PREAMBLE OF INVENTION- This invention in particular describes 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 material and advanced method of making a bacterial formation using Pseudomonas fluorescens for controlling various diseases of crop plant.

PRIOR ART

In the existing system as given in WIPO Patent Application No. PCT/IN2007/000064, wherein, the 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 separately, followed by mixing both the bio-pesticides in the proportion of 1-2: 1-2: preferably 1 : 1 to get the final combination, formulation..

In the existing system as given in Indian Patent Application No. 1019/DEL/2007, 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

HI liquid fermentation process in 5-10% pongamia cake aqueous extract, 5-10% neem cake aqueous extract, 0.3-0.5% sugarcane molasses and King"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, wherein, the invention relates to 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. Biocomp-X is a Biofunginemacide of Pseudomonas fluorescens to control wilt disease complex caused by Meloidogyne and Fusarium spp. jointly. ^'

In the existing system as given in Indian Patent Application No. 2197/DEL/2006, 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 United Indian Patent Application No. 2621/CHENP/2004, wherein, the invention relates to 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, 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 that 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.

PARENT CASE DATE:

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application number 341 /MUM/2010, filed February, 02, 2010, now pending.

BACKGROUND OF THE INVENTION

Soil borrie 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.

This invention relates to a method for utilizing bacterial based product containing Pseudomonas fluoresence 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.

OBJECT OF THE INVENTION

Hence, the present invention has been made with the foregoing background in mind and its object resides in providing a material and method of rationally producing a high quality of method for utilizing bacterial based product containing Pseudomonas fluoresence to control various soil borne diseases at the highest economical level using raw material which can be easily procured at the most inexpensive cost.

STATEMENT OF INVENTION-

The inventor has invented a material and method for improving crop yields, utilizing bacterial based product containing Pseudomonas fluoresence 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.

DETAILED DESCRIPTION OF INVENTION-

The subject invention concerns an advanced combination of bacterial based product containing Pseudomonas fluoresence 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 fluorescens) 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 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 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 media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl 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 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 Bacterial Separation

Final out put from big fermentor is centrifuge by using advance centrifugation technology. By this separated bacteria are use for formulation of product whereas remaining liquid ..is throughout.

Step-7 Formulation

Recovered bacteria form centrifuge is mix in a set of ration with suitable carrier composed of zero TDS water etc and than homogenized it for equivalently of formulation.

Step-8 Packging

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

Step-09 - Distrubution 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 ml per seed required per hector

For soil application add 100 ml to 250 ml per hector