WO2011099027A1

WO2011099027A1 - Composition and method of preparation of bio fungicide based on trichoderma viride for controlling soil borne diseases

Info

Publication number: WO2011099027A1
Application number: PCT/IN2010/000185
Authority: WO
Inventors: Chetan S. Patel
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-02-09
Filing date: 2010-03-24
Publication date: 2011-08-18
Anticipated expiration: 2012-08-09

Abstract

This invention is based upon composition and method of preparing a Bio Fungicide containing Trichoderma viride 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, and club rot.

Description

Composition and Method of Preparation of Bio Fungicide based on Trichoderma Viride for Controlling Soil Borne 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 fungal based product containing Trichoderfna viride for controlling various soil born disease.

PRIOR ART-

In the existing system as given in Indian Patent Application No. 163/MAS/2002 - Granted Patent wherein the invention relates to a method for preparation of a bioformulation comprising selecting the effective bio-control agents from Paecilomyces lalacinus Bainier, Verticillium chlamydosporium Goddard, Trichoderma harzianum Rifai (TH-1) and TH-2, T. viride Pers. Ex. S. F. Grey, Laetisaria arvalis Burdsall, Gliocladium virens Miller, Giddens & Foster and Bacillus subtilis, preparing a medium by mixing thoroughly sourghum flour, river sand and distilled water, sterilizing the medium in an autoclave, shaking the medium to avoid clogging of the substrates, cooling the medium, inoculating the medium with said effective biocontrol agents in a flask, incubating said flask for few days, collecting the biomass of biocontrol agent alongwith media, mixing the said biomass of biocontrol agent and media with a carrier, drying blending the said mixtures air drying the contents and packing them in polythene bags.- In the existing system as given in Indian Patent Application No. 0083/CAL/2000 - Granted Patent 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 pathogenic fungal strains namely Corticium invisum and Corticium theae.. The preparation of the biocide is made by mining the four ingredients strains at the ratio of 4:3:2:1 and the culture, are prepared in the following manners a) Transferring the Bacillus subtilis and Pseudomanas putida cultures into mother culture scandium, b) allowing the another cultures to grow for 3— 7 day., c) transferring the grown mother cultures to main culture m4dium and allowing the main cultures to grow for 3— 7 days, d) transferring the Tnichoderma viridi and Trichoderma harzianum cultures in the mother culture medium, e) allowing the mother cultures to grow for 15 days, (f) transferring the mother cultures 4~ to the main culture, medium and allowing the main cultures to grow for 15 days, g) mixing the said ready cultures— Bacillus subtilis, 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. 4489161 - Granted Patent wherein a new strain of the fungus Trichoderma viride designated as T- 1 -R9 is described. The new strain is an effective biocontrol agent for fusarium wilt of chrysanthemum.

In the existing system as given in United States Patent Application No. 015498 - Granted Patent wherein the invention relates to ATCC 74084: ##STR1## which are squalene synthase inhibitors and thus useful as cholesterol lowering agents. These compounds are also potent antifungal agents. Additionally, they inhibit farnesyl protein transferase and famesylation of the oncogene protein Ras and are thus useful in treating cancer. This invention also relates to a process for obtaining compounds of structural formula (I).

In the existing system as given in United States Patent Application No. 015498 - Granted Patent wherein the invention relates generally to biological control of plant disease. The invention discloses an antagonistic Trichoderma fungal strain that targets soil-born fungi from the genus Fusaria and the genus Rhizoctonia.

In the existing system as given in WIPO Patent Application No. PCT/ES 1996/000206 - Granted Patent wherein the invention relates to liquid formulation based on strains of the stringy fungi Trichoderma harzianum and Trichoderma viride.- It comprises sources of carbon, mineral salts and traces of elements with organic and - inorganic nutrients, and vegetative structures reproducing strains of the stringy fungus Trichoderma harzianum Rifai which have been identified as Trichoderma harzianum IMI 352940, Trichoderma harzianum IMI 352941 , and Trichoderma harzianum CECT 20179; and Trichoderma viride Pers.: Fr identified as Trichoderma viride CECT 20178. The formulation is useful as a biological control agent for combatting diseases of plants and plant material and biodamage produced or transmitted by stringy fungi and other pests of plants, and as a biolixiviation agent.

In the existing system as given in Canadian Patent Application No. 1 106201- Granted Patent wherein the invention concerns a mycofungicidal product consisting of immunizing commensals (:IC) and cereal flour where the immunizing commensal is Scytalidium lignicola ATCC 16,675, Trichoderma polysporum Rifai ATCC 20,475, Trichoderma viride sensu Bisby ATCC 20,476 or Trichoderma viride sensu Bisby strain CG BINAB.INRA T 030(Pasteur Institute, Paris). It is aimed also at uses of this product for the treatment of trees, wood and plants.

In the existing system as given in WIPO Patent Application No. PCT/US1988/002011- Granted Patent wherein the invention relates to A rhizosphere-competent $i(Trichoderma polysporum), (ATCC 20852) and a rhizosphere-competent $i(Trichoderma viride) (ATCC 20853) are described. They are . respectively, mutants of rhyzosphere-incompetent $i(Trichoderma polysporum) ATCC i20475 and rhizosphere- incompetent $i(Trichoderma viride) ATCC 20476. The rhizosphere-competent strains are particularly effective biocontrol agents for plant and/or treeidiseases associated with various fungi e.g., Pythium spp., $i(Sclerotium), spp., $i(Rhizoctonia solani) and basidiomycetes such as $i(Chondrostereum purpureum).

In the existing system as given in WIPO Patent Application No. PCT/JP 1997/003268- Granted Patent wherein the invention relates to Establishment of a system for mass- producing proteins or peptides, particularly a system for mass-producing cellulase in molds such as $i(Trichoderma viride). As the regulatory sequence of cellulase cbhl genes originating in $i(Trichoderma viride) can highly express objective proteins, objective proteins, particularly cellulase, can be amply expressed with this sequence. Thus an endoglucanase originating in $i(Humicola insolens) has now been successfully produced in a quantity of 15 g/1.

In the existing system as given in Canadian Patent Application No. 1054541- Granted Patent wherein the invention states that Fermentation parameters are improved by the addition of certain polymers to the fermentation broth. Small quantities of carboxypolymethylene in Aspergillus niger, Trichoderma viride and Penicillium notatum cultures are particularly effective. OBJECT OF THE INVENTION Traditionally, the known fungicides have different method of its preparation and composition. The present invention is based upon novel composition and method of making fungal based product containing Trichoderma viride for controlling various soil born diseases like root rot, set rot,; wilt, collar rot, red rot, rhizome rot, damping off, stem rot and club rot.

STATEMENT OF INVENTION- The inventor has invented a composition and method of making a fungal based product containing Trichoderma viride for controlling various soil born diseases like root rot, set rot, wilt, collar rot, red rot, rhizome rot, damping off, stem rot and club rot.

DETAILED DESCRIPTION OF INVENTION-

Background of Invention: This invention relates to a composition and method for utilizing fungal based product containing Trichoderma viride for controlling various soil born diseases like root rot, set rot, wilt, collar rot, red rot, rhizon e rot, damping off, stem rot and club rot.

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 an innovative combination of fungal based product containing Trichoderma viride with enzymes, fats and growth promoting molecules to control soil borne diseases like root rot, set rot, wilt, collar rot, red rot, r izome rot, damping off, stem rot, club rot and alike.

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

Pure culture of fungus named Trichoderma viride is inoculated aseptically on plate having 20 ml PAC-00 agar Media; such two to three plates are generally inoculated. Such inoculated plate is maintained in BOD incubator for at 26 ± 1 °C for 6 to 7 days. (Composition for one liter of PAC-00 Media contains 300 gm potato, 20 gm dextrose, 20 gm agar agar and 1000 ml distilled water)

Step-2 Starter culture (In flask)

Culture grown on plate is inoculated aseptically in a flask (1000 ml capacity) having 500 ml PAC-00 broth media via cork borer (having 5 mm diameter cutting capacity), and allow to grow for at 26 ± 1 °C for at least 3 to 4 days. For superior growth, flask is put on shaker with agitation of media at 150 RPM. Such two, flasks are maintained. (Composition for one liter of PAC-00 broth Media contains 300 gm potato, 20 gm dextrose and 1000 ml distilled water)

Step-3 Seed Fermentor (In small fermentor)

One (1) lit culture grown in step-2 is inoculated aseptically in small fermenter having 10 litter PAC-00 media (Composition for one liter of PAC-00 broth Media contains 300 gm potato, 20 gm dextrose and 1000 ml distilled water), and allow it for further growth at 26 ± 1 °C for at least 3 to 4 days with agitation by motor at 100 RPM Step-4 Add to solid substrate (poly bags)

20 ml culture form fermenter is inoculated aseptically in previously sterilized poly propelin polybag (12 x 10 inch) having 180 gm broken sorghum gain. After inoculation of culture mouth opening of poly bag seal with sponge and allowed to develop. Such 500 bag get inoculated from 10 lit broth recovered from fermentor

Step-5 Allow to develop

After inoculation each and every bag transfer to growth room and allows developing at 26 ± 1 °C for 9-10 days. During growth (four days after growth) all bag are crush smoothly by hand to break the clump of mycelium develops on grains. After 10 days of growth all content of bags are removed in trays and transfer to cold room where they maintain at 5 °C till the grinding of grain or submerging of grain having fungal growth in Ground nut oil.

Step-6 Grind

After sufficient cooling and drying (in cold room at 5 °C) for 10 to 15 days all the grain with the growth of microbes are grind in grinder or they were brought to submerge technique whereby all the grain were directly submerge in definite quantity of Ground nut oil and then oil-grain mixture is agitate by using commercial in tank agitator. And in case of submerge technique oil-grain mixture sieve from 300 micron sieve and collected material which have fungal spore and mycelium in oil brought to homogenizer.

Step-7 Sieving

Grinding material sieve from 75 micron net with the help of vibro shifter and powder was collected by using cyclone separator, especially design for collecting of spore of microbes. And in case of submerge technique oil-grain mixture sieve from 300 micron sieve and collected material which have fungal spore and mycelium in oil brought to homogenizer.

Step-8 Mixing (Liquid carrier, emulsifier)

10 % fungal spore powder is than mixing in mineral oil (ground nut oil) and tween- 80 emulsifier mixture (90 % ground nut oil + 10 % tween-80) or in case of submerge technique homogenize mixture having spore of fungus where again homogenized by adding 10 to 15 % tween-80 which act as a emulsifier.

Step-9 Curing

Mix formulation is allow to cure for two to three days in drums and than send for storage or for packing

Step-10 Packaging

From above prepared mixture 50 ml, 100 ml pouch is prepared by using pouch packing machine

Step-11 - Distribution to farmer, retailer, distributor, etc. The final material prepared has to be used in following form: Dose in one Acre: Liquid Form: 50 ml to 100 ml

Claims

CLAIMS I claim,

1. A composition and method of preparing a bio fungicide containing Trichoderma viride 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 and club rot, the method comprising steps of nucleus culture, starter culture, seed fermentor, addition to solid substrate, allowing to develop, grinding, sieving, mixing, curing and packing the final output.

2. A composition and method of preparing bio fungicide according to claim 1, by inoculating aseptically pure culture of fungus named Trichoderma viride on two to three plates having 20 ml PAC-00 agar Media and maintaining in BOD incubator for at 26 ± 1 °C for 6 to 7 days, wherein composition for one liter of PAC-00 Media contains 300 gm potato, 20 gm dextrose, 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 in two flask (1000 ml capacity) having 500 ml PAC-00 broth media via cork borer (having 5 mm diameter cutting capacity), and allowing it to grow for at 26 ± 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. Such two flasks are maintained, wherein composition for one liter of PAC-00 broth Media contains 300 gm potato, 20 gm dextrose and 1000 ml distilled water.

4. A composition and method of preparing bio fungicide according to claim 1 , by inoculating aseptically one (1) lit culture grown as per method cited in claim 3 in small fermenter having 10 litter PAC-00 media, wherein composition for one liter of PAC-00 broth Media contains 300 gm potato, 20 gm dextrose and 1000 ml distilled water, and allowing it for further growth at 26 ± 1 °C for at least 3 to 4 days with agitation by motor at 100 RPM.

5. A composition and method of preparing bio fungicide according to claim 1, wherein 20 ml culture form fermenter is inoculated aseptically in previously sterilized poly propelin polybag (12 x 10 inch) having 180 gm broken sorghum gain and after inoculation of culture mouth opening of poly bag seal with sponge and allowed to develop, wherein such 500 bag get inoculated from 10 lit broth recovered from fermentor.

6. A composition and method of preparing bio fungicide according to claim 1, wherein, after inoculation each and every bag is transferred to growth room and allowed to develop at 26 ± 1 °C for 9-10 days and during growth (four days after growth) all bag are crushed smoothly by hand to break the clump of mycelium develops on grains and after 10 days of growth all content of bags are removed in trays and transfer to cold room where they maintain at 5 °C till the grinding of grain or submerging of grain having fungal growth in Ground nut oil.

7. A composition and method of preparing bio fungicide according to claim 1, wherein, after sufficient cooling and drying (in cold room at 5 0C) for 10 to 15 days all the grain with the growth of microbes are grind in grinder or they were brought to submerge technique whereby all the grain were directly submerge in definite quantity of Ground nut oil and then oil-grain mixture is agitate by using commercial in-tank agitator.

8. A composition and method of preparing bio fungicide according to claim 1, wherein, grinding material is sieved from 75 micron net with the help of vibro shifter and powder was collected by using cyclone separator, especially design for collecting of spore of microbes. And in case of submerge technique oil-grain mixture sieve from 300 micron sieve and collected material which have fungal spore and mycelium in oil brought to homogenizer.

9. A composition and method of preparing bio fungicide according to claim 1, wherein 10 % fungal spore powder is then mixed in mineral oil (ground nut oil) and tween- 80 emulsifier mixture (90 % ground nut oil + 10 % tween-80) Or in case of submerge technique homogenize mixture having spore of fungus where again homogenized by adding 10 to 15 % tween-80 which act as a emulsifier.

10. A composition and method of preparing bio fungicide according to claim 1, mixed formulation is allow to cure for two to three days in drums and than send for storage or for packing in 50 ml, 100 ml pouch by using pouch packing machine as final bio fungicide.