WO2011154963A1

WO2011154963A1 - Advance material and method of preparation of bacterial formulation using phosphorus solubilizing bacteria that makes phosphorous available to plant which are unavailable due to higher soil ph

Info

Publication number: WO2011154963A1
Application number: PCT/IN2010/000391
Authority: WO
Inventors: Chetan S. Patel
Original assignee: Patel, Babubhai C.
Priority date: 2010-06-09
Filing date: 2010-06-09
Publication date: 2011-12-15

Abstract

The subject invention concerns materials and methods for providing or enhancing phosphorous availability in plants from pure culture bacteria like Bacillus megaterium, Pseudomonas putida that makes phosphorous available to plant, which are unavailable due to higher soil PH. It also increases efficiency of chemical fertilizer.

Description

TITLE- Advance Material And Method Of Preparation Of Bacterial Formulation Using Phosphorus Solubilizing Bacteria That Makes Phosphorous Available To Plant Which Are Unavailable Due To Higher Soil Ph.

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 method of preparing a bacterial formulation using phosphorus solubilizing bacteria that makes phosphorus available to plant which are unavailable due to higher soil PH.

PRIOR ART-

In the existing system as given in United States Patent Application No. 20100093538, wherein, the invention relates to a new isolate of a Pseudomonas spp, DSM 21663, has been shown to possess unique properties. This Pseudomonas is a plant growth-promoting rhizobacterium (PGPR). Among its modes of action involved in plant growth-promotion are anti-biotic production (2,4-diacetylphloroglucinol, (DAPG); pyrrolnitrin, PRN and others), indole-3-acetic acid (IAA) production and phosphate solubilization, and production of unique volatiles. The strain is fluorescent, oxidase-positive, and has the ability to suppress soil-borne root and foliar pathogens of both fungal and bacterial origin.

[i] In the existing system as given in United States Patent Application No. 5026417, wherein, the invention relates to a method and composition for increasing the amounts of phosphorus and/or micronutrients available for uptake by plants from the soil. The invention involves introducing an inoculum of the fungus Penicillium bilaji into (or onto) the soil. This has the effect of increasing the solubility of phosphates and micronutrient sources which may be either native to the soil or added to it, e.g. in the form of insoluble rock phosphate or manufactured phosphate fertilizer. The invention can be used to increase the health, growth rates and yields of plants, especially crop plants grown on nutrient-deficient soils, while eliminating or minimizing the need for expensive manufactured fertilizers.

In the existing system as given in United States Patent Application No. 20040248738, wherein, the invention relates to a synergistic composition useful as plant and soil health enhancer, comprising urine, neem and garlic, individually or in all possible combinations, with the treatment showing it has the ability to stimulate accumulation of nutrients in the plant biomass, proliferation of plant growth promoting, phosphorous solubilizing, abiotic stress tolerant and antagonists towards plant pathogenic fungi, control phytopathogenic fungi in the rhizosphere of plants, and enhances the total phenolic contents of the plants.

In the existing system as given in United States Patent Application 20090308121, wherein, the invention relates to eco-friendly compositions and methods for providing plant growth enhancing formulations comprising mixtures of microbial isolates. In particular, numerous bacterial and fungal strains were isolated from a variety of soil types, from rhizospheres and from root nodules of leguminous plants, in designed combinations, for providing plant growth and plant productivity enhancing formulations. These specifically designed polymicrobial formulations would further provide protection against plant pathogens lowering the need for nitrogen containing fertilizers, solubilize minerals, protect plants against pathogens, and make available to the plant valuable nutrients, such as phosphate, thus reducing and eliminating the need for using chemical fertilizers and chemical pesticides.

In the existing system as given in Indian Patent Application No. 290/DELNP/2005, wherein, the invention relates to a composition for biologically controlling the cryptogamic vine diseases by application of compositions of microorganisms, comprising in a mixture one or several bacteria from the Bacillus megaterium species and/or from the Bacillus subtilis species and one or several yeasts, the bacterium or bacteria and the yeast or yeasts being non-toxic to the plant, together with an inert vehicle, wherein the concentration of yeast being of 0.5 to 0.75x1010 and of bacteria of 0.30 to 0.50x1010 .

In the existing system as given in Indian Patent Application No. 00183 OLNP/2004, wherein, the invention relates to product(s) containing alive micro-organism(s) suitable for soil treatment, microorganisms multiplying under different climatic and natural circumstances, as well as the procedure for the production of the products, furthermore procedure for the treatment of the soil and plants with the products. More detailed, the invention relates to a procedure for preparing the products from any of the micro-organisms specified below, or from the mixture thereof. Furthermore, the invention relates to a procedure for the creation of the cultures of the micro-organisms to be used. Subject of the invention are the micro-organisms themselves, as well. More detailed, the invention relates to a procedure for the treatment of the soil and the plants with a product containing at least one of the micro-organisms Azospirillum brasilense ssp. SW51 (NCAIM /P/ B 001293), Azotobacter vinelandii spp. M657 (NCAIM P/ B 001291), Pseudomonas fluorescens var. SW1 1 (NCAIM I? I B 001296), Bacillus polymyxa var. SW17 (NCAIM I? I B 001295/, Bacillus megaterium var. M326 (NCAIM ΓΡΙ B 001301/ -, furthermore the products multiplying and existing in the environment of the plant in question,containing the listed micro-organisms and their production.

In the existing system as given in Indian Patent Application No. 85/CAL/2000, wherein, the invention relates to a process for preparing a novel biofertiliser strains of nitrogen fixer and phosphate solubiliser to promote soil health in particular for growth of tea plants comprising of nitrogen fixer and phosphate solubiliser in equal proportion wherein the nitrogen fixer is prepared by the steps of a) transferring the stock culture Azotobacter Chroococcum (BAH) into mother culture modified Ashley's medium, b) allowing the mother culture medium to grow for 3-7 days, c) transferring the grown mother culture to main culture medium and ^fd) allowing the main culture medium to grow ge for another 3-7 days to obtain Azotobacter choococcum (BAH) strains and in which the phosphate solubiliser is prepared from as a mixture of Bacillus subtilis (BB15), Pseudomonas Putida (BP 19) Aspergillus awamori (FA 16) and Aspergitlus flavus (FA 18) in a ratio of 5:2:2:1 in Pikovskaya's medium wherein Bacillus Subtilis (BB15) and Pseudomonas putida (BP 19) are prepared as in a manner of preparing Azotobacter Choococcum and Aspergillus awamori (FA 16) and Aspergillus flavus (FA 18) (molasses or yeast medium) are prepared by transferring the stock culture to mother culture medium, allowing the mother culture to grow for 15 days, transferring the grown mother culture to main culture medium and allowing it to grow again for another 15 days to obtain Aspergillus awamori (FA 16) and Aspergillus flavus (FA 18) strains.

In the existing system as given in Indian Patent Application No. 0083/CAL/2000, wherein, the invention relates to a process for preparing a novel biocide comprising a consortium of two bacterial and two fungal strains for controlling 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 subtil 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) al lowing the mother ϋμΐπατεβ 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 subti l is Pseudomonas putida, Trichoderma viridi and Trichoderma harzianum in a ratio of 4:3 :2: 1 to obtain the biocide.

In the existing system as given in United States Patent Application No. 7053025, wherein, the invention relates to novel strains of Pseudomonas putida, more specifically, to a novel strain of Pseudomonas putida which are tolerant to organic solvents including hydrocarbons, alcohols, ethers, ketones and their derivatives or mixtures thereof, and a mutant strain of said microorganism having the availability of genetic manipulation. The said Pseudomonas putida strains can be used in biotransformation in the presence of organic solvents and bioremediation of toxic organic compounds. Furthermore, they are useful as supply sources of resistance genes and cell fusion of organic solvent-tolerant microorganisms producing useful substances and the breeding of said microorganisms can be practiced in the art, which allows their universal use in the fields of bioreactor, liquid- waste treatment, protein engineering, etc.

In the existing system as given in WIPO Application No. PCT/US2003/027388, wherein, ? the invention is directed to isolated Bacillus subtilis, Pseudomonas putida, and

Sporobolomyces roseus which are useful as a biocontrol agent. These organisms are useful in a method of imparting to plants protection against plant pathogens by applying them to plants, plant seeds, or soil surrounding plants under conditions effective to impart disease protection to the plants or plants produced from the plant seeds. The biocontrol agents are also useful in a method of enhancing plant growth which involves applying them to plants, plant seeds, or soil surrounding plants under conditions effective to enhance growth in the plants or plants produced from the plant seeds.

PARENT CASE DATE:

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application number 338 MUM/2010, filed February, 02, 2010, now pending.

BACKGROUND OF THE INVENTION

The ability of a few soil microorganisms to convert insoluble forms of phosphorus to an accessible form is an important trait in plant growth-promoting bacteria for increasing plant yields. The use of phosphorous solubilizing bacteria as inoculants increases the P uptake by plants

The phosphorous solubilizing bacteria have the capability to change the insoluble form of phosphorus into soluble one.

As is well known by any expert in the art, there have been hitherto made a number of proposals as to a method of producing phosphorus solubulizing fertilizer. To practice the conventional methods various kinds of raw materials and processes were employed but each of them has drawbacks in terms of procurement of raw material and cost.

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 phosphorous solubulizing organic fertilizer 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, involving plant growth promoting bacteria such as Bacillus megaterium and Pseudomonas putida that makes phosphorous available to plant, which are unavailable due to higher soil PH.

Material and Method used in preparing phosphorous solubilizing bacteria is as under Step-1 Nucleus culture (Maintain culture)

Pure culture of bacteria {Bacillus megaterium and Pseudomonas putida) 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 output 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 mixed in a set of ration with suitable carrier composed of zero TDS water etc and then homogenized it for equivalently of formulation.

Step-8 Packaging

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

Step-9 - 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

Claims

1. A material and method of preparing an improved phosphorous solubilizing bacterial based product from Bacillus megathrium and Pseudomonas putida that makes phosphorous available to plant, which are un available due to higher soil PH, the method comprising steps of nucleus culture, starter culture, growth culture, seed fermentor, production fermentor, bacterial separation, formulation and packing the final output.

2. A material and method of preparing improved phosphorous solubilizing bacterial product according to claim 1, by inoculating aseptically pure culture of Bacillus megathrium and Pseudomonas putida on two or three plate having 20 ml PAC-07 Agar media and maintaining it 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 material and method of preparing improved phosphorous solubilizing bacterial product according to claim 1, wherein culture grown on plate is inoculated aseptically in a flask of 250 ml capacity having 100 ml PAC-07 broth media with the help of inoculating loop, and allowing to grow it at 27 ± 1 °C for at least 3 to 4 days, where as 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 and 1000 ml distilled water.

4. A material and method of preparing improved phosphorous solubilizing bacterial product according to claim 1, wherein after sufficient growth in 250 ml flask, 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, wherein 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.

5. A material and method of preparing improved phosphorous solubilizing bacterial product according to claim 1, One (1) lit grown culture, is inoculated aseptically in small fermenter having 10 litter PAC-07 broth media, and allowing it 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 and 1000 ml distilled water) '

6. A material and method of preparing improved phosphorous solubilizing bacterial product according to claim 1, ten (10) lit culture grown 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 and 1000 ml distilled water, and it is then allowed for further growth at 27 ± 1 °C for at least 3 to 4 days with agitation by motor at 100 RPM.

7. A material and method of preparing improved phosphorous solubilizing bacterial product according to claim 1 , wherein, final output from big fermentor Final output from big fermentor is centrifuge by using advance centrifugation technology and thereby this separated bacteria are further used for formulation of product whereas remaining liquid is discarded

8. A material and method of preparing improved phosphorous solubilizing bacterial product according to claim 1 , wherein, Recovered bacteria form centrifuge is mixed in a set of ration with suitable carrier composed of zero TDS water etc and then homogenized it for equivalently of formulation.

9. A material and method of preparing bacterial based product according to claim 1 , further comprising of packing the final output in 100 ml, 250 ml and 500 ml packing by using bottle or pouch packing machine for distribution to farmer, retailer, distributor, etc.

10. A material and method of preparing bacterial based product according to claim 1, wherein 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.