Title of Invention	"A PROCESS FOR PRODUCING A BIO-PESTICIDE COMPOSITION CONTAINING Trichoderma harzianum AND Pseudomonas fluorescens"
Abstract	This invention relates to a process for the production of organic and combination formulation of bio-pesticide containing Trichoderma harzianum and Pseudomonas fluorescens comprising preparation of mother culture, liquid fermentation as well as solid fermentation of T. harzianum, preparation of mother culture liquid fermentation as well as solid fermentation of P. fluorescens sepreately, followed by mixing both the bio-pesticides in the proportion of 1-2:1-2 to get the final combination formulation.

Title of Invention

"A PROCESS FOR PRODUCING A BIO-PESTICIDE COMPOSITION CONTAINING Trichoderma harzianum AND Pseudomonas fluorescens"

Abstract

This invention relates to a process for the production of organic and combination formulation of bio-pesticide containing Trichoderma harzianum and Pseudomonas fluorescens comprising preparation of mother culture, liquid fermentation as well as solid fermentation of T. harzianum, preparation of mother culture liquid fermentation as well as solid fermentation of P. fluorescens sepreately, followed by mixing both the bio-pesticides in the proportion of 1-2:1-2 to get the final combination formulation.

Full Text	Field of Invention; This invention relates to a process for producing a bio-pesticide composition containing Trichoderma harzianum and Pseudomonas fluorescens. Background of Invention; A standardized process for mass production of Trichoderma harzianum Rifai and Pseudomonas fluorescens (Migula) separately is known in the art. Such a process included liquid fermentation step and not solid fermentation. As it is, liquid fermentation process alone would result in the end product of bio-control agent formulation of inferior quality. Hence, by this process it is not economical to produce a bio-pesticide of T. harzianum or P. fluorescens. For the management of various soil borne pathogens, formulation of T. harzianum is being used widely on the various agricultural crops. Similarly formulation of P. fluorescens is also widely used for the management of various soil borne pathogens infecting various agricultural crops. There are also several reports on the efficacy of these two potential bio-agents against various soil borne pathogens infecting various agricultural crops. However, there is no organic product of combination formulation of these two bio-agents.OBJECTS OF THE INVENTION An object of this invention is to propose a process for liquid and solid fermentation of Trichoderma harzianum using organic materials. Another object of the present invention is to propose a process for liquid and solid fermentation of Pseudomonas fluorescens using organic material. Yet another object of this invention is to propose a preparation containing T.harzianum and P.fluorescens which is highly effective for the management of various soil borne pathogens on agricultural crops. STATEMENT OF INVENTION According to this invention there is provided a process for the production of organic and combination formulation of bio-pesticide containing Trichoderma harzianum and Pseudomonas fluorescens comprising preparation of mother culture, liquid fermentation as well as solid fermentation of T.harzianum, preparation of mother culture liquid fermentation as well as solid fermentation of P. fluorescens sepreately, followed by mixing both the bio-pesticides in the proportion of 1-2:1-2 to get the final combination formulation. DETAILED DESCRIPTION OF THE INVENTION; The process of mass production of combination formulation of T.harzianum and P.fluorescens include (a) liquid as well as solid fermentation of T.harzianum (b) liquid as well as solid fermentation of P. fluorescens separately. 1. Fermentation of Trichoderma harzianunv Preparation of Mother culture: The isolate is sub cultured on potato dextrose agar medium, which is incubated under dark condition. The discs are cut and inoculate into a conical flask containing Potato Dextrose Broth (PDB) (Hi-medical), and incubated, which can be used for the liquid fermentation. Liquid fermentation for the production of Trichoderma harzianum In the liquid fermentation process, organic materials (4-8%, preferably 5% neem (Azadirachta indica) cake suspension + 4-8%, preferably 5% pongamia cake extract (Pongamia pinnata) + 0.02-0.05%, preferably 0.03% sugar cane molasses in the ratio of 1:1:1) are used as substrate. The substrate is autoclaved at 121° C (15 PSI) for 20 min. for its use in the liquid fermentation process. This is unique feature in this process. The liquid concentration of neem and pongamia aqueous extracts are prepared by pre-soaking the oil cakes and later decanted and filtered using muslin cloth. The required concentration of sugar cane molasses (organic material) is prepared by dissolving the molasses (v/v) in a known amount of tap water. All the ingredients are mixed and used as the substrate in the liquid fermentation process. Such as substrate is sterilized in the fermenter. After it is cooled, it is added with the mother culture of the bio-agent prepared using Potato Dextrose Broth (PDB) (Hi-media) (the preparation of the mother culture is mentioned above). The fungal mat is harvested in the aseptic conditions and it is used for the solid fermentation. Solid fermentation for the production of Trichoderma harzianum Solid fermentation is an important step and contributes tremendously for the mass production process making it more efficient and economical. In this stage, organic materials [(neem cake (de oiled cake) + pongamia cake (de oiled cake) + wheat bran in the ratio of 1-3:1-3:1-2, preferably 1:1:2] are used as substrate in the solid fermentation of Trichoderma harzianum. The substrate is autoclaved at 121°C (15 PSI) for 20 min. for use in the solid fermentation process. The fungal mat obtained through the liquid fermentation is mixed and filled in a tray with the lid and should be left for 7 days for the solid fermentation. The solid substrate enables the bio-control fungus to produce the maximum number of spores per gram of substrate. Liquid fermentation Pseudomonas fluorescens: Preparation of Mother culture of P. fluorescens The isolate has to be sub cultured on King's B- medium. It should be incubated for 24-36 hours 30 + 1° C. Later with the help of cork borer (7mm) the discs should be cut and inoculate into the 500 ml conical flask containing 250 ml of King's B broth. The has to be incubated at 30 + 1°C for 24-36 hours and this can be used in the liquid fermentation process. Liquid fermentation of Pseudomonas fluorescens: Liquid fermentation of P. fluorescens is carried out using a fermenter. Initially, the medium be used will be required quantity of King's B broth. The medium is sterilized and culture of P. fluorescens is inoculated to the sterilized broth. Afterwards the liquid fermentation is allowed. Later on this harvested under aseptic conditions and used for solid fermentation. Composition of King's B medium (Table Removed) Solid fermentation of P. fluorescens: Pseudomonas fluorescens obtained through the liquid fermentation has to be used for the solid fermentation using organic materials. The composition of the substrate (organic materials) to be used in the solid fermentation Pongamia (de oiled) cake: Neem (de oiled) cake: Wheat Bran (1:1:2). These materials have to be autoclaved at 121°C (15 PSI) for 20 min. Inoculation of P. fluorescens on to organic materials in the solid fermentation; Pseudomonas fluorescens obtained through the liquid fermentation has to be mixed at rate of 25 ml per kg of the above substrate filled in a tray with the lid and should be left for 10 days for the solid fermentation. Production of the organic combination formulation (final product) of the bio-agents T. harzianum and P. fluorescens: The material obtained through the solid fermentation of P. fluorescens can be packed in one or five or ten kg pockets in aseptic conditions. Hence the organic materials (Neem (de oiled) cake + Pongamia (de oiled) cake Wheat Bran) will become carrier material of the formulation or product of this bio-agent. Characteristics of formulation: • Neem and pongamia cakes were used as the carrier of the formulation of the product. This is also unique since this improve the establishment of the bio-control agent in the soil, it will be economical, eco-friendly and fits in the organic farming practices. • The final product will have Colony Forming Units (CFU) of T. harzianum to a level of 2.5 x 109/g and Colony Forming Units (CFU) of P. fluorescens to a level of 2.7 x 109/g. (Structure Removed) • The final product will have Colony Forming Units (CFU) of T. harzianum to a level of 2.5 x 109/g and Colony Forming Units (CFU) of P. fluorescens to a level of 2.7 x 109/g. • Moisture percentage - 7.5% • Shelf life of the product - 12 months • In the process, organic materials [neem cake (de oiled cake): pongamia cake (de oiled cake): Wheat Bran in the ratio of 1:1:2] are used as substrate in the solid fermentation of Pseudomonas fluorescens. This is a unique feature in this process. • After the solid fermentation of T. karzianum and P. fluorescens separately, both the bio-pesticides are mixed at in the proportion of 1-2:1-2, preferably 1:1 to get the final combination formulation of these two bio-agents. These two bio-agents are compatible and will have a shelf life of 12 months. • Organic materials [neem cake (de oiled cake) + pongamia cake (de oiled cake) + wheat Bran in the ratio of 1:1:2] act as the carrier of the formulation of final product. This is also an unique feature in this process. 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 the production of organic and combination formulation of bio-pesticide containing Trichoderma harzianum and Pseudomonas fluorescens comprising preparation of mother culture, liquid fermentation as well as solid fermentation of T.harzianum, preparation of mother culture liquid fermentation as well as solid fermentation of P. fluorescens sepreately, followed by mixing both the bio-pesticides in the proportion of 1-2:1-2 to get the final combination formulation. 2. The process as claimed in claim 1, wherein liquid fermentation of T-harzianum is done by mixing neem cake suspension 4-8%, pongamia cake suspension 4-8%, sugarcane molasses 0.02-0.05%. 3. The process as claimed in claim 1, wherein solid fermentation of T.harzianum is done by using neem cake, pongamia cake and wheat bran in 1:1:2 ratio as sinstrate. 4. The process as claimed in claim 1, wherein liquid fermentation of P.fluorescens is done in kings' B broth. 5. The process as claimed in claim 1, wherein solid fermentation of P.fluorescens is done by using neem cake, pongamia cake and wheat bran in 1:1:2 ratio as substrate. 6. The process as claimed in claim 1, wherein the combination formulation of the two bio-pesticides is done by mixing T.harzianum and P. fluorescens after solid fermentation in the proportion of 1-2:1-2. 7. The process as claimed in claim 1, wherein organic materials like de oiled neem cake, de oiled pongamia cake and wheat bran in the ratio of 1:1:2 act as the carrier of the formulation of the final product.

Full Text

Field of Invention;
This invention relates to a process for producing a bio-pesticide composition containing Trichoderma harzianum and Pseudomonas fluorescens.
Background of Invention;
A standardized process for mass production of Trichoderma harzianum Rifai and Pseudomonas fluorescens (Migula) separately is known in the art. Such a process included liquid fermentation step and not solid fermentation. As it is, liquid fermentation process alone would result in the end product of bio-control agent formulation of inferior quality. Hence, by this process it is not economical to produce a bio-pesticide of T. harzianum or P. fluorescens.
For the management of various soil borne pathogens, formulation of T. harzianum is being used widely on the various agricultural crops. Similarly formulation of P. fluorescens is also widely used for the management of various soil borne pathogens infecting various agricultural crops. There are also several reports on the efficacy of these two potential bio-agents against various soil borne pathogens infecting various agricultural crops. However, there is no organic product of combination formulation of these two bio-agents.OBJECTS OF THE INVENTION
An object of this invention is to propose a process for liquid and solid fermentation of Trichoderma harzianum using organic materials.
Another object of the present invention is to propose a process for liquid and solid fermentation of Pseudomonas fluorescens using organic material.
Yet another object of this invention is to propose a preparation containing T.harzianum and P.fluorescens which is highly effective for the management of various soil borne pathogens on agricultural crops.
STATEMENT OF INVENTION
According to this invention there is provided a process for the production of organic and combination formulation of bio-pesticide containing Trichoderma harzianum and Pseudomonas fluorescens comprising preparation of mother culture, liquid fermentation as well as solid fermentation of T.harzianum, preparation of mother culture liquid fermentation as well as solid fermentation of P. fluorescens sepreately, followed by mixing both the bio-pesticides in the proportion of 1-2:1-2 to get the final combination formulation.
DETAILED DESCRIPTION OF THE INVENTION;
The process of mass production of combination formulation of T.harzianum and P.fluorescens include (a) liquid as well as solid fermentation of T.harzianum (b) liquid as well as solid fermentation of P. fluorescens separately.
1. Fermentation of Trichoderma harzianunv
Preparation of Mother culture:
The isolate is sub cultured on potato dextrose agar medium, which is incubated under dark condition. The discs are cut and inoculate into a conical flask containing Potato Dextrose Broth (PDB) (Hi-medical), and incubated, which can be used for the liquid fermentation.
Liquid fermentation for the production of Trichoderma harzianum
In the liquid fermentation process, organic materials (4-8%, preferably 5% neem (Azadirachta indica) cake suspension + 4-8%, preferably 5% pongamia cake extract (Pongamia pinnata) + 0.02-0.05%, preferably 0.03% sugar cane molasses in the ratio of 1:1:1) are used as substrate. The substrate is autoclaved at 121° C (15 PSI) for 20 min. for its use in the liquid fermentation process. This is unique feature in this process.
The liquid concentration of neem and pongamia aqueous extracts are prepared by pre-soaking the oil cakes and later decanted and filtered using muslin cloth.
The required concentration of sugar cane molasses (organic material) is prepared by dissolving the molasses (v/v) in a known amount of tap water. All the ingredients are mixed and used as the substrate in the liquid fermentation process. Such as substrate is sterilized in the fermenter. After it is cooled, it is added with the mother culture of the bio-agent prepared using Potato Dextrose Broth (PDB) (Hi-media) (the
preparation of the mother culture is mentioned above). The fungal mat is harvested in the aseptic conditions and it is used for the solid fermentation.
Solid fermentation for the production of Trichoderma harzianum
Solid fermentation is an important step and contributes tremendously for the mass production process making it more efficient and economical. In this stage, organic materials [(neem cake (de oiled cake) + pongamia cake (de oiled cake) + wheat bran in the ratio of 1-3:1-3:1-2, preferably 1:1:2] are used as substrate in the solid fermentation of Trichoderma harzianum. The substrate is autoclaved at 121°C (15 PSI) for 20 min. for use in the solid fermentation process.
The fungal mat obtained through the liquid fermentation is mixed and filled in a tray with the lid and should be left for 7 days for the solid fermentation.
The solid substrate enables the bio-control fungus to produce the maximum number of spores per gram of substrate.
Liquid fermentation Pseudomonas fluorescens:
Preparation of Mother culture of P. fluorescens
The isolate has to be sub cultured on King's B- medium. It should be incubated for 24-36 hours 30 + 1° C. Later with the help of cork borer (7mm) the discs should be cut and inoculate into the 500 ml conical flask containing 250 ml of King's B broth. The has to be incubated at
30 + 1°C for 24-36 hours and this can be used in the liquid fermentation process.
Liquid fermentation of Pseudomonas fluorescens:
Liquid fermentation of P. fluorescens is carried out using a fermenter. Initially, the medium be used will be required quantity of King's B broth. The medium is sterilized and culture of P. fluorescens is inoculated to the sterilized broth. Afterwards the liquid fermentation is allowed. Later on this harvested under aseptic conditions and used for solid fermentation.
Composition of King's B medium
(Table Removed)
Solid fermentation of P. fluorescens:
Pseudomonas fluorescens obtained through the liquid fermentation has to be used for the solid fermentation using organic materials.

The composition of the substrate (organic materials) to be used in the solid fermentation Pongamia (de oiled) cake: Neem (de oiled) cake: Wheat Bran (1:1:2).
These materials have to be autoclaved at 121°C (15 PSI) for 20 min.
Inoculation of P. fluorescens on to organic materials in the solid fermentation;
Pseudomonas fluorescens obtained through the liquid fermentation has to be mixed at rate of 25 ml per kg of the above substrate filled in a tray with the lid and should be left for 10 days for the solid fermentation.
Production of the organic combination formulation (final product) of the bio-agents T. harzianum and P. fluorescens:
The material obtained through the solid fermentation of P. fluorescens can be packed in one or five or ten kg pockets in aseptic conditions. Hence the organic materials (Neem (de oiled) cake + Pongamia (de oiled) cake Wheat Bran) will become carrier material of the formulation or product of this bio-agent.
Characteristics of formulation:
• Neem and pongamia cakes were used as the carrier of the formulation of the product. This is also unique since this improve the establishment of the bio-control agent in the soil, it will be economical, eco-friendly and fits in the organic farming practices.
• The final product will have Colony Forming Units (CFU) of T.
harzianum to a level of 2.5 x 109/g and Colony Forming Units
(CFU) of P. fluorescens to a level of 2.7 x 109/g.
(Structure Removed)
• The final product will have Colony Forming Units (CFU) of T.
harzianum to a level of 2.5 x 109/g and Colony Forming Units
(CFU) of P. fluorescens to a level of 2.7 x 109/g.
• Moisture percentage - 7.5%
• Shelf life of the product - 12 months
• In the process, organic materials [neem cake (de oiled cake):
pongamia cake (de oiled cake): Wheat Bran in the ratio of 1:1:2] are
used as substrate in the solid fermentation of Pseudomonas
fluorescens. This is a unique feature in this process.
• After the solid fermentation of T. karzianum and P. fluorescens separately, both the bio-pesticides are mixed at in the proportion of 1-2:1-2, preferably 1:1 to get the final combination formulation of these two bio-agents. These two bio-agents are compatible and will have a shelf life of 12 months.
• Organic materials [neem cake (de oiled cake) + pongamia cake (de oiled cake) + wheat Bran in the ratio of 1:1:2] act as the carrier of the formulation of final product. This is also an unique feature in this process.
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 the production of organic and combination formulation of bio-pesticide containing Trichoderma harzianum and Pseudomonas fluorescens comprising preparation of mother culture, liquid fermentation as well as solid fermentation of T.harzianum, preparation of mother culture liquid fermentation as well as solid fermentation of P. fluorescens sepreately, followed by mixing both the bio-pesticides in the proportion of 1-2:1-2 to get the final combination formulation.
2. The process as claimed in claim 1, wherein liquid fermentation of T-harzianum is done by mixing neem cake suspension 4-8%, pongamia cake suspension 4-8%, sugarcane molasses 0.02-0.05%.
3. The process as claimed in claim 1, wherein solid fermentation of T.harzianum is done by using neem cake, pongamia cake and wheat bran in 1:1:2 ratio as sinstrate.
4. The process as claimed in claim 1, wherein liquid fermentation of P.fluorescens is done in kings' B broth.
5. The process as claimed in claim 1, wherein solid fermentation of P.fluorescens is done by using neem cake, pongamia cake and wheat bran in 1:1:2 ratio as substrate.
6. The process as claimed in claim 1, wherein the combination formulation of the two bio-pesticides is done by mixing T.harzianum and P. fluorescens after solid fermentation in the proportion of 1-2:1-2.
7. The process as claimed in claim 1, wherein organic materials like de oiled neem cake, de oiled pongamia cake and wheat bran in the ratio of 1:1:2 act as the carrier of the formulation of the final product.

Documents:

434-DEL-2006-Abstract-(09.09.2010).pdf

434-del-2006-abstract.pdf

434-DEL-2006-Claims-(09.09.2010).pdf

434-del-2006-claims.pdf

434-del-2006-Correspondence Others-(05-07-2011).pdf

434-del-2006-Correspondence-Others-(06-04-2011).pdf

434-DEL-2006-Correspondence-Others-(09-09-2010).pdf

434-del-2006-correspondence-others.pdf

434-del-2006-correspondence-po.pdf

434-DEL-2006-Description (Complete)-(09.09.2010).pdf

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

434-del-2006-description (provisional).pdf

434-del-2006-form-1.pdf

434-del-2006-form-18.pdf

434-del-2006-form-2.pdf

434-del-2006-Form-3-(05-07-2011).pdf

434-del-2006-form-5.pdf

434-del-2006-GPA-(05-07-2011).pdf

434-del-2006-Petition-137-(05-07-2011).pdf

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Patent Number

250779

Indian Patent Application Number

434/DEL/2006

PG Journal Number

04/2012

Publication Date

27-Jan-2012

Grant Date

27-Jan-2012

Date of Filing

16-Feb-2006

Name of Patentee

INDIAN INSTITUTE OF HORTICULTURAL RESEARCH

Applicant Address

HESSARGHATTA LAKE(PO), BANGALORE-580089

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. MAHENDRAKAR SREENIVASA RAO	INDIAN INSTITUTE OF HORTICULTURAL RESEARCH, HESSARGHATTA LAKE (PO)., BANGALORE-560 089, INDIA
2	DR. N. RAMACHANDRAN	INDIAN INSTITUTE OF HORTICULTURAL RESEARCH, HESSARGHATTA LAKE (PO)., BANGALORE-560 089, INDIA

PCT International Classification Number

A01N 65/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA