Title of Invention	"A PROCESS FOR MASS MULTIPLICATION OF PSEUDOMONAS FLUORESCENS ON COW DUNG"
Abstract	This invention relates to a process for mass multiplication of Pseudomonas fluorescens on cow dung comprising the steps of air drying of cow dung by spreading to form layer of 2.5 thickness under open shade for 5 to 7 days, maintaining the moisture content to 40% by weight of air dried cow dung by adding water, maintaining the pH of cow dung between 7 to 7.5, autoclaving the cow dung of step b) at 15 lbs for 30 minutes, 2 ml to 5 ml suspension of Pseudomonas fluorescens PBAP-27 talc-based formulation (lg/ 100 ml; population 106 cfu/ml) was added and mixed thoroughly under aseptic condition, after 2 weeks of incubating at 30+2°C the Pseudomonas fluorescens multiplied to 1014 cfu/g air dried cow dung, Pseudomonas fluorescens colonized cow dung was dried, powdered and formulated with talc containing carboxymethyl cellulose (1% w/w) or mineral oil Paraffinic Petroleum Oil to obtain final concentration of ≥ 2 x 108 cfu/g or per ml formulation.

Title of Invention

"A PROCESS FOR MASS MULTIPLICATION OF PSEUDOMONAS FLUORESCENS ON COW DUNG"

Abstract

This invention relates to a process for mass multiplication of Pseudomonas fluorescens on cow dung comprising the steps of air drying of cow dung by spreading to form layer of 2.5 thickness under open shade for 5 to 7 days, maintaining the moisture content to 40% by weight of air dried cow dung by adding water, maintaining the pH of cow dung between 7 to 7.5, autoclaving the cow dung of step b) at 15 lbs for 30 minutes, 2 ml to 5 ml suspension of Pseudomonas fluorescens PBAP-27 talc-based formulation (lg/ 100 ml; population 106 cfu/ml) was added and mixed thoroughly under aseptic condition, after 2 weeks of incubating at 30+2°C the Pseudomonas fluorescens multiplied to 1014 cfu/g air dried cow dung, Pseudomonas fluorescens colonized cow dung was dried, powdered and formulated with talc containing carboxymethyl cellulose (1% w/w) or mineral oil Paraffinic Petroleum Oil to obtain final concentration of ≥ 2 x 108 cfu/g or per ml formulation.

Full Text	FIELD OF INVENTION This invention relates to the process for mass multiplication of Pseudomonas fluorescens on cow dung. BACKGROUND OF INVENTION Pseudomonas fluorescens is one of the most important biocontrol agents and biofertilizers for the management of plant health. Success of a biocontrol agent depends not only on its bioefficacy or shelf life but also the easiness with which it can be mass multiplied on a suitable substrate, which is easily obtainable and comparatively inexpensive. Pseudomonas spp. have been grown on a wide range of liquid media both for small scale and mass multiplication (King et al. 1954, Wollum 1982, Slininger and Shea-Wilbur 1995). However, ingredients of these media are relatively expensive. There is no report to grow this bacterium on solid substrate. In present .invention Pseudomonas fluorescens was mass multiplied on an easily available and inexpensive substrate i.e. cow dung as solid fermentation. There is no other ingredient involved. Yield is very high (>1014 cfu per g air dried cow dung). Bioefficacy of P. fluorescens multiplied on cow dung was better than any other reported media. Cow dung supports Pseudomonas fluorescens even after its application to soil, seed or foliage. A number of commercial formulations are available in the market for multiplication of Pseudomonas fluorescens but present technology will greatly help in its mass production on a most easily available and inexpensive substrate, which is available and used throughout the world as organic manure. OBJECTS OF THE INVENTION The main object of the invention is to propose a process for mass multiplication of Pseudomonas fluorescens on cow dung which uses solid substrate. Another object of the invention is to propose a process for mass multiplication of Pseudomonas fluorescens on cow dung which gives very high quantity (approximately ≥ 1014cfu per g air dried cow dung). Yet another object of this invention is to propose a process for mass multiplication of Pseudomonas fluorescens on cow dung wherein substrate cow dung is inexpensive and easily available. Another object of this invention is to propose a process for mass multiplication of Pseudomonas fluorescens on cow dung wherein temperature and moisture requirements are standardized for better multiplication. Yet another object of this invention is to propose a process for mass multiplication of Pseudomonas fluorescens on cow dung which does not use expensive ingredients like yeast extract, peptone etc.DRAW BACKS IN THE EXISTING STATE-OF-ART Most of the substrates/culture media that are presently in use for mass culture of Pseudomonas fluorescens contain expensive ingredients (e.g. yeast extract, peptone etc.) and they do not support the biocontrol agent after its application in the yield. Since these media are highly enriched, inoculum produced on them has relatively poor parasite fitness. STATEMENT OF INVENTION According to this invention there is provided a process for mass multiplication of Pseudomonas fluorescens on cow dung comprising the steps of air drying of cow dung by spreading to form layer of 2.5 thickness under open shade for 5 to 7 days, maintaining the moisture content to 40% by weight of air dried cow dung by adding water, maintaining the pH of cow dung between 7 to 7.5, autoclaving the cow dung of step b) at 15 lbs for 30 minutes, 2 ml to 5 ml suspension of Pseudomonas fluorescens PBAP-27 talc-based formulation (1g/ 100 ml; population 106 cfu/ml) was added and mixed thoroughly under aseptic condition, after 2 weeks of incubating at 30+2°C the Pseudomonas fluorescens multiplied to 1014 cfu/g air dried cow dung, Pseudomonas fluorescens colonized cow dung was dried, powdered .and formulated with talc containing carboxymethyl cellulose (1% w/w) or mineral oil Paraffinic Petroleum Oil to obtain final concentration of ≥ 2 x 108 cfu/g or per ml formulation. Figure-1. Multiplication of Pseudomonas fluorescens on cow dung in flask. Figure-2. Multiplication of Pseudomonas fluorescens on poly bags. DETAILED DESCRIPTION OF THE INVENTION: The antagonist Pseudomonas fluorescens isolate PBAT-27 was maintained and sub-cultured on King's B agar medium at 28°C. It was multiplied on King's B broth (King et al. 1954) for 5 days at 28°C and then mixed with talcum powder containing carboxymethyl cellulose (CMC)(@ 1%, w/w), air dried, ground and sieved to obtain the commercial powder formulation. This powder was suspended in sterilized distilled water (@ 1 g/ 100 ml; approx. population 106 cfu/ ml) and used for inoculation of cow dung. Multiplication of Pseudomonas fluorescens on cow dung in flasks: Fresh cow dung was collected and air dried by spreading as an approximately 2.5 cm thick layer under open shade for 5 to 7 days. Fifty g of air-dried (moisture content~30% when estimated by oven drying for 24 h at 40°C) cow dung was taken in 250 ml Erlenmeyer flasks. pH of the cow dung was 7 to 7.5. Moisture content of cow dung was adjusted to 40% by weight of air-dried cow dung by adding water. Flasks were then autoc laved at 15 lbs psi for 30 minutes. Two ml suspension of Pseudomonas fluorescens PBAP-27 formulated powder (@ 1 g/ 100 ml) was added in each flask and mixed thoroughly under aseptic conditions. Inoculated flasks were incubated in a BOD incubator at 30±2°C (Figure 1). After two weeks of incubation the population of Pseudomonas fluorescens could go as high as (≥ 14 cfu/g of air dried sample). Colonized cow dung was air dried and powdered with the help of mixer and grinder. Powdered cow dung was mixed with required quantity of talc powder containing 1% carboxymethyl cellulose (CMC) by weight to obtain final population of ≥ 2 x 108 cfu/g formulation and moisture content of ≤ 12%. Its oil based formulation can be also prepared by suspending colonized FYM in mineral oil (Paraffinic Petroleum Oil) containing emulsifier, stabilizer and anti-oxidant present in the final product at a concentration of 1.2%). Multiplication of Pseudomonas fluorescens on cow dung in Poly bags: Fresh cow dung was collected and air dried by spreading as an approximately 2.5 cm thick layer under open shade for one week. Two hundred g of air-dried (moisture content approx. 30% when estimated by oven drying for 24 h at 40°C) cow dung was taken in one kg capacity transparent autoclavable polypropylene bags. pH of the cow dung was 7 to 7.5. Moisture content of cow dung was adjusted to 40% by weight of air-dried cow dung by adding water. The bags were then autoclaved at 15 lbs psi for 30 minutes. Five ml suspension of Pseudomonas fluorescens PBAP-27 spore powder (@ 1 g / 100 ml) was injected in each bag with the help of a disposable syringe and the point of injection was sealed with cellophane tape (Figure 2). Inoculated bags were incubated in a BOD incubator at 30+2 °C. After two weeks of incubation the population of Pseudomonas fluorescens could go as high as 10 x 1013 cfu per g of air dried sample. Colonized cow dung was air dried and powdered with the help of mixer and grinder. Powdered cow dung was mixed with required quantity of talc powder containing 1% carboxymethyl cellulose (CMC) by weight to obtain final population of ≥ 2 x 108 cfu per g formulation and moisture content of ≤ 12%. Its oil based formulation can be also prepared by suspending colonized FYM in mineral oil (Paraffinic Petroleum Oil) containing emulsifier, stabilizer and anti-oxidant present in the final product at a concentration of 1.2%). It is to be noted that the formulation of the present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant formulations are intended to be within the scope of the present invention which is further set forth under the following claims:- WE CLAIM: 1. A process for mass multiplication of Pseudomonas fluorescens on cow dung comprising the steps of:- a) air drying of cow dung by spreading to form layer of 2.5 thickness under open shade for 5 to 7 days, maintaining the moisture content to 40% by weight of air dried cow dung by adding water, b) maintaining the pH of cow dung between 7 to 7.5, c) autoclaving the cow dung of step b) at 15 lbs for 30 minutes, d) 2 ml to 5 ml suspension of Pseudomonas fluorescens PBAP-27 talc-based formulation (lg/ 100 ml; population 106 cfu/ml) was added and mixed thoroughly under aseptic condition, e) after 2 weeks of incubating at 30+2°C the Pseudomonas fluorescens multiplied to 1014 cfu/g air dried cow dung, f) Pseudomonas fluorescens colonized cow dung was dried, powdered and formulated with talc containing carboxymethyl cellulose (1% w/w) or mineral oil Paraffinic Petroleum Oil to obtain final concentration of ≥ 2 x 108 cfu/g or per ml formulation. 2. The process for mass multiplication of Pseudomonas fluorescens on cow dung substantially as herein described with reference to, and as illustrated by, the examples.

Full Text

FIELD OF INVENTION
This invention relates to the process for mass multiplication of Pseudomonas fluorescens on cow dung.
BACKGROUND OF INVENTION
Pseudomonas fluorescens is one of the most important biocontrol agents and biofertilizers for the management of plant health. Success of a biocontrol agent depends not only on its bioefficacy or shelf life but also the easiness with which it can be mass multiplied on a suitable substrate, which is easily obtainable and comparatively inexpensive. Pseudomonas spp. have been grown on a wide range of liquid media both for small scale and mass multiplication (King et al. 1954, Wollum 1982, Slininger and Shea-Wilbur 1995). However, ingredients of these media are relatively expensive. There is no report to grow this bacterium on solid substrate.
In present .invention Pseudomonas fluorescens was mass multiplied on an easily available and inexpensive substrate i.e. cow dung as solid fermentation. There is no other ingredient involved. Yield is very high (>1014 cfu per g air dried cow dung). Bioefficacy of P. fluorescens multiplied on cow dung was better than any other reported media. Cow dung supports Pseudomonas fluorescens even after its application to soil, seed or foliage.
A number of commercial formulations are available in the market for multiplication of Pseudomonas fluorescens but present technology will
greatly help in its mass production on a most easily available and inexpensive substrate, which is available and used throughout the world as organic manure.
OBJECTS OF THE INVENTION
The main object of the invention is to propose a process for mass multiplication of Pseudomonas fluorescens on cow dung which uses solid substrate.
Another object of the invention is to propose a process for mass multiplication of Pseudomonas fluorescens on cow dung which gives very high quantity (approximately ≥ 1014cfu per g air dried cow dung).
Yet another object of this invention is to propose a process for mass multiplication of Pseudomonas fluorescens on cow dung wherein substrate cow dung is inexpensive and easily available.
Another object of this invention is to propose a process for mass multiplication of Pseudomonas fluorescens on cow dung wherein temperature and moisture requirements are standardized for better multiplication.
Yet another object of this invention is to propose a process for mass multiplication of Pseudomonas fluorescens on cow dung which does not use expensive ingredients like yeast extract, peptone etc.DRAW BACKS IN THE EXISTING STATE-OF-ART
Most of the substrates/culture media that are presently in use for mass culture of Pseudomonas fluorescens contain expensive ingredients (e.g. yeast extract, peptone etc.) and they do not support the biocontrol agent after its application in the yield. Since these media are highly enriched, inoculum produced on them has relatively poor parasite fitness.
STATEMENT OF INVENTION
According to this invention there is provided a process for mass multiplication of Pseudomonas fluorescens on cow dung comprising the steps of air drying of cow dung by spreading to form layer of 2.5 thickness under open shade for 5 to 7 days, maintaining the moisture content to 40% by weight of air dried cow dung by adding water, maintaining the pH of cow dung between 7 to 7.5, autoclaving the cow dung of step b) at 15 lbs for 30 minutes, 2 ml to 5 ml suspension of Pseudomonas fluorescens PBAP-27 talc-based formulation (1g/ 100 ml; population 106 cfu/ml) was added and mixed thoroughly under aseptic condition, after 2 weeks of incubating at 30+2°C the Pseudomonas fluorescens multiplied to 1014 cfu/g air dried cow dung, Pseudomonas fluorescens colonized cow dung was dried, powdered .and formulated with talc containing carboxymethyl cellulose (1% w/w) or mineral oil Paraffinic Petroleum Oil to obtain final concentration of ≥ 2 x 108 cfu/g or per ml formulation.
Figure-1. Multiplication of Pseudomonas fluorescens on cow dung in flask.
Figure-2. Multiplication of Pseudomonas fluorescens on poly bags.
DETAILED DESCRIPTION OF THE INVENTION:
The antagonist Pseudomonas fluorescens isolate PBAT-27 was maintained and sub-cultured on King's B agar medium at 28°C. It was multiplied on King's B broth (King et al. 1954) for 5 days at 28°C and then mixed with talcum powder containing carboxymethyl cellulose (CMC)(@ 1%, w/w), air dried, ground and sieved to obtain the commercial powder formulation. This powder was suspended in sterilized distilled water (@ 1 g/ 100 ml; approx. population 106 cfu/ ml) and used for inoculation of cow dung.
Multiplication of Pseudomonas fluorescens on cow dung in flasks:
Fresh cow dung was collected and air dried by spreading as an approximately 2.5 cm thick layer under open shade for 5 to 7 days. Fifty g of air-dried (moisture content~30% when estimated by oven drying for 24 h at 40°C) cow dung was taken in 250 ml Erlenmeyer flasks. pH of the cow dung was 7 to 7.5. Moisture content of cow dung was adjusted to 40% by weight of air-dried cow dung by adding water. Flasks were then autoc laved at 15 lbs psi for 30 minutes. Two ml suspension of Pseudomonas fluorescens PBAP-27 formulated powder (@ 1 g/ 100 ml) was added in each flask and mixed thoroughly under aseptic conditions. Inoculated flasks were incubated in a BOD incubator at 30±2°C (Figure 1). After two weeks of incubation the population of Pseudomonas fluorescens could go as high as (≥ 14 cfu/g of air dried sample). Colonized cow dung was air dried and powdered with the help of mixer and grinder. Powdered cow dung was mixed with required quantity of talc powder containing 1% carboxymethyl cellulose (CMC) by weight to obtain final population of ≥ 2 x 108 cfu/g formulation and moisture
content of ≤ 12%. Its oil based formulation can be also prepared by suspending colonized FYM in mineral oil (Paraffinic Petroleum Oil) containing emulsifier, stabilizer and anti-oxidant present in the final product at a concentration of 1.2%).
Multiplication of Pseudomonas fluorescens on cow dung in Poly bags:
Fresh cow dung was collected and air dried by spreading as an approximately 2.5 cm thick layer under open shade for one week. Two hundred g of air-dried (moisture content approx. 30% when estimated by oven drying for 24 h at 40°C) cow dung was taken in one kg capacity transparent autoclavable polypropylene bags. pH of the cow dung was 7 to 7.5. Moisture content of cow dung was adjusted to 40% by weight of air-dried cow dung by adding water. The bags were then autoclaved at 15 lbs psi for 30 minutes. Five ml suspension of Pseudomonas fluorescens PBAP-27 spore powder (@ 1 g / 100 ml) was injected in each bag with the help of a disposable syringe and the point of injection was sealed with cellophane tape (Figure 2). Inoculated bags were incubated in a BOD incubator at 30+2 °C. After two weeks of incubation the population of Pseudomonas fluorescens could go as high as 10 x 1013 cfu per g of air dried sample. Colonized cow dung was air dried and powdered with the help of mixer and grinder. Powdered cow dung was mixed with required quantity of talc powder containing 1% carboxymethyl cellulose (CMC) by weight to obtain final population of ≥ 2 x 108 cfu per g formulation and
moisture content of ≤ 12%. Its oil based formulation can be also prepared by suspending colonized FYM in mineral oil (Paraffinic Petroleum Oil) containing emulsifier, stabilizer and anti-oxidant present in the final product at a concentration of 1.2%).
It is to be noted that the formulation of the present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant formulations are intended to be within the scope of the present invention which is further set forth under the following claims:-

WE CLAIM:
1. A process for mass multiplication of Pseudomonas fluorescens on
cow dung comprising the steps of:-
a) air drying of cow dung by spreading to form layer of 2.5 thickness under open shade for 5 to 7 days, maintaining the moisture content to 40% by weight of air dried cow dung by adding water,
b) maintaining the pH of cow dung between 7 to 7.5,
c) autoclaving the cow dung of step b) at 15 lbs for 30 minutes,
d) 2 ml to 5 ml suspension of Pseudomonas fluorescens PBAP-27 talc-based formulation (lg/ 100 ml; population 106 cfu/ml) was added and mixed thoroughly under aseptic condition,
e) after 2 weeks of incubating at 30+2°C the Pseudomonas fluorescens multiplied to 1014 cfu/g air dried cow dung,
f) Pseudomonas fluorescens colonized cow dung was dried, powdered and formulated with talc containing carboxymethyl cellulose (1% w/w) or mineral oil Paraffinic Petroleum Oil to obtain final concentration of ≥ 2 x 108 cfu/g or per ml formulation.
2. The process for mass multiplication of Pseudomonas fluorescens on
cow dung substantially as herein described with reference to, and
as illustrated by, the examples.

Documents:

2197-DEL-2006-Abstract-(23-07-2010).pdf

2197-del-2006-abstract.pdf

2197-DEL-2006-Claims-(18-05-2009).pdf

2197-DEL-2006-Claims-(23-07-2010).pdf

2197-del-2006-claims.pdf

2197-DEL-2006-Correspondence-Others-(11-03-2010).pdf

2197-DEL-2006-Correspondence-Others-(18-05-2009).pdf

2197-DEL-2006-Correspondence-Others-(23-07-2010).pdf

2197-del-2006-correspondence-others-1.pdf

2197-del-2006-correspondence-others.pdf

2197-DEL-2006-Description (Complete)-(18-05-2009).pdf

2197-DEL-2006-Description (Complete)-(23-07-2010).pdf

2197-del-2006-description (complete).pdf

2197-DEL-2006-Form-1-(18-05-2009).pdf

2197-del-2006-form-1.pdf

2197-del-2006-form-18.pdf

2197-DEL-2006-Form-2-(18-05-2009).pdf

2197-del-2006-form-2.pdf

2197-del-2006-form-26.pdf

2197-DEL-2006-Form-3-(18-05-2009).pdf

2197-DEL-2006-Form-5-(23-07-2010).pdf

2197-DEL-2006-Petition 137-(23-07-2010).pdf

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Patent Number

242067

Indian Patent Application Number

2197/DEL/2006

PG Journal Number

33/2010

Publication Date

13-Aug-2010

Grant Date

09-Aug-2010

Date of Filing

05-Oct-2006

Name of Patentee

G.B. PANT UNIVERSITY OF AGRICULTURE AND TECHNOLOGY

Applicant Address

PANTNAGAR 263145, INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. UMA SHANKAR SINGH	PROFESSOR, DEPARTMENT OF PLANT PATHOLOGY, COLLEGE OF AGRICULTURE, G.B. PLANT UNIVERSITY OF AGRICULTURE AND TECHNOLOGY, PANTNAGAR, 263145, U.S. NAGAR, UTTARANCHAL
2	DR. NAJAM WARIS ZAIDI	PROFESSOR, DEPARTMENT OF PLANT PATHOLOGY, COLLEGE OF AGRICULTURE, G.B. PLANT UNIVERSITY OF AGRICULTURE AND TECHNOLOGY, PANTNAGAR, 263145, U.S. NAGAR, UTTARANCHAL

PCT International Classification Number

C12N

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA