WO2011099025A1

WO2011099025A1 - Preparation of bio fungicide based on pseudomonas fluorescens for controlling foilage and soil born diseases

Info

Publication number: WO2011099025A1
Application number: PCT/IN2010/000183
Authority: WO
Inventors: Chetan S. Patel
Original assignee: Patel, Babubhai, C.
Priority date: 2010-02-09
Filing date: 2010-03-24
Publication date: 2011-08-18

Abstract

This invention is based upon composition and method of preparing a Bio Fungicide from innovative combination of pure culture of bacteria named Pseudomonas florescense with enzymes, fats and growth promoting molecules to control foliage and soil borne diseases like root rot, set rot, wilt, collar rot, red rot, rhizome rot, damping off, stem rot, club rot and alike and as well as managing disease like blight, blast, all leaf spot, anthracnose and alike.

Description

PREPARATION OF BIO FUNGICIDE BASED ON PSEUDOMONAS FLUORESCENS FOR CONTROLLING FOILAGE AND SOIL BORN DISEASES

PREAMBLE OF INVENTION- This invention is particular described the nature of the invention and the manner in which it is to be performed.

FIELD OF INVENTION- The present invention relates generally to a composition and method of making bacterial based product containing Pseudomonas fluoresceins for controlling various Foliage and soil born disease.

PRIOR ART-

In the existing system as given in India Patent Application No. 1019/DEL/2007 - Patent Application, wherein the invention relates to a process for the production of organic formulation of bio pesticide containing Pseudomonas Fluorescens comprising preparation of Mother culture of P. fluorescens and inoculating in King"s B broth, at 30 + 1° C for 24-36 hours followed by liquid fermentation process in 5-10% pongamia cake aqueous extract, 5-10% neem cake aqueous extract, 0.3-0.5% sugarcane molasses and ing"s B broth followed by solid fermentation using sterile Pongamia deoiled cake, Neem deoiled cake, Wheat Bran.

In the existing system as given in Indian Patent Application No. 1621/DEL/2005 - Patent Application wherein disclosed is a novel process for commercial production of biopesticides has been invented. The process involves two steps. In the first step, mass culture or stock culture of biocontrol fungi and bacteria is prepared on saw dust, soil and molasses mixture. The second step involves immobilization of the bioagents on a fly ash based carrier. Using the process following three biopesticides based on Trichoderma harzianum, Pochonia chlamydosporia and Pseudomonas fluorescens have been developed. The three biopesticides with their names and the diseases they control are as follows. 1. Biowilt-X is a Biofungicide of Trichoderma harzianum to control fusarial wilt disease caused by fusarium spp. 2. Bionem-X is a Bionematicide of Pochonia chlamydosporia to control root-knot disease caused by Meloidogyne spp. 3.

In the existing system as given in Indian Patent Application No. 2197/DEL/2006 - Patent Application wherein the 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; approx. 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 to obtain final concentration of £ 2 x 108 cfu/g or per ml formulation.

In the existing system as given in Indian Patent Application No. 2621/CHENP/2004, - Patent Application wherein the invention relates to a low cost method of producing peptides, including antimicrobial peptides (AMPs), by using microbes. The subject methods enable greatly improved yields of the peptide/AMP as compared to those heretofore known in the art. The subject methods also surprisingly enable the use of Pseudomonas fluorescens to produce AMPs and other peptides. There are several components of the subject invention, which can be used alone or in combination. The subject invention provides for the production of peptides/AMPs in concatemeric precursors. The subject invention also provides novel methods of assembling monomers into multimers, and of cleaving the multimers to yield active monomers. The subject invention also relates to the use of these multimers fused to carrier peptides to produce fusion proteins. Preferably, both the multimers and the fusion proteins (multimers with the carrier polypeptides) lack charge balancing. It has been surprisingly determined that it is not necessary to offset the positive charges of multiple copies of AMPs in multimeric constructs. Thus, the subject invention enables the use of a wider range of multimers and carrier peptides.

In the existing system as given in United States Patent Application No. 5348742 - Granted Patent wherein the invention relates to Purified bacterial strains that are effective for the inhibition of plant pathogens, including the fungi Rhizoctonia solani and Pythium ultimum have been isolated. These strains are useful as biocontrol agents, and can be used to produce antifungal metabolites, such as antibiotic compounds, active against the plant pathogenic fungi Rhizoctonia solani and Pythium ultimum. Both the purified bacterial strains and the antibiotic compounds can be used as active agents for biocontrol compositions.

OBJECT OF THE INVENTION Traditionally, the known insecticides or fungicides have different method of its preparation and composition. The present invention is based upon novel composition and method of making bacterial based product containing Pseudomonas fluorescens for controlling various foliage and soil born disease. STATEMENT OF INVENTION- The inventor has invented a composition and method of making a bacterial based product to control various foliage and soil born disease.

DETAILED DESCRIPTION OF INVENTION-

Background of Invention: This invention relates to a method for utilizing bacterial based product containing Psendomonas fluorescens to control various soil borne diseases like root rot, set rot, wilt, collar rot, red rot, rhizome rot, damping off, stem rot, club rot and alike and as well as managing disease like blight, blast, all leaf spot, anthracnose and alike.

Soil borne disease have until recently, received less attention than plant diseases affecting the shoot and foliage. However, this is not a reflection of their economic importance, but rater of the difficulties in investigating and detecting pathogens below soil level. Many soil born diseases are stress related and it is in the tropics where crop growth is particularly limited by environmental stress, predisposing crops to infection by soil borne pathogen.

The present invention is art innovative combination of bacterial based product containing Pseudomonas fluorescens with enzymes, fats and growth promoting molecules to control soil borne diseases and manage disease like blight, blast, all leaf spot, anthracnose and alike.

Material and Method used in preparing the above said innovative combination is as under Step-1 Nucleus culture (Maintain culture)

Pure culture of bacteria Pseudomonas florescense) is inoculated aseptically on plate having 20 ml PAC-07 Agar media; such two to three plates are generally inoculated. Such inoculated plate is maintained in BOD incubator at 27 ± 1 °C for 6 to 7 days with 12/12 hr lighting cycle. (Composition for one liter of PAC-07 agar media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water)

Step-2 Starter culture (In 100 ml flask)

Culture grown on plate is inoculated aseptically in a flask (250 ml capacity) having 100 ml PAC-07 broth media with the help of inoculating loop, and allow to grow at 27 ± 1 °C for at least 3 to 4 days. For superior growth, flask is put on shaker with agitation of media at 150 RPM. (Composition for one liter of PAC-07 broth media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water)

Step-3 Growth culture (In 1000 ml flask)

After sufficient growth in 250 ml flask (step-2), 50 ml culture from this grown culture is inoculated aseptically in 500 ml PAC-07 broth media in a 1000 ml capacity flask for further growth at 27 ± 1 °C for at least 3 to 4 days on shaker with 150 RPM, such two flasks is prepared. (Composition for one liter of PAC-07 broth media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water)

Step-4 Seed Fermentor (In small fermentor of 10 lit capacities)

One (1) lit grown culture in step-3 is inoculated aseptically in small fermenter having 10 litter PAC-07 broth media, and allow for further growth at 27 ± 1 °C for at least 3 to 4 days with agitation by motor at 100 RPM. (Composition for one liter of PAC-07 broth W media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water)

Step-5 Production Fermentor (In Big fermentor of 200 lit capacities)

Ten (10) lit culture grown in step-4 is inoculated aseptically in Big fermenter having 200 litter PAC-07 media (Composition for one liter of PAC-07 broth media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water), and allowed for further growth at 27 ± 1 °C for at least 3 to 4 days with agitation by motor at 100 RPM

Step-6 Mixing

Final out put from big fermentor is mixed with starch @ 4 to 5 % (which act as a stabilizer) and melto dextrin @ 1 to 2 % (which acts as a drying agent) or with 5 % skim milk powder and agitated continuously by using agitator.

Step-7 Drying

Mix culture prepared as per step-6, is then Spray dried at definite 160 °C in let and 70 to 72 °C out let temp in spray dryer unit

Step-7 Collecting dry bacterial powder

Material dried in spray dryer unit is then collected in clean stainless steel vessels and stored at 5 °C up to further use.

Step-8 Mix with suitable carrier

At the time of formulation material previously dried is mixed with dextrose @ 10 % (10 gm bacterial powder with 90 gm dextrose powder). Step-9 Packging

Material prepared as above is then packed in 100 gm, 250 gm and 500 gm packing by using pouch packing machine.

Step- 10 - Distribution to farmer, retailer, distributor etc The final material prepared has to be used in following form: Dose in one Acre: In case of Seed treatment add 100 gm per seed required per hector For soil application add 100 gm to 250 gm per hector

Claims

1. A composition and method of preparing a bio fungicide from innovative combination of pure culture of bacteria named Pseudomonas florescense with enzymes, fats and growth promoting molecules to control soil borne diseases like root rot, set rot, wilt, collar rot, red rot, rhizome rot, damping off, stem rot, club rot and alike and as well as managing disease like blight, blast, all leaf spot, anthracnose and alike, the method comprising steps of nucleus culture, starter culture, growth culture, seed fermentor, production fermentor, mixing, drying, collecting, mixing with detrose, and packing the final output.

2. A composition and method of preparing bio fungicide according to claim 1, by inoculating aseptically pure culture of bacteria Pseudomonas florescense on two to three plate having 20 ml PAC-07 Agar media and maintaining in BOD incubator at 27 ± 1 °C for 6 to 7 days with 12/12 hr lighting cycle, wherein composition for one liter of PAC-07 agar media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water.

3. A composition and method of preparing bio fungicide according to claim 1, by inoculating aseptically culture grown on plate as per method cited in claim 2 in a flask of 250 ml capacity having 100 ml PAC-07 broth media with the help of inoculating loop, and allowing it to grow at 27 ± 1 °C for at least 3 to 4 days, wherein for superior growth, flask is put on shaker with agitation of media at 150 RPM, wherein composition for one liter of PAC-07 broth media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water.

4. A composition and method of preparing bio fungicide according to claim 1 , wherein after sufficient growth in 250 ml flask as cited in claim 3, 50 ml culture from this grown culture is inoculated aseptically in 500 ml PAC-07 broth media in two 1000 ml capacity flask for further growth at 27 ± 1 °C for at least 3 to 4 days on shaker with 150 RPM, wherein composition for one liter of PAC-07 broth media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water.

5. A composition and method of preparing bio fungicide according to claim 1, wherein, one (1) lit grown culture as mentioned in claim 4 is inoculated aseptically in small fermenter having 10 litter PAC-07 broth media, and allowed for further growth at 27 ± 1 °C for at least 3 to 4 days with agitation by motor at 100 RPM, wherein composition for one liter of PAC-07 broth media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water.

6. A composition and method of preparing bio fungicide according to claim 1, wherein, ten (10) lit culture grown in as per method cited in claim 5 is inoculated aseptically in Big fermenter having 200 litter PAC-07 media, wherein composition for one liter of PAC-07 broth media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water, and is allowed for further growth at 27 ± 1 °C for at least 3 to 4 days with agitation by motor at 100 RPM

7. A composition and method of preparing bio fungicide according to claim 1, wherein, final out put from big fermentor is mixed with starch @ 4 to 5 % (which act as a stabilizer) and melto dextrin @ 1 to 2 % (which acts as a drying agent) or with 5 % skim milk powder and agitated continuously by using agitator.

8. A composition and method of preparing bio fungicide according to claim 1, wherein, mixed culture prepared as per claim 7, is then Spray dried at definite 1 0 °C in let and 70 to 72 °C out let temp in spray dryer unit

9. A composition and method of preparing bio fungicide according to claim 1, wherein, material dried in spray dryer unit is then collected in clean stainless steel vessels and stored at 5 °C up to further use

10. A composition and method of preparing bio fungicide according to claim 1, wherein, at the time of formulation material previously dried is mixed with dextrose @ 10 % (10 gm bacterial powder with 90 gm dextrose powder) and the final out put is packed as a bio fungicide.