U.S. patent application number 09/923586 was filed with the patent office on 2002-03-28 for process of its application against lepidopteran insects using albizzia lebbeck plant extract and bacilus thuriengiensis delta-endotoxin.
This patent application is currently assigned to Council of Scientific and Industrial Research, India corporation. Invention is credited to Arya, Jai Shankar, Darokar, Mahendra Pandurang, Khanuja, Suman Preet Singh, Kumar, Sushil, Kumar, Tiruppadiripuliyur Ranganathan Santha, Satapathy, Sarita, Shasany, Ajit Kumar, Singh, Subhash Chandra, Tripathy, Arun Kumar.
Application Number | 20020037330 09/923586 |
Document ID | / |
Family ID | 24159234 |
Filed Date | 2002-03-28 |
United States Patent
Application |
20020037330 |
Kind Code |
A1 |
Khanuja, Suman Preet Singh ;
et al. |
March 28, 2002 |
Process of its application against lepidopteran insects using
Albizzia lebbeck plant extract and Bacilus thuriengiensis
delta-endotoxin
Abstract
The present invention provides a novel synergistic composition
comprising extract obtained from the plant Albizzia lebbeck
together with Bacillus thuringiensis .delta.-endotoxin, useful in
controlling lepidopteran insects, methods for the preparation of
the composition and application of the insecticidal composition to
standing crops.
Inventors: |
Khanuja, Suman Preet Singh;
(Lucknow, IN) ; Satapathy, Sarita; (Lucknow,
IN) ; Singh, Subhash Chandra; (Lucknow, IN) ;
Kumar, Tiruppadiripuliyur Ranganathan Santha; (Lucknow,
IN) ; Arya, Jai Shankar; (Lucknow, IN) ;
Tripathy, Arun Kumar; (Lucknow, IN) ; Shasany, Ajit
Kumar; (Lucknow, IN) ; Darokar, Mahendra
Pandurang; (Lucknow, IN) ; Kumar, Sushil;
(Lucknow, IN) |
Correspondence
Address: |
JOHN W. FREEMAN, ESQ.
Fish & Richardson P.C.
225 Franklin Street
Boston
MA
02110-2804
US
|
Assignee: |
Council of Scientific and
Industrial Research, India corporation
|
Family ID: |
24159234 |
Appl. No.: |
09/923586 |
Filed: |
August 7, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09923586 |
Aug 7, 2001 |
|
|
|
09541357 |
Mar 31, 2000 |
|
|
|
Current U.S.
Class: |
424/725 ;
424/93.461 |
Current CPC
Class: |
A01N 65/20 20130101;
A01N 65/00 20130101; A01N 2300/00 20130101; A01N 65/20 20130101;
A01N 63/23 20200101; A01N 63/50 20200101 |
Class at
Publication: |
424/725 ;
424/93.461 |
International
Class: |
A01N 063/00; A01N
065/00 |
Claims
1. An insecticidal composition comprising alcoholic extract
obtained from the plant Albizzia lebbeck together with Bacillus
thuringiensis .delta.-endotoxin, useful in controlling insects of
the class lepidoptara.
2. A composition as claimed in claim 1 wherein the composition is
useful in controlling insects selected from the class lepidoptara
comprising Spilarctia obliqua, Spodoptera litura and Heliothis
armigera.
3. A composition as claimed in claim 1 wherein the concentration of
the plant extract in the composition is about 2.5 mg/ml alcohol for
killing insects.
4. A composition as claimed in claim 1 wherein the alcoholic
extract is used in combination with Bacillus thuringiensis
.delta.-endotoxin at a concentration of about LC 5 or more of both
the constituents.
5. A composition as claimed in claim 1 wherein .delta.-endotoxin
from Bacillus thuringiensis is used as acetone powder.
6. A process for the preparation of an insecticidal composition as
claimed in claim 1, said process comprising the steps of
collecting, drying, pulverizing part of the plant Albizzia lebbeck,
treating the pulverized plant parts with alcohol, filtering and
evaporating the alcohol, drying in a freeze drier, and dispersing
the alcoholic extract in Bacillus thuringiensis .delta.-endotoxin
acetone powder.
7. A process as claimed in claim 5 wherein the plant parts used for
preparation of extract are selected from stem, leaves or seeds.
8. A process as claimed in claim 5 wherein the alcohol is selected
from Ethanol, Methanol and Isopropanol.
9. A process as claimed in claim 5 wherein the pulverized plant
parts are kept in about 100 ml to 5 litre alcohol per 100 gram of
plant material overnight for 7 days at room temperature.
10. A process as claimed in claim 5 wherein the alcohol is filtered
out using Whatman no.1 filter paper and evaporated naturally or in
a rotavapour at 40-60.degree. C.
11. A process as claimed in claim 5 wherein the extract is dried in
a freeze drier at -110.degree. C.
12. A process as claimed in claim 5 wherein the insecticidal
composition is capable of controlling in insects selected from the
class Lepidoptera comprising Spilarctia obliqua, Spodoptera litura
and Heliothis armigera.
13. A process as claimed in claim 5 wherein the concentration of
the plant extract is about 2.5 mg/ml and the concentration of
.delta.-endotoxin is about LC 5 or more of both the
constituents.
14. A method of application of the insecticidal composition as
claimed in claim 1 said method comprising the step of spraying the
insecticidal composition on standing crops.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a novel insecticidal
composition comprising extract(s) obtained from the plant Albizzia
lebbeck and .delta.-endotoxin from Bacillus thuringiensis, useful
in effectively controlling the lepidopteran crop damages insects.
The invention also provides a process for the preparation of the
said composition and a method for the application of the
composition.
BACKGROUND OF THE INVENTION
[0002] Insecticides have long been used against harmful insects,
pests in plants. Insects of the order `Lepidoptera` particularly
cause maximum damage to the field crops, drastically reducing the
economic yield of cultivated plants. Globally, the estimates put
crop loss due to insect attack somewhere between 18 to 30%. The
pesticides and repellents available in the prior art, however,
suffer from various deficiencies. Often, compositions provided are
insect repellents and not insecticidal. Furthermore, many
compositions are either toxic or generally unpleasant to the human
beings and animals. Still others require complicated process to
provide active ingredients for efficient protection.
[0003] Many synthetic prior art compositions have been proposed as
insect repellents, but have later been determined to be unsuitable
for safe use by humans, as these are not selective and cause grave
concern by damaging the environment. Moreover, the use of
repellents is not feasible practically for agricultural use.
[0004] Various crude extracts and essential oils from plants, such
as Neem extract and compounds from Azadirachta indica, citronella
oil obtained from Cymbopogon species, or eucalyptus oil obtained
from Eucalyptus species etc., have been reported to be useful in
insect pest management but mostly as pest repellents. These also
suffer from limited activity, unpleasant odor, inconsistency and
unreliable composition. Most of the art for plant based insecticide
formulation deals with complex compositions with several
ingredients required in heavy dosages. The need of the hour is
effective insecticidal composition, which is easier to consistently
produce and monitor. In the art there are many examples of
production and application of different preparations from Bacillus
thuringiensis .delta.-endotoxin for plant protection. but the
apprehension is of the resistance development in the insect
population due to continuous monotonous exposure of this toxin to
the insects. Therefore, the applicants studied the combination of
microbial pesticides with botanical insecticidal compounds and
found that the formulation consisting of diverse compounds with
novel and different modes of action is capable of reducing the risk
and probability of simultaneous resistance development.
[0005] Accordingly, the applicants have developed a plant based
insecticidal composition which when combined with other biological
insecticide(s) including Bacillus thuringiensis .delta.-endotoxin
restricts resistance development against the endotoxin. Further,
the composition is environmentally safe and economically effective
with significantly lower dosage.
OBJECTS OF THE INVENTION
[0006] The main object of the invention is to provide a novel
insecticidal composition comprising alcoholic extract obtained from
the plant Albizzia lebbeck together with Bacillus thuringiensis
.delta.-endotoxin.
[0007] Yet another object of the invention is to provide an
insecticidal combination that is effective at very low dosage
against plant pests, such as lepidopteran insects.
[0008] Another object of the invention is to provide a composition
that may be used for these insects as spray.
[0009] Still another object is to provide a composition that
exhibits synergistic properties and is capable of enhancing the
effect of the endotoxin and killing the resistance developed by
insects due to sole application of .delta.-endotoxin.
[0010] Another object is to provide process for the preparation of
the novel insecticidal composition of the invention.
[0011] Yet another object of the invention is to provide a method
of application of the composition in terms of sequence of repeat
treatments effective in controlling insect population below the
threshold level of economic damage.
SUMMARY OF THE INVENTION
[0012] The present invention provides a novel synergistic
composition comprising alcoholic extract obtained from the plant
Albizzia lebbeck together with Bacillus thuringiensis
.delta.-endotoxin acetone powder. The compositions can be sprayed
on the infested standing crops. The said composition exhibits
potency at very low dosage against lepidopteran insects. The
invention also describes a method for the preparation and
application of the insecticidal composition.
DETAILED DESCRIPTION OF THE INVENTION
[0013] The composition and the method of application as described
in the present invention is intended to be used against all plant
pests, which succumb to the lethal properties of the two biocontrol
agents and their synergistic combination thereof.
[0014] The invention provides novel insecticidal composition
comprising alcoholic extract obtained from the plant Albizzia
lebbeck together with Bacillus thuringiensis .delta.-endotoxin
useful in controlling insects of the class lepidoptara.
[0015] In an embodiment the composition is useful in controlling
insects selected from the class lepidoptara comprising Spilarctia
obliqua, Spodoptera litura and Heliothis armigera.
[0016] Yet another embodiment the concentration of the plant
extract in the composition is about 2.5 mg/ml alcohol for killing
insects.
[0017] In another embodiment the .delta.-endotoxin obtained from
Bacillus thirungiensis is obtained as acetone powder.
[0018] In still another embodiment the alcoholic extract is used in
combination with Bacillus thuringiensis .delta.-endotoxin at a
concentration of about LC 5 or more of both the constituents.
[0019] One more embodiment relates to a process for the preparation
of the insecticidal composition, said process comprising the steps
of collecting, drying, pulverizing part of the plant Albizzia
lebbeck, treating the pulverized plant parts with alcohol,
filtering and evaporating the alcohol, drying in a freeze drier,
and dispersing the alcoholic extract in Bacillus thuringiensis
.delta.-endotoxin acetone powder.
[0020] In an embodiment the plant parts used for preparation of
extract are selected from stem, leaves or seeds.
[0021] Yet another embodiment the alcohol is selected from Ethanol,
Methanol and Isopropanol.
[0022] Still another embodiment the pulverized plant parts are kept
in about 100 ml to 5 litre alcohol per 100 gram of plant material
overnight for 7 days at room temperature.
[0023] In an embodiment the alcohol is filtered out using Whatman
no.1 filter paper and evaporated naturally or in a rotavapour at
40-60.degree. C.
[0024] Yet another embodiment the extract is dried in a freeze
drier at -110.degree. C.
[0025] Still another embodiment the insecticidal composition is
capable of controlling in insects selected from the class
Lepidoptera comprising Spilarctia obliqua, Spodoptera litura and
Heliothis armigera.
[0026] In an embodiment the concentration of the plant extract is
about 2.5 mg/ml and the concentration of .delta.-endotoxin is about
LC 5 or more of both the constituents.
[0027] One more embodiment a method of application of the
insecticidal composition said method comprising the step of
spraying the insecticidal composition on standing crops.
[0028] The Present Invention in its Disclosure Provides the Process
for the Preparation of the Extract from the Plant
[0029] The processing of plant parts to obtain usable extract as
biocontrol agent can be achieved in the following sequence of
steps:
[0030] Step 1: The plant material (leaf stem and seed) are
collected/harvested from the plant preferably in the month of
[0031] Step 2: The material is shade dried carefully by regular
respreading in a clean environment such that no microbial
infestation occur.
[0032] Step 3: Percolation: An alcohol, preferably ethanol
sufficient to dip the plant material (1.0 to 5.0 liter per kilogram
of plant material) 1 is added to the plant material in a percolator
and kept overnight to 7 days at room temperature.
[0033] Step 4: Alcohol part is filtered out by Whatman no.1 filter
paper and evaporated naturally or in a rotavapour at 40-60.degree.
C. The leftover plant material is reprocessed with fresh alcohol in
similar fashion.
[0034] Step 5: Then these extracts are pooled.
[0035] Step 6: Further drying of the pooled extract is performed in
a freeze drier at -100.degree. C. (optional).
[0036] Step 7: The final extract thus obtained can be powdered and
used for spraying after dissolving in suitable solvent or can be
combined with any other insecticidal preparations like Bacillus
thuringiensis .delta.-endotoxin.
[0037] Insecticidal Bioassay
[0038] These preparations were dissolved in ethanol or water and
coated on castor (Ricinus communis) leaf in a series of
concentrations mentioned in Table 1. In case of ethanol the coated
leaf was dried at ambient temperature such that the leaf did not
lose its freshness and fed to 6 to 10 days old larvae of Spilarctia
obliqua in petriplates. The experiments were conducted in
replicated trial by taking 30 larvae per treatment with 5
replications. The treated plant materials were changed every second
day. The larvae were observed for growth and feeding behavior
everyday and % mortality was noted on 9.sup.th day of the
experiment.
1TABLE 1 Percentage larval mortality (after 9 days) upon feeding
different concentrations of the extract*. Control coated Extract No
with Concentration of the extract in .mu.l ethanol from coating
solvent 1.0 0.5 0.25 0.125 0.0625 0.0312 0.0165 0.0082 Stem 0 0 100
100 100 100 100 100 50 25 Leaf 0 0 100 100 100 100 50 20 10 5 Seed
0 0 100 100 100 100 100 50 25 10 *400 .mu.l of given concentration
of treatment was coated on leaf of 12 square centimeter.
[0039] From the above table it was observed that the extract from
stem was more potent as larvicidal because it kills 50% of the
insects at a concentration of 0.0165 .mu.g/.mu.l compared to the
extract from leaf (0.0625 .mu.g/.mu.l) and seed (0.0312
.mu.g/.mu.l). LC50 was estimated as the concentration at which 50%
mortality of the larvae was observed and similarly the other lethal
doses were determined.
[0040] Lifecycle Disruption Assays
[0041] In the next step we studied the effect of the extract on
pupation, adult emergence and hatching of eggs upon continuously
feeding the larvae on non-lethal or sub-lethal doses of the
extracts. The results obtained were interesting and as described in
Table 2. In this experiment 0.0312 .mu.g/.mu.l concentration of the
extracts was found effective.
2TABLE 2 Lifecycle disruption in Spilarctia obliqua by the extract
of Albizzia lebbeck at sub-lethal concentration (0.0312
.mu.g/.mu.l) Extract Extract Extract from from from Control Control
Activity stem leaf seed solvent water Larvicidal 100 100 100 0 0
Pupicidal 80 80 70 0 0 Adult Eggs Eggs Eggs Eggs Eggs did not
hatched did not hatched hatched hatch hatch
[0042] So it was evident from the above experiment that the extract
front stem was more potent than leaf and seed and disrupts the
lifecycle of the insect at every stages of the lifecycle.
[0043] Defining LC Values for Bt Acetone Powder
[0044] In the next step we carried out feeding the larvae with
different concentrations of Bacillus thuringiensis
.delta.-endotoxin protein preparation in form of acetone powder
which was prepared in the following manner. Bacillus thuringiensis
cells were grown in MGM broth for 62 h. the pH of the culture was
brought down to 7.0 using 1 N HCl. Cells were pelleted by
centrifugation at 8000rpm for 20 min. Cell pellet was suspended in
6% lactose (0.1-0.2 volume) by stirring for 30 min. on magnetic
stirrer and acetone (4 volumes) was added slowly while stirring
which was continued for another 30 min. Suspension was allowed to
stand for 10 min. and th4en filtered through filter paper (Whatman
no. 1) under suction. Residue was resuspended in 25 ml of acetone
and stirred for 30 min. This process was repeated three times.
Finally residue was dried overnight in a vacuum desicator at
25.degree. C. This residue (also called acetone powder) contained
the crude ednotoxin. Mortality observed for the larvae on 9.sup.th
day of treatment is given in Table 3.
3TABLE 3 Percent larval mortality on 9.sup.th day in Spilarctia
obliqua against Bt acetone powder. Concentra- tions of Acteone 400
.mu.g/ 200 .mu.g/ 100 .mu.g/ 40 .mu.g/ 25 .mu.g/ powder Control 400
.mu.l 400 .mu.l 400 .mu.l 400 .mu.l 400 .mu.l Percent Nil 70 65 45
20 10 mortality
[0045] From this experiment the LC10 and LC50 values were
calculated as 25 .mu.g/400 .mu.l and 125 .mu.g/400 .mu.l
respectively by plotting a standard curve of Acetone Powder Vs.
Mortality.
[0046] Development of Synergistic Combination
[0047] To check whether the plant extract has an enhancing effect
on the .delta.-endotoxin we fed the larvae with different
concentrations of the stem extract and the endotoxin (Table 4).
4TABLE 4 Activity of the extract and .delta.-endotoxin in
combination. LC5 LC LC25 LC LC B - 12.5 B B - Control 10 10 LC5
LC25 LC25 - LC LC50 LC50 LC25 coated %Effect 42 E E B E 12.5 E B E
E Control solvent Larvicidal 10 10 10 26 25 62 45--50 45--31 66-75
0 0 Pupicidal 20 20 50 52 50 60 55 48 70 0 0 Hatching 0 33 0 0 0 0
0 0 0 100 100
[0048] As described in the table the pupicidal activity increases
significantly to 20%. in the combination at a dose of LC 5 each, in
comparison to, when applied separately at a concentration of LC 10.
Similarly for the larvicidal and pupicidal activity at a
concentration of LC 12.5 and LC 25 in the combinations, is much
higher than LC 25 and LC 50 when taken individually. So the
conclusion was that, the plant extract and Bacillus thuringiensis
.delta.-endotoxin have synergistic effect enhancing the
effectiveness of one another. Even the larvae escape mortality
after feeding on the combination, the adult may not emerge from the
pupae or the eggs will not hatch. This combination is
environmentally safe as tested earlier for .delta.-endotoxin. The
plant also produces many compounds, which are used for medication
for human.
[0049] We started our study keeping in view the Lepidopteran insect
Spilarctia obliqua which cause wide spread damage to Mentha
species. This is an insect of first magnitude with world wide in
distribution. It is polyphagous and major pest of several crops
like radish, soybean, groundnut, blackgram, bengalgram, cowpea,
sunflower, cabbage, rye, jute, mint, turmeric, cotton, in India.
But after observing the effect we were encouraged to test the
effect on other lepidopteran plant insects like Spodoptera litura
and Heliothis armigera with similar results. So the present
invention provides insecticidal compositions comprising a plant
extract in alcohol, as applied as a lone insecticide or the plant
extract with Bacillus thuringiensis .delta.-endotoxin as a
combination insecticide with synergistic effect. The composition of
plant extract and Bacillus thuringiensis .delta.-endotoxin can be
prepared in alcohol at required concentrations. properly dispersed
and sprayed on the infested plants. These combinations as described
in the tables were tested on the plants of Mentha arvensis infested
with Spilarctia obliqua in the glasshouse and in the field with
complete dis-infestation within 15 days.
* * * * *