Note: Descriptions are shown in the official language in which they were submitted.
21182G7
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
21182G7
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.
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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
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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.
21 18267
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
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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
21 1 8267
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.
_:'
.
21182G7
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.,
2118267
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
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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
- 14 -
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
21 1 8267
- 15 -
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
- 16 -
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
KCTC 0107BP KCTC 0108BP
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 ~
- 18 -
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.
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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~
- 20 -
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
- 21 -
within the scope of the invention as defined by the claims
that follow.
