID=19366376
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002118267A Expired - Fee Related CA2118267C (en) | 1993-10-22 | 1994-10-17 | Bioencapsulated biopesticides and process for the manufacture thereof |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR970007083B1 (en) |
| CA (1) | CA2118267C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109497050A (en) * | 2018-11-28 | 2019-03-22 | 浙江大学 | A kind of Paenibacillus polymyxa fermentation material and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19990041258A (en) * | 1997-11-21 | 1999-06-15 | 구광시 | Islands-in-the-sea hollow fiber |
| KR20010045115A (en) * | 1999-11-03 | 2001-06-05 | 구광시 | A thermoplastic ultra fine fiber |
| KR100470232B1 (en) * | 2002-06-04 | 2005-02-05 | 서형원 | Delivery medium for a microorganism and process for producing a biopesticide containing the same |
-
1993
- 1993-10-22 KR KR1019930022037A patent/KR970007083B1/en not_active IP Right Cessation
-
1994
- 1994-10-17 CA CA002118267A patent/CA2118267C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109497050A (en) * | 2018-11-28 | 2019-03-22 | 浙江大学 | A kind of Paenibacillus polymyxa fermentation material and its preparation method and application |
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| Publication number | Publication date |
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| KR950010747A (en) | 1995-05-15 |
| KR970007083B1 (en) | 1997-05-02 |
| CA2118267A1 (en) | 1995-04-23 |
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