Title of Invention	A PROCESS FOR PREPARING A BIOCIDE USED FOR CONTROLLING BLACK ROT DISEASE IN TEA PLANT .
Abstract	A process for preparing a novel biocide comprising a consortium of two bacterial and two fungal strains for control1ing a severe disease of tea plants caused by pathoganic fungal strains namely corticium invisum and corticium theae. The preparation of the biocide is made by mixing the four ingredients strains at the ratio of 4:3 : 2:1 and the cultures are prepared in the following manner: a) Transferring the Bacillus subti1 is and Pseudomanas putida cultures into mother culture memdium, b) allowing the mother cultures to grow for 3-7 days, c) transferring the grown mother cultures to main culture medium and a 1 lowing the main cultures to grow for 3-7 clays. d) transferring the Trichoderma viridi and Trichoderma harzianum cultures in the mother culture memdiumi e) a1 lowing the mother cultures to grow for 15 days, f) transferring the mother cultures to the mam culture medium and allowing the main cultures to grow for 15 days. g) mixing the said ready cultures - Bacillus subti1is Pseudomonas putida, Trichoderma viridi and Trichoderma harzianum in a ratio of 4:3:2:1 to obtain the biocide.

Title of Invention

A PROCESS FOR PREPARING A BIOCIDE USED FOR CONTROLLING BLACK ROT DISEASE IN TEA PLANT .

Abstract

A process for preparing a novel biocide comprising a consortium of two bacterial and two fungal strains for control1ing a severe disease of tea plants caused by pathoganic fungal strains namely corticium invisum and corticium theae. The preparation of the biocide is made by mixing the four ingredients strains at the ratio of 4:3 : 2:1 and the cultures are prepared in the following manner: a) Transferring the Bacillus subti1 is and Pseudomanas putida cultures into mother culture memdium, b) allowing the mother cultures to grow for 3-7 days, c) transferring the grown mother cultures to main culture medium and a 1 lowing the main cultures to grow for 3-7 clays. d) transferring the Trichoderma viridi and Trichoderma harzianum cultures in the mother culture memdiumi e) a1 lowing the mother cultures to grow for 15 days, f) transferring the mother cultures to the mam culture medium and allowing the main cultures to grow for 15 days. g) mixing the said ready cultures - Bacillus subti1is Pseudomonas putida, Trichoderma viridi and Trichoderma harzianum in a ratio of 4:3:2:1 to obtain the biocide.

Full Text	The present invention relates to process for preparing a blooide which is used for controlling the black rot disease in tea plant particularly in tea leaves. Tea plants are generally affected with various types of diseases. It has been a serious problem both within India and other tea growing countries to control the most common disease associated with tea. In the prior art, black rot disease of tea was treated with the help of conventional fungicides and not biocides. The applicant for the first time has found that a biocide can effectively control the black rot disease of tea. Moreover, the concept of bioproduct in controlling the black rot disease of tea has no precedent. The use of conventional fungicide which includes copper fungicides naaely Bavistem, Fytolan etc. has some inherent drawbacks. The fungicide if sprayed with a lower concentration, the causative organise of the disease develops resistance against the fungicide. Further it is harmful to the user also. In case of the bioeide which inc lades four aiorobial strains also soaetiees is difficult to establish in the field. - 2 - Moreover, the toxic metabolltes production by microorganisms vary from time to time according to the abiotic factors which includes aablent temperature, the moisture content and the intensity of the light* Further it is found that the development of the alcroorganlsas in the field is rather slow as compared to its requirement in controlling the black rot disease of tea* One of the objects of the present invention is to mitigate the aforesaid drawbacks of the conventional processes of establishment of microorganisms in the field. Another object of the present invention is to establish a biocide which effectively controls th" black rot disease of tea. A stlII further object of the present invention is to reduce the load of chemical pesticide in the environment. A further object of the present Invention is to produce a biocide which can effectively control or prevent spreading of the black rot disease of tea which ie economical. A still farther object of the present Invention is to control the black rot disease of tea with a biocide which Is not harmful for the human beings and no adverse effect on the person handling it. - 3 - With the above objects in view, it has been the endeavour of the applicant to produce a biocide which will satisfy all the above requirements and objectives. The applicant has utilised four strains of microbes in producing the bIOCIdes which effectively controls or prevents the disease in tea leaves. This invention provides preparing a new biocide which comprises the following steps: (i) transferring Baci1lus suht i1 is (BB 15) to mother culture medium containing trypton, yeast extract and dextrose f (ii) allowing the mother culture to grow fro 3 to 7 days; (III) transferr i ng the grown mother culture to main culture medium essentially containing molasses and yeast; (iv) a1 lowing the main culture to grow for another 3 to 7 days to obtain (BB 15) cultures (v) transferring Pseudomonas -putida (BP 20) to mother culture medium. - 4 - vi) allowing it to. grow for 3 to 7 day*; vti) transferring the grown mother culture to main culture medium viii) allowing it to grow for 3 to 7 days to produce the ready culture; ix) transferring Trichoderma viride (FT1) to mother culture medium; x) allowing it to grow for 15 days; xi) transferring the grown mother culture to main culture medium; xii) allowing the main culture to grow for another 1S days to prepare the ready culture. xiii) transferring Trichoderma harzianum (FT12) to mother culture Medium. xiv) allowing it to grow for 15 days; xv) transferring the grown mother culture to main culture medium. - 5 - xvi) allowing it to grow tor another 15 days to obtain the ready cultures and xvii) mixing the said ready stranis 1 ,2,3 and 4 in a ratio of 4:3:2:1 to obtain the desired bxocide. In the above process of preparing the biocide, mother culture medium is subjected to agitation in a rotary shakar at 100 rpm while the main culture is preferably contained by passing sterile air through the main culture medium. To produce the FT11 and FT 12 cultures, mother culture is maintained essentially in yeast extract and glucose medium. Boil ing of main culture medium water for 15-20 minutes will help to produce better quality product. In the aforesaid process the stock culture grows in the mother culture and also in the mam culture medium preferably at a o temperature between 25-35 C and atmospheric pressure. The applicant has found that the biocide of the present inventionis very effective against Corticium invisum and C. theae which is the causative agent of black rot disease in tea. Although the medium has been selected for molasses and yeast? other suitable medium may also be employed for the culture in the preparation of biocide of the present invention. 6 - The applicant has found that the bioproduct can be applied foliar spray using knapsack spray at a rate of 4 litres per 2 litres of water ie about 2% in strength between 1 and 3% of biocide in water is also effective in control ling or preventing the black rot disease of tea leaves. In the present invention - Mother culture medium: Microbial strains are cultivated in artificial culture medium either non-synthetic or synthetic in nature- The present invention process contains both synthetic as wel1 as non-synthetic medium comprising inorganic and organic ingredients. Mother culture medium can be defined as the culture medium in which a large "inoculum" is prepared aseptically under laboratory conditions using an autoclave for pre sterilization. The memdium comprises various inorganic/organic compounds depending an the microbial strain to be cultured/cultivated" it can also be termed as "inoculum". Main culture mediums Main culture medium is the commercial medium prepared under semisterile condition which is used for larga scale aproduction of the desired microorganisms for product preparation. The medium comprises commercial grade yeast cake and molasses at definite proportion depending on the microbial cultures to be produced in large scale. Therefore" this can also be termed as "mass culture medium"- -7- Experiments conducted in Tea Gardens where mature tea bushes were severally attack by black rot disease found to be very effective in controlling the same and bush recovery was tremendous. 1) A block of tea bushes each were considered one unit and five units were taken against each dilution. 2) Mix consortium of Baci1lus subti1 is, Pseudomonas putida, Tnchoderma viride and Trichoderma harzianum cultures were diluted to 0.5 %., 1.10 %, 2.0% and 3.0 % suspensions. 3) Three consecutive sprays using a back pak sprayer with berbicide nozzle was performed at an interval of 10 days. 4) Observations and data collection was made in alternate days to fmdout the efficacy of the biocide. 5) Observations revealed that recovery of effected bushes from the second round took palce in dilution 1-0 %. to 3.0 % New fresh shoots stated emerging from the plucking point giving a fresh loot- to the black rot effected bushes. But in 0.5 7. dilution sprayed bushes recovery was quite slow and not encouraging. - 8 - WE CLAIM: 1. A process for preparing a new biocide used for controlling the black rot disease in tea plant which comprises the following steps: (i) transferring Baci1lus sufati1 is (BB 15) to mother culture medium as herein described containing trypton, yeast extract and dextrose; (ii) allowing the mother culture to grow fro 3 to 7 days; (iii) transferring the grown mother culture to main culture medium as herein described essentially containing molasses and yeast; (iv) allowing the main culture to grow for another 3 to 7 days to obtain (BB 15) culture; (v) transferring Pseudomonas -putida (BP 20) to said mother culture medium. (vi) allowing it to grow for 3 to 7 days5 (vii) transferring the grown mother culture to said main culture medium; (viii) allowing it to grow for 3 to 7 days to produce the ready culture; (ix) transferring Trichoderma viride (FT1l) to mother culture medium; : 9 : (x) allowing it to grow for 15 days; (xi) transferring the grown mother culture to main culture medium; (xii) allowing the main culture to grow for another 15 days to prepare the ready culture; (xiii) transferring Trichoderma harzianum (FT12) to mother culture medium; (xiv) allowing it to grow for 15 days; (xv) transferring the grown mother culture to main culture; medium; (xvi) allowing it to grow for another 15 days to obtain the ready culture; and (xvii) mixing the said ready cultures 1,2,3 and 4 in a ratio of 4:3:2:1 to obtain the desired biocide. /2. A process as claimed in Claim 1 wherein said mother culture medium is subjected to agitation in a rotary shakar at 100 rpm while the main culture is preferably contained by passing sterile air through the main culture medium. 10 3. A process as claimed in Claim 1 or 2 wherein said mother culture is maintained essentially in yeast extract and glucose medium, box 1 ing water to main culture medi urn for 15-20 minutes to produce FT11 and FT12 cultures, 4. A process as claimed in any of the preceding claims wherein the said stock culture is allowed to grow in the mother culture as herein described and also in the main culture medium as herein described preferably at a temperature between 25 and o 35 C and at atmospheric pressure. 11 5. A process for preparing a new biocide used for controlling the blacfc rot disease in tea plant substantially as herein described. A process for preparing a novel biocide comprising a consortium of two bacterial and two fungal strains for control1ing a severe disease of tea plants caused by pathoganic fungal strains namely corticium invisum and corticium theae. The preparation of the biocide is made by mixing the four ingredients strains at the ratio of 4:3 : 2:1 and the cultures are prepared in the following manner: a) Transferring the Bacillus subti1 is and Pseudomanas putida cultures into mother culture memdium, b) allowing the mother cultures to grow for 3-7 days, c) transferring the grown mother cultures to main culture medium and a 1 lowing the main cultures to grow for 3-7 clays. d) transferring the Trichoderma viridi and Trichoderma harzianum cultures in the mother culture memdiumi e) a1 lowing the mother cultures to grow for 15 days, f) transferring the mother cultures to the mam culture medium and allowing the main cultures to grow for 15 days. g) mixing the said ready cultures - Bacillus subti1is Pseudomonas putida, Trichoderma viridi and Trichoderma harzianum in a ratio of 4:3:2:1 to obtain the biocide.

Full Text

The present invention relates to process for preparing a blooide which is used for controlling the black rot disease in tea plant particularly in tea leaves.
Tea plants are generally affected with various types of diseases. It has been a serious problem both within India and other tea growing countries to control the most common disease associated with tea. In the prior art, black rot disease of tea was treated with the help of conventional fungicides and not biocides. The applicant for the first time has found that a biocide can effectively control the black rot disease of tea. Moreover, the concept of bioproduct in controlling the black rot disease of tea has no precedent.
The use of conventional fungicide which includes copper fungicides naaely Bavistem, Fytolan etc. has some inherent drawbacks. The fungicide if sprayed with a lower concentration, the causative organise of the disease develops resistance against the fungicide. Further it is harmful to the user also.
In case of the bioeide which inc lades four aiorobial strains also soaetiees is difficult to establish in the field.
- 2 -

Moreover, the toxic metabolltes production by microorganisms vary from time to time according to the abiotic factors which includes aablent temperature, the moisture content and the intensity of the light* Further it is found that the development of the alcroorganlsas in the field is rather slow as compared to its requirement in controlling the black rot disease of tea*
One of the objects of the present invention is to mitigate the aforesaid drawbacks of the conventional processes of establishment of microorganisms in the field.
Another object of the present invention is to establish a biocide which effectively controls th" black rot disease of tea.
A stlII further object of the present invention is to reduce the load of chemical pesticide in the environment.
A further object of the present Invention is to produce a biocide which can effectively control or prevent spreading of the black rot disease of tea which ie economical.
A still farther object of the present Invention is to control the black rot disease of tea with a biocide which Is not harmful for the human beings and no adverse effect on the person
handling it.
- 3 -

With the above objects in view, it has been the endeavour of the applicant to produce a biocide which will satisfy all the above requirements and objectives.
The applicant has utilised four strains of microbes in producing
the bIOCIdes which effectively controls or prevents the disease in tea leaves.
This invention provides preparing a new biocide which comprises the following steps:
(i) transferring Baci1lus suht i1 is (BB 15) to mother culture medium containing trypton, yeast extract and dextrose f
(ii) allowing the mother culture to grow fro 3 to 7
days;
(III) transferr i ng the grown mother culture to main
culture medium essentially containing molasses and yeast;
(iv) a1 lowing the main culture to grow for another 3 to 7 days to obtain (BB 15) cultures
(v) transferring Pseudomonas -putida (BP 20) to mother culture medium.
- 4 -

vi) allowing it to. grow for 3 to 7 day*;
vti) transferring the grown mother culture to main culture medium
viii) allowing it to grow for 3 to 7 days to produce the ready culture;
ix) transferring Trichoderma viride (FT1) to mother culture medium;
x) allowing it to grow for 15 days;
xi) transferring the grown mother culture to main culture medium;
xii) allowing the main culture to grow for another 1S days to prepare the ready culture.
xiii) transferring Trichoderma harzianum (FT12) to mother culture Medium.
xiv) allowing it to grow for 15 days;
xv) transferring the grown mother culture to main culture medium.
- 5 -

xvi) allowing it to grow tor another 15 days to obtain the ready cultures and
xvii) mixing the said ready stranis 1 ,2,3 and 4 in a ratio of 4:3:2:1 to obtain the desired bxocide.
In the above process of preparing the biocide, mother culture medium is subjected to agitation in a rotary shakar at 100 rpm while the main culture is preferably contained by passing sterile air through the main culture medium. To produce the FT11 and FT 12 cultures, mother culture is maintained essentially in yeast extract and glucose medium. Boil ing of main culture medium water for 15-20 minutes will help to produce better quality product.
In the aforesaid process the stock culture grows in the mother
culture and also in the mam culture medium preferably at a
o temperature between 25-35 C and atmospheric pressure.
The applicant has found that the biocide of the present inventionis very effective against Corticium invisum and C. theae which is the causative agent of black rot disease in tea. Although the medium has been selected for molasses and yeast? other suitable medium may also be employed for the culture in the preparation of biocide of the present invention.
6 -

The applicant has found that the bioproduct can be applied foliar spray using knapsack spray at a rate of 4 litres per 2 litres of water ie about 2% in strength between 1 and 3% of biocide in water is also effective in control ling or preventing the black
rot disease of tea leaves.
In the present invention -
Mother culture medium:
Microbial strains are cultivated in artificial culture medium either non-synthetic or synthetic in nature- The present invention process contains both synthetic as wel1 as non-synthetic medium comprising inorganic and organic ingredients.
Mother culture medium can be defined as the culture medium in which a large "inoculum" is prepared aseptically under laboratory conditions using an autoclave for pre sterilization. The memdium comprises various inorganic/organic compounds depending an the microbial strain to be cultured/cultivated" it can also be termed as "inoculum".
Main culture mediums
Main culture medium is the commercial medium prepared under semisterile condition which is used for larga scale aproduction of the desired microorganisms for product preparation. The medium comprises commercial grade yeast cake and molasses at definite proportion depending on the microbial cultures to be produced in large scale. Therefore" this can also be termed as "mass culture medium"-
-7-

Experiments conducted in Tea Gardens where mature tea bushes were severally attack by black rot disease found to be very effective in controlling the same and bush recovery was tremendous.
1) A block of tea bushes each were considered one unit and five
units were taken against each dilution.
2) Mix consortium of Baci1lus subti1 is, Pseudomonas putida,
Tnchoderma viride and Trichoderma harzianum cultures were
diluted to 0.5 %., 1.10 %, 2.0% and 3.0 % suspensions.
3) Three consecutive sprays using a back pak sprayer with
berbicide nozzle was performed at an interval of 10 days.
4) Observations and data collection was made in alternate days
to fmdout the efficacy of the biocide.
5) Observations revealed that recovery of effected bushes from
the second round took palce in dilution 1-0 %. to 3.0 %
New fresh shoots stated emerging from the plucking point giving a fresh loot- to the black rot effected bushes. But in 0.5 7. dilution sprayed bushes recovery was quite slow and not encouraging.
- 8 -

WE CLAIM:
1. A process for preparing a new biocide used for controlling the black rot disease in tea plant which comprises the following steps:
(i) transferring Baci1lus sufati1 is (BB 15) to mother culture medium as herein described containing trypton, yeast extract and dextrose;
(ii) allowing the mother culture to grow fro 3 to 7 days;
(iii) transferring the grown mother culture to main culture medium as herein described essentially containing molasses and yeast;
(iv) allowing the main culture to grow for another 3 to 7 days to obtain (BB 15) culture;
(v) transferring Pseudomonas -putida (BP 20) to said mother culture medium.
(vi) allowing it to grow for 3 to 7 days5
(vii) transferring the grown mother culture to said main culture medium;
(viii) allowing it to grow for 3 to 7 days to produce the ready culture;
(ix) transferring Trichoderma viride (FT1l) to mother culture medium;
: 9 :

(x) allowing it to grow for 15 days;
(xi) transferring the grown mother culture to main culture medium;
(xii) allowing the main culture to grow for another 15 days to prepare the ready culture;
(xiii) transferring Trichoderma harzianum (FT12) to mother culture medium;
(xiv) allowing it to grow for 15 days;
(xv) transferring the grown mother culture to main culture; medium;
(xvi) allowing it to grow for another 15 days to obtain the ready culture; and
(xvii) mixing the said ready cultures 1,2,3 and 4 in a ratio of 4:3:2:1 to obtain the desired biocide.
/2. A process as claimed in Claim 1 wherein said mother culture medium is subjected to agitation in a rotary shakar at 100 rpm while the main culture is preferably contained by passing sterile air through the main culture medium.
10

3. A process as claimed in Claim 1 or 2 wherein said
mother culture is maintained essentially in yeast extract and
glucose medium, box 1 ing water to main culture medi urn for 15-20
minutes to produce FT11 and FT12 cultures,
4. A process as claimed in any of the preceding claims
wherein the said stock culture is allowed to grow in the mother
culture as herein described and also in the main culture medium
as herein described preferably at a temperature between 25 and
o 35 C and at atmospheric pressure.
11
5. A process for preparing a new biocide used for
controlling the blacfc rot disease in tea plant substantially as
herein described.
A process for preparing a novel biocide comprising a consortium of two bacterial and two fungal strains for control1ing a severe disease of tea plants caused by pathoganic fungal strains namely corticium invisum and corticium theae. The preparation of the biocide is made by mixing the four ingredients strains at the ratio of 4:3 : 2:1 and the cultures are prepared in the following manner:
a) Transferring the Bacillus subti1 is and Pseudomanas
putida cultures into mother culture memdium,
b) allowing the mother cultures to grow for 3-7 days,
c) transferring the grown mother cultures to main culture
medium and a 1 lowing the main cultures to grow for 3-7 clays.
d) transferring the Trichoderma viridi and Trichoderma
harzianum cultures in the mother culture memdiumi
e) a1 lowing the mother cultures to grow for 15 days,
f) transferring the mother cultures to the mam culture
medium and allowing the main cultures to grow for 15 days.
g) mixing the said ready cultures - Bacillus subti1is
Pseudomonas putida, Trichoderma viridi and Trichoderma harzianum
in a ratio of 4:3:2:1 to obtain the biocide.

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Patent Number

208121

Indian Patent Application Number

0083/CAL/2000

PG Journal Number

28/2007

Publication Date

13-Jul-2007

Grant Date

12-Jul-2007

Date of Filing

17-Feb-2000

Name of Patentee

GOODRICKE GROUP LIMITED

Applicant Address

CAMELLIA HOUSE, 14 GURUSADAY ROAD, CALCUTTA - 700 019, WEST BENGAL, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	TAMULI DR. A. K.	RESEARCH OFFICER, DANGUAJHAR T.G., P.O. DANGUAJHAR - 735 121, JALPAIGURI, WEST BENGAL, INDIA.

PCT International Classification Number

C12N 1/16

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA