|Title of Invention||
"A NOVEL INSECTICIDAL COMPOSITION COMPRISING EXTRACT(S) OBTAINED FROM THE PLANT ALBIZZIA LEBBECK AND δ- ENDOTOXIN FROM BACILLUS THURINGIENSIS"
|Abstract||A novel insecticidal composition comprising: (a) alcoholic extract obtained from one or more parts of the plant Albizzia lebbeck; together with (b) Bacillusthuringiensis delta-endotoxin, useful in controlling insects of the class lepidopaara, ingredients (a) and (b) together being present in amounts that, taken together, are insecticidally effective.|
|Full Text||FIELD OF THE INVENTION
The present invention relates to a novel insecticidal composition comprising extract(s) obtained from the plant Albizzia lebbeck and delta- endotoxin form Bacillus thuringiensis. The composition useful in effectively controlling the lepidopteron crop damages insects. The invention also provides a process for the preparation for the said composition and a method for the application of the composition.
BACKGROUND OF THE INVENTION
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 arts, 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.
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.
Various crude extracts and essential oils from plants, such as Neem extract and compounds from Azadirecta 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 composition 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 8- 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. The combination of microbial pesticides with botanical anti-insect compounds can provide a formulation consisting of diverse compounds with novel and different modes of actions and thus reducing the risk and probability of simultaneous resistance development. Objects of the invention
The main object of the invention is to provide a novel insecticidal
composition comprising extract (s) obtained from the plant Albizzia lebbeck and delta- endotoxin form Bacillus thuringiensis.
Another object of the invention is to provide a process of preparation of novel insecticidal composition comprising extract(s) obtained from the plant Albizzia lebbeck and delta- endotoxin form Bacillus thuringiensis.
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.
Another object of the invention is to provide a composition that may be used for these insects as spray.
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 insect due to sole application of delta- endotoxin.
.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
The present invention provides a novel synergistic combination comprising alcoholic extract obtained from the plant albizzia lebbeck together with Bacillus thuringiensis 6- 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
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.
Accordingly the invention provides a novel insecticidal composition
(a) alcoholic extract obtained from one or more parts of the plant Albizzia lebbeck; together with
(b) Bacillus thuringiensis. delta.-endotoxin, useful in controlling insects of the class lepidoptara, ingredients (a) and (b) together being present in amounts that, taken together, are insecticidally effective.
In the first embodiment of the invention the composition contains alcoholic
extract in powder form 0.1 to 500 mg and 8- endotoxin acetone powder 3 to
500 mg to make a concentrate for dispersing in a volume of 1 liter solvent
for final application.
In an embodiment of the invention the alcohol used for extraction is selected
from Ethanol, Methanol and Isopropanol.
In another embodiment of the invention the plant parts are leaf, stem or seed.
In yet another embodiment of the invention the lepidopteran insects killed
are Spilarctia obliqua, Spodoptera litura and Heliothis armigera.
In yet another embodiment of the invention describes a method of killing
lepidopteran insects by applying the alcoholic extract of the plant Albizia
lebbeck in which Bacillus thuringiensis 8- endotoxin is dispersed in different
concentrations, to the infested plants.
In still another embodiment of the invention the alcoholic extract should be used in combination with Bacillus thuringiensis 8- endotoxin at a concentration of minimum LC 5 (0.001 ug/ul and 0.03 |ig/ul) or
The present invention in its disclosure provides the process for the preparation of the extract from the plant.
The processing of plant parts to obtain usable extract as biocontrol agent can
be achieved in the following sequence of steps.
Step 1: The plant material (leaf, stem and seed) are collected / harvested
from the plant preferably in the month of.
Step 2: The material is shade dried carefully by regular respreading in a
clean environment such that no microbial infestation occur.
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
Step 4: Alcohol part is filtered out by Whatman no. 1 filter paper and
evaporated naturally or in a rotavapour at 40-60°C.The leftover plant
material is reprocessed with fresh alcohol in similar fashion.
Step 5: Then these extracts are pooled.
Step 6 : Further drying of the pooled extract is performed in a freeze drier at-110°C (optional).
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. £7f9^/>te - 2.
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 9th day of the experiment.
400 (j,l of given concentration of treatment was coated on leaf of 12 square centimeter.
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 M,g/ul compared to the extract from leaf (0.0625 |ig/ul) and seed (0.0312 u.g/ul). LC50 was estimated as the concentration at which 50% mortality of the larvae was observed and similarly the other lethal doses were determined.
Lifecycle disruption assays
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 ug/ul
concentration of the extracts was found effective.
So it was evident from the above experiment that the extract from stem was more potent than leaf and seed and disrupts the lifecycle of the insect at all stages of the lifecycle.
Defining LC values for Bt acetone powder
In the next step we carried out feeding the larvae with different concentrations of Bacillus thuringiensis 8- 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 douwn to 7.0 using IN HC1. Cells were pelleted by centrifugation (8,000 rpm, 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 then
filtered through filter paper (Whatman no.l) 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° C. This residue (also called acetone powder) contained the crude endotoxin. Mortality observed for the larvae on 9th day of treatment is given in Table
From this experiment the LC10 and LC50 values were calculated as 25|iig/400^il and 125ug/400|il respectively by plotting a standard curve of
acetone powder concentration vs. mortality. pxa^ph- 5" Development of synergistic combinations
To check whether the plant extract has an enhancing effect on the 8-endotoxin we fed the larvae with different concentrations of the stem extract and the endotoxin (Table 4).
Table 4As described in the table the pupicidal activity increases significantly to 20%, in the combinatin 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 and LC 50 when taken individually. So the conclusin was that, the plant extract and Bacillus thuringiensis 5- 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. The combination is environmentally safe as tested earlier for 8-m endotoxin. The plant also produces many compounds, which are used for medicatin for human.
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 an alcoholic plant extract applied as a lone insecticide or the plant extract with Bacillus thuringiensis 8- endotoxin as a combination insecticide with synergistic effect.
The composition of plant extract and Bacillus thuringiensis 6- endotoxin can be prepared in aqueous solution 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.
1. A novel insecticidal composition comprising:
(a) alcoholic extract obtained from one or more parts of the plant Albizzia lebbeck; together with
Bacillus thuringiensis delta.-endotoxin, useful in controlling insects of the class lepidoptara, ingredients (a) and (b) together being present in amounts that, taken together, are insecticidally effective.
2. A composition as claimed in claim 1 wherein the composition is useful in controlling insects selected from the class lepidoptara comprising Spilarctia oblique, Sopodoptera 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 novel insecticidal composition comprising extract(s) obtained from the plant Albizzia lebbeck and delta-endotoxin form Bacillus thuringiensis substantially as herein described with reference to the examples.
|Indian Patent Application Number||1140/DEL/2003|
|PG Journal Number||37/2008|
|Date of Filing||11-Sep-2003|
|Name of Patentee||COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH|
|Applicant Address||RAFI MARG, NEW DELHI-110 001, INDIA.|
|PCT International Classification Number||A01N 63/00|
|PCT International Application Number||N/A|
|PCT International Filing date|