Title of Invention	DEVELOPMENT OF LOW COST LIQUID FORMULATION OF NATIVE STRAINS OF BACILLUS THURINGIENSIS AGAINST HELICOVERPA ARMIGERA
Abstract	A low cost liquid formulation of native strains of Bacillus thuringiensis against Helicoverpa armigera comprising step of growing, the Bacillus thuringiensis strains on media containing 2-14 ml /litre potato extract as carbon source, 2-14 gm/litre cotton seed meal as nitrogen source, 0.5 g/1 - 2.5 g/1, preferably 2.0 gm peptone, 0.5 g/1 - 2.5 g/1 preferably 1.5 g Dextrose, 0.5 g/1 - 2.5 g/ 1 preferably 2.0 gm yeast extract, 0.03 gm MgSO4, 7H2O, ,O2g FeSO4, 7H2O, .02g ZnSO4, 7H2O, 5.0g Nacl, 1ml Tween 60 and distilled water to make volume to 1 litre for 48 hrs at 30-40°C preferably at 30°C harvested cells are poured into the bottle containing sterile mustard oil, preferably -1012 cells/100 ml of liquid.

Title of Invention

DEVELOPMENT OF LOW COST LIQUID FORMULATION OF NATIVE STRAINS OF BACILLUS THURINGIENSIS AGAINST HELICOVERPA ARMIGERA

Abstract

A low cost liquid formulation of native strains of Bacillus thuringiensis against Helicoverpa armigera comprising step of growing, the Bacillus thuringiensis strains on media containing 2-14 ml /litre potato extract as carbon source, 2-14 gm/litre cotton seed meal as nitrogen source, 0.5 g/1 - 2.5 g/1, preferably 2.0 gm peptone, 0.5 g/1 - 2.5 g/1 preferably 1.5 g Dextrose, 0.5 g/1 - 2.5 g/ 1 preferably 2.0 gm yeast extract, 0.03 gm MgSO4, 7H2O, ,O2g FeSO4, 7H2O, .02g ZnSO4, 7H2O, 5.0g Nacl, 1ml Tween 60 and distilled water to make volume to 1 litre for 48 hrs at 30-40°C preferably at 30°C harvested cells are poured into the bottle containing sterile mustard oil, preferably -1012 cells/100 ml of liquid.

Full Text	Field of Invention; The invention relates to the development of low cost liquid formulation of native strains of Bacillus thuringiensis against Helicoverpa arrnigera. Background of the Invention; H. armigera, a major pest of various cropplants worldwide has developed resistance against chemicals. Therefore there is a need to explore BT formulations so that losses due to this pest could be minimized. The formulation should be viable at high temperatures prevailing in that area. The bioinsecticides formulation in controlling Helicoverpa armigera larvae in the local area are mainly imported from Europe and China. The formulations available in market is very costly which increases the cost of input. The formulations available do not contain the native strains, so are less effective. Also these formulations cannot tolerate high temperature in the field and hence are less persistent in the field. Low cost Agro industrial byproducts are used for the production of Bacillus thuringiensis formulations. Therefore the cost of production is 7-9 times lower than the previously available formulations. Since the native strains are used for the preparation of these formulations, they are more effective in controlling the pests in local area. Also as the native strains are isolated from the fields of local area, they can resist the high temperature prevailing in the area. Objects of Invention; The object of this invention is to control the Helicoyerpa armigera larvae in the local area. Other object is to prepare liquid formulation of Bacillus thuringiensis strains Btm and Se. Another objection is to use the native strains for making effective formulations. Yet another object is to use a formulation that can tolerate high temperature in the field. Another object is to make a formulation that is persistent in the field. Other object is to develop a formulation wherein the cost of production is much lower than the previously available formulations. Statement of the Invention; According to this invention there is provided a low cost liquid formulation of native strains of Bacillus thuringiensis BtIII and S6 against Helicoverpa armigera, the stabilizing agent used being sterile mustard oil preferably 100 ml and non food grade starch @ of 0.01% as a spreading material before spraying wherein the Bacillus thuringiensis strains are grown on production media containing 2-14 ml/litre potato extract as carbon source, 2-14 gm/litre cotton seed meal as nitrogen source, 0.5 g/1 - 2.5 g/1, preferably 2.0 gm peptone, 0.5 g/1 - 2.5 g/1 preferably 1.5 g Dextrose, 0.5 g/1 - 2.5 g/1 preferably 2.0 gm yeast extract, 0.03 gm MgSO4, 7H2O, .02g FeSO4, 7H2O, .02g ZnSO4, 7H2O, 5.0g Nacl, 1rnl Tween 60 and distilled water to make volume to 1 litre for 48 hrs at 30- 40°C preferably at 30°C and harvested cells are poured into the bottle containing mustard oil, so that each bottle has got ~1012 cells/100 ml of liquid. Brief description of the accompanying Figures; The figure 1 shows the effect of different nitrogen sources on the growth of B. thuringiensis strain. The figure 2 shows the effect of different carbon sources on the growth of B. thuringiensis strain. The figure 3 shows that 1012 cells/g of carrier material showed the highest viability of cells. The figure 4 shows that Evaluation of persistence of B. thuringiensis Btm cells in different stabilizers based liquid formulations. The figure 5 shows that Persistence of Bt cells in mustard oil based formulation. Detailed description of the Invention; The formulation is prepared by using native strains of Bacillus thuringiensis i.e. Btm and Se. The stabilizing material used for the liquid formulation is Mustard Oil. Non food grade starch @ 0.01% is added as a spreading material before spraying. Low cost production medium for Bacillus thuringiensis contained potato extract 12 ml/1, cotton seed meal 10 gm/1, peptone 2 g/1, Dextrose 1.5 g/1, yeast extract 2.0 g/1, MgSO4, 7H2O .03 g/1, FeSO4, 7H2O 0.02 g/1, ZnSO4, 0.02 g/1, Nacl 5.0 g/1, Tween 60, 1.0 ml/1. The production medium was inoculated with Bt native strains in the Erlenmeyer flasks as well as in the Roux bottles. The Biomass obtained after 48 hours of growth was collected for preparing the liquid formulation of Bt. One hundred milliliter of mustard oil was put in autoclavable plastic bottles for preparation of the liquid formulation. These bottles were sterilized at 15 1b/sq inch for 20 minutes. The biomass of Bt obtained after 48 hours was poured into the bottles such that each bottle should get 1012 cell/ml of the liquid. These formulations were kept at room temperature. The bioefficacy of these formulations were determined by applying these formulations in the fields infested with Helicoverpa armigera and the bio efficacy was determined by calculating % larval mortality. Various Agro industrial residues & their concentrations used in the formulation For biomass production of B. thuringiensis subsp. kurstaki, different agro-industrial by-products viz. potato extract, corn starch, wheat flour, sugarcane molasses, barley flour, soluble starch (carbon source) cotton seed meal, soya meal, sunflower meal, urea, ammonium sulfate and potassium nitrate(nitrogen source) were used as carbon and nitrogen sources (Table 1, 2) Table 1 Various Carbon sources used for Production of Bt Biomass (Table Removed) Table 2 Various Nitrogen sources used for Production of Bt Biomass (Table Removed) The concentration of carbon & nitrogen sources varied from 2g to 14 grams in basal medium (Table 3) Table 3 (Table Removed) log number of total viable cells Table 3B: Effect of Dextrose concentration on production of B. thuringiensis (Table Removed) log number of total viable cells The concentration of MgSO4.7H2O, FeSO4.7H2O, ZnSO4.7H2b, NaCl, Tween 60 was taken @ 0.003%, 0.002%, 0.002%, 0.5% and 0.1% respectively as per recommendation of Morris et. al. (1997) for the basal medium for the growth of B. thuringiensis isolates. Table 4: Effect of potato extract concentration on production of B. thuringiensis Biomass (Table Removed) log number of total viable cells * potato extract was added @10ml/l in all the media except LB From these studies, it is clear that potato extract and cotton seed meal were the best carbon and nitrogen sources respectively, for maximum biomass production. The composition of optimized medium for production of Bt. Biomass is given below (Table-6). Table 6: Optimized production media for production of B. thuringiensis biomass (Table Removed) Table 7: Production of B. thuringiensis isolates Btm and Se biomass (Table Removed) * viable cell number/ml. Harvesting of cells/Centrifugation for the preparation of Bt culture packets Production of Bt biomass; The Bt cultures were inoculated into OPM in Roux bottles or in flasks and incubated at 30°C for 48 hours. From Roux bottles, cells were harvested by scraping off the cells with sterilized distilled water using a glass rod. Cells from culture broth in flasks, were harvested by two methods. i) Cells were collected by centrifugation at 10,000 rpm for 10 minutes ii) To culture broth, bentonite: agar mix (1:1) @ 0.1% was added aseptically and kept for 24 h. at room temperature. Cells settled down, culture supernatant was removed by decanting off, cells were collected and absorbance was measured. The cell suspension was injected aseptically into each packet at a concentration of ~1012 cells/g of the carrier material. The contents of the culture packets were mixed thoroughly to prevent clump formation. The cells were used as 1012 cells/g of carrier material so that the final spray suspension would contain 108 cells/ml of the spray suspension (international standard). Tolerance at High temperature The growth of B. thuringiensis Btm and standard culture B. thuringiensis subsecies kurstaki (isolated from Dipel) were examined at different temperatures viz. 30°C, 35°C, 40°C. It was observed that both the strains showed same growth at 30°C. But at 35°C and 40 °C, the growth of B. thuringiensis subsecies kurstaki was less than 50 % in terms of biomass as compared to B. thuringiensis Btm • Table 8: Bioefficacy of Liquid formulations of B. thuringiensis against Helicoverpa armigera larvae in cotton field (Table Removed) Table 9: Estimation of cost of production of B. thuringiensis formulation for one acre (Table Removed) We claim: 1. A low cost liquid formulation of native strains of Bacillus thuringiensis against Helicoverpa armigera comprising step of growing, the Bacillus thuringiensis strains on media containing 2-14 ml/litre potato extract as carbon source, 2-14 gm/litre cotton seed meal as nitrogen source, 0.5 g/1 - 2.5 g/1, preferably 2.0 gm peptone, 0.5 g/1 - 2.5 g/1 preferably 1.5 g Dextrose, 0.5 g/1 - 2.5 g/1 preferably 2.0 gm yeast extract, 0.03 gm MgSO4, 7H2O, .02g FeSO4, 7H2O, .02g ZnSO4, 7H2O, 5.0g Nacl, 1ml Tween 60 and distilled water to make volume to 1 litre for 48 hrs at 30-40°C preferably at 30°C harvested cells are poured into the bottle containing sterile mustard oil, preferably ~1012 cells/100 ml of liquid. 2. The formulation as claimed in claim 1, wherein by products like potato extract and cotton seed meal are used as carbon and nitrogen sources respectively.

Full Text

Field of Invention;
The invention relates to the development of low cost liquid formulation of native strains of Bacillus thuringiensis against Helicoverpa arrnigera.
Background of the Invention;
H. armigera, a major pest of various cropplants worldwide has developed resistance against chemicals. Therefore there is a need to explore BT formulations so that losses due to this pest could be minimized. The formulation should be viable at high temperatures prevailing in that area. The bioinsecticides formulation in controlling Helicoverpa armigera larvae in the local area are mainly imported from Europe and China. The formulations available in market is very costly which increases the cost of input. The formulations available do not contain the native strains, so are less effective. Also these formulations cannot tolerate high temperature in the field and hence are less persistent in the field.
Low cost Agro industrial byproducts are used for the production of Bacillus thuringiensis formulations. Therefore the cost of production is 7-9 times lower than the previously available formulations. Since the native strains are used for the preparation of these formulations, they are more effective in controlling the pests in local area. Also as the native strains are isolated from the fields of local area, they can resist the high temperature prevailing in the area.

Objects of Invention;
The object of this invention is to control the Helicoyerpa armigera larvae in the local area.
Other object is to prepare liquid formulation of Bacillus thuringiensis strains Btm and Se.
Another objection is to use the native strains for making effective formulations.
Yet another object is to use a formulation that can tolerate high temperature in the field.
Another object is to make a formulation that is persistent in the field.
Other object is to develop a formulation wherein the cost of production is much lower than the previously available formulations.
Statement of the Invention;
According to this invention there is provided a low cost liquid formulation of native strains of Bacillus thuringiensis BtIII and S6 against Helicoverpa armigera, the stabilizing agent used being sterile mustard oil preferably 100 ml and non food grade starch @ of 0.01% as a spreading material before spraying wherein the Bacillus thuringiensis strains are grown on production media containing 2-14 ml/litre potato extract as carbon source, 2-14 gm/litre cotton seed meal as nitrogen source, 0.5 g/1 - 2.5 g/1, preferably 2.0 gm peptone, 0.5 g/1 - 2.5 g/1 preferably 1.5 g Dextrose, 0.5 g/1 - 2.5 g/1 preferably 2.0 gm yeast extract, 0.03 gm MgSO4, 7H2O, .02g FeSO4, 7H2O, .02g ZnSO4, 7H2O, 5.0g Nacl, 1rnl

Tween 60 and distilled water to make volume to 1 litre for 48 hrs at 30-
40°C preferably at 30°C and harvested cells are poured into the bottle
containing mustard oil, so that each bottle has got ~1012 cells/100 ml of
liquid.
Brief description of the accompanying Figures;
The figure 1 shows the effect of different nitrogen sources on the growth of B. thuringiensis strain.
The figure 2 shows the effect of different carbon sources on the growth of B. thuringiensis strain.
The figure 3 shows that 1012 cells/g of carrier material showed the highest viability of cells.
The figure 4 shows that Evaluation of persistence of B. thuringiensis Btm cells in different stabilizers based liquid formulations.
The figure 5 shows that Persistence of Bt cells in mustard oil based
formulation.
Detailed description of the Invention;
The formulation is prepared by using native strains of Bacillus thuringiensis i.e. Btm and Se. The stabilizing material used for the liquid formulation is Mustard Oil. Non food grade starch @ 0.01% is added as a spreading material before spraying.
Low cost production medium for Bacillus thuringiensis contained potato extract 12 ml/1, cotton seed meal 10 gm/1, peptone 2 g/1, Dextrose 1.5 g/1, yeast extract 2.0 g/1, MgSO4, 7H2O .03 g/1, FeSO4, 7H2O 0.02 g/1, ZnSO4, 0.02 g/1, Nacl 5.0 g/1, Tween 60, 1.0 ml/1. The production medium was inoculated with Bt native strains in the Erlenmeyer flasks

as well as in the Roux bottles. The Biomass obtained after 48 hours of growth was collected for preparing the liquid formulation of Bt. One hundred milliliter of mustard oil was put in autoclavable plastic bottles for preparation of the liquid formulation. These bottles were sterilized at 15 1b/sq inch for 20 minutes. The biomass of Bt obtained after 48 hours was poured into the bottles such that each bottle should get 1012 cell/ml of the liquid. These formulations were kept at room temperature. The bioefficacy of these formulations were determined by applying these formulations in the fields infested with Helicoverpa armigera and the bio efficacy was determined by calculating % larval mortality.
Various Agro industrial residues & their concentrations used in the formulation
For biomass production of B. thuringiensis subsp. kurstaki, different agro-industrial by-products viz. potato extract, corn starch, wheat flour, sugarcane molasses, barley flour, soluble starch (carbon source) cotton seed meal, soya meal, sunflower meal, urea, ammonium sulfate and potassium nitrate(nitrogen source) were used as carbon and nitrogen sources (Table 1, 2)
Table 1 Various Carbon sources used for Production of Bt Biomass
(Table Removed)

Table 2 Various Nitrogen sources used for Production of Bt Biomass
(Table Removed)
The concentration of carbon & nitrogen sources varied from 2g to 14 grams in basal medium (Table 3)
Table 3
(Table Removed)

*log number of total viable cells

Table 3B: Effect of Dextrose concentration on production of B. thuringiensis

(Table Removed)
*log number of total viable cells
The concentration of MgSO4.7H2O, FeSO4.7H2O, ZnSO4.7H2b, NaCl, Tween 60 was taken @ 0.003%, 0.002%, 0.002%, 0.5% and 0.1% respectively as per recommendation of Morris et. al. (1997) for the basal medium for the growth of B. thuringiensis isolates.
Table 4: Effect of potato extract concentration on production of B. thuringiensis Biomass

(Table Removed)

*log number of total viable cells
** potato extract was added @10ml/l in all the media except LB
From these studies, it is clear that potato extract and cotton seed meal were the best carbon and nitrogen sources respectively, for maximum biomass production. The composition of optimized medium for production of Bt. Biomass is given below (Table-6).
Table 6: Optimized production media for production of B. thuringiensis biomass
(Table Removed)
Table 7: Production of B. thuringiensis isolates Btm and Se biomass

(Table Removed)
* viable cell number/ml.
Harvesting of cells/Centrifugation for the preparation of Bt culture packets
Production of Bt biomass;
The Bt cultures were inoculated into OPM in Roux bottles or in flasks and incubated at 30°C for 48 hours.
From Roux bottles, cells were harvested by scraping off the cells with
sterilized distilled water using a glass rod.
Cells from culture broth in flasks, were harvested by two methods.
i) Cells were collected by centrifugation at 10,000 rpm for 10 minutes
ii) To culture broth, bentonite: agar mix (1:1) @ 0.1% was added aseptically and kept for 24 h. at room temperature. Cells settled down, culture supernatant was removed by decanting off, cells were collected and absorbance was measured. The cell suspension was injected aseptically into each packet at a concentration of ~1012 cells/g of the carrier material. The contents of the culture packets were mixed thoroughly to prevent clump formation. The cells were used as 1012 cells/g of carrier material so that the final spray suspension would contain 108 cells/ml of the spray suspension (international standard).
Tolerance at High temperature
The growth of B. thuringiensis Btm and standard culture B. thuringiensis subsecies kurstaki (isolated from Dipel) were examined at different temperatures viz. 30°C, 35°C, 40°C. It was observed that both the strains showed same growth at 30°C. But at 35°C and 40 °C, the growth of B. thuringiensis subsecies kurstaki was less than 50 % in terms of biomass as compared to B. thuringiensis Btm •
Table 8: Bioefficacy of Liquid formulations of B. thuringiensis against Helicoverpa armigera larvae in cotton field
(Table Removed)
Table 9: Estimation of cost of production of B. thuringiensis formulation for one acre

(Table Removed)

We claim:
1. A low cost liquid formulation of native strains of Bacillus
thuringiensis against Helicoverpa armigera comprising step of growing, the
Bacillus thuringiensis strains on media containing 2-14 ml/litre potato extract
as carbon source, 2-14 gm/litre cotton seed meal as nitrogen source, 0.5 g/1 -
2.5 g/1, preferably 2.0 gm peptone, 0.5 g/1 - 2.5 g/1 preferably 1.5 g
Dextrose, 0.5 g/1 - 2.5 g/1 preferably 2.0 gm yeast extract, 0.03 gm MgSO4,
7H2O, .02g FeSO4, 7H2O, .02g ZnSO4, 7H2O, 5.0g Nacl, 1ml Tween 60 and
distilled water to make volume to 1 litre for 48 hrs at 30-40°C preferably at
30°C harvested cells are poured into the bottle containing sterile mustard oil,
preferably ~1012 cells/100 ml of liquid.
2. The formulation as claimed in claim 1, wherein by products like potato extract and cotton seed meal are used as carbon and nitrogen sources respectively.

Documents:

0888-del-2007-abstract.pdf

0888-del-2007-claims.pdf

0888-del-2007-correspondence-others.pdf

0888-del-2007-description (complete).pdf

0888-del-2007-drawings.pdf

0888-del-2007-form-1.pdf

0888-del-2007-form-2.pdf

888-DEL-2007-Abstract-(25-08-2011).pdf

888-DEL-2007-Claims-(25-08-2011).pdf

888-DEL-2007-Correspondence Others-(25-08-2011).pdf

888-del-2007-form-18 (25-02-2008).pdf

888-DEL-2007-Form-3-(25-08-2011).pdf

888-DEL-2007-GPA-(25-08-2011).pdf

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Patent Number

253532

Indian Patent Application Number

888/DEL/2007

PG Journal Number

31/2012

Publication Date

03-Aug-2012

Grant Date

27-Jul-2012

Date of Filing

23-Apr-2007

Name of Patentee

CCS HARYANA AGRICULTURAL UNIVERSITY

Applicant Address

HISAR-125 004, AN INDIAN INSTITUTION

Inventors:

#	Inventor's Name	Inventor's Address
1	KAMLA CHAUDHARY,HARISH DHINGRA & K.S.BOORA	DEPARTMENT OF BIOTECHNOLOGY & MOLECULAR BIOLOGY, CCS HARYANA AGRICULTURAL UNIVERSITY, HISAR-125 004, HARYANA,INDIA

PCT International Classification Number

C07H

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA