Title of Invention

A METHOD OF PREPARING A BIOFORMULATION

Abstract

This 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 alon.gwith 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.

Full Text

FIELD OF INVENTION This invention relates to a method of preparing a bioformulation which prevents root knot caused by a nematode meloidogyne incognita in mulberry. This invention also relates to the use of the bioformulation in the treatment of plants. BACKGROUND OF INVENTION The disease is reported from almost all mulberry growing areas of Karnataka, A.P., T.N., Keral and West Bengal. Severely affected plants show stunted growth, marginal necrosis and chlorosis of leaves. Under ground symptoms include formation of galls on roots. Various cultural and chemical methods have been recommended for the control of this disease but single method is not much effective in controlling the disease. Moreover, chemical control is expensive and toxic to silkworm as well as affects the beneficial microbes in soil, therefore, emphasis is being given on the use of ecofriendly methods using a biocontrol agents for the control of root knot nematode disease in mulberry. OBJECTS OF THIS INVENTION An object of the invention is to propose a method of preparing a bioformulation which prevents root knot caused by nematode. A further object of the invention is to propose a method of preparing a bioformulation M^hich do not affect the microbes in soil. A still further object of the invention is to propose a method of preparing a bioformulation which is ecofriendly. DESCRIPTION OF INVENTION Accordingly to this invention there is provided 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 mixture, air drying the contents and packing them in polythene bags. Further according to this invention there is provided a method of applying the bioformulation into the said comprising mixing talc based bioformulation with Farm Yard Manure (FYM) in combination with neem oil cake applying the said mixture around the root zone of the plant at proper intervals. In accordance with this invention, eight biocontrol agents were grown in Potato Dextrose broth to obtain the culture filtrates and tested for hatching of M. incognita eggs. Among them, culture filtrates of V. chlamydosporium, P. lilacinus, T. harzianum (TH-2), T. harzianum (TH-1) and T. viride were found effective (Table-1) and tested in pots to know their comparative efficacy with neem oil cake and Rugby 10 G against the disease. Results revealed that two biocontrol agents, V. chlamydosporium and T. harzianum (TH-2) were found effective (Table-2). However, Rugby, was found to be significantly superior compared to biocontrol agents and neem oil cake against the disease. Hence, the integration of biocontrol agents with neem oil cake was essential for the effective and eco-friendly management of nematode disease. The compatibility of nematicide (Rugby), Neem oil cake and chemical fertilizers were investigated and these were found compatible with V. chlamydosporium and T. harzianum and can be used along with these biocontrol agents for better management of the disease. Neem oil cake does not affect the beneficial microbes in soil. Further objects and advantages of this invention will be more apparent fi"om the ensuing examples. However, it is to be understood that the examples are not intended to imply any limitation on the scope of the invention. EXAMPLE 1 Development of bioformulation: For quick growth of V. chlamydosporium, the following medium was used. Sorghum flour (Sorghum vulgare) .... 1 kg River sand (after sieving in 60 mesh) .... 1 kg Distilled water ... .450-500 ml For preparation of media, the sorghum flour and river sand was mixed thoroughly with addition of distilled water. The solid medium was taken in 500 ml Elerlmeyer flasks @ 200 g/flask and sterilized in autoclave at 15 lbs pressure for 30 minutes. Immediately after sterilization of media it was thoroughly shaked to lose the materials in order to avoid the clogging of the substrates and after cooling inoculated with 4 mycelial discs (8 mm diameter) of effective biocontrol/flask. These flasks were kept for incubation at 28 ± 2°C. After 10 days of incubation, the biomass of biocontrol agents along with media was collected in one place and mixed thoroughly with carrier like talc (2:1 w/w). This was kept for air drying and after drying blended in a mixie and packed in opaque polythene bags. Rhizosphere study and field testing of bioformulation: A talc based bioformulation produced by V. chlamydosporium contains initial Colony Forming Unit/g as 4.5 x lO^i. Rhizosphere studies indicate that after application, it maintained sufficient Colony Forming Unit/g (30 X 103) in the soil for about 120 days. Due to perennial nature of mulberry crops and endoparasitic habit of nematode, it is necessary to maintain the sufficient Colony Forming Unit/g of V. chalmydosporium in soil to stop the further hatching/multiplication of nematode. Therefore, the present study clearly indicates that at least three doses/yr of bioformulation are required for better management of the root knot disease. EXAMPLE 2 A field experiment on the biological control of root knot disease using two antagonistic microbes, V. chalmydosporium and T. hazianum (TH-2) were conducted in severely infested field with nematode (350-370 larvae/250 cc soil) at CSRTI, Mysore. Talc based bioformulation of these microbes were developed and applied alone after mixing with Farm Yard Manure (1:200) and in combination with neem oil cake (1:200:24) around the root zone of plant @ 200 g/plant at 15 cm depth in 3 doses/yr at an interval of 4 months during cultural operations/fertilizer application and irrigated. A similar quantity of Farm Yard Manure @ 200 g/plant was applied in plots treated with Rugby, neem oil cake and also in check. Among them application of V. chlamydosporium along with neem oil cake have shown significant reduction in disease severity and reduced the number of galls, egg masses and nematode population in soil by 85.7, 88.5 and 90.8 per cent respectively and 23.7% leaf yield was increased over the check (Table-3). Bio-assay study Bio-assay study showed that silkworm larvae fed with the leaves obtained from Bionema (V. chlamydosporium) along with neem oil cake treated plants significantly improved in all the commercial characters of cocoons, which was on par with the nematode free (without any application) treatment. It does not show any residual toxicity on silkworms even after 2 days of application. Example 3 The invention also relates to the use of talc based bioformulation named as Bionema using the biocontrol agent V. chlamydosporium and neem oil cake. One kg Bionema is mixed with 200 kg Farm Yard Manure and 24 kg neem oil cake (for 1000 plants). The mixture is kept under shade for a week to enhance multiplication of biocontrol agent. The mixture is applied @ 200 g/plant around the roots at 15 cm depth after removing the bunches of galls. Annually 3 doss are to be applied at an intervals of 4 months during cultural operations/fertilizer application and irrigate the garden. For one ha/yr, 80 kg Bionema, 20 mt Farm Yard Manure and 2 mt neem oil cake are required. The shelf life of Bionema produced by V.chlamydosporium was determined as 180 days under room temperature (25-30°C) and 10 months under low temperature (4-6"*C). Advantages and precautions: The formulation is ecofriendly and effective. It reduces the disease severity by 85-90% and increases the leaf yield by 23.7%. Whereas in recommended cultural or chemical method (use of neem oil cake or Rugby 10 G), the severity of disease was reduced by 63-78% and increased the leaf yield by 16-18% (Table 3). Bionema produced by V. chlamydosporium is not phytotoxic to mulberry and does not cause any skin disorders in human. It does not show any residual toxicity on silkworm Treatment should be given for 2-3 years. Do not apply yearly dose of FYM (20 MT.), straight use the same for the application of Bionema. Reference is made to the following chart to describe the steps of the process. FLOWCHART (I) Selection of biocontrol agents: Collection of root knot (Meloidogyne incognita) infected mulberry roots from field Isolation of biocontrol agents from nematode eggs In vitro screening of various biocomrol agents against root knot disease Selection of effective biocontro agents from pot culture studies Selected Verticillum chlamydosponum for preparation of bio formulation (II) Preparation of bioformuiation (Bionema): Testing of various substrates/media for niass multiplication of V. chlamydosponum Selected sorghum-sand medium (1:1) Sterilization of medium in Elerlmeyer flasks Inoculation of V.chlamydosponum and incubation for 10 days at 28°C Collection of biomass and mixing with talcum power as a carrier (2:1) 4^ Shade drying and packing in opaque polythene bags (III) Method of application at field: Mix Bionema with FYM and neem oil cake (1:200:24) Store the mixture with 30% moisture for one week under shade for enhance the colonies \/ Apply the mixture @ 200 g/plant in the root zone at 15 cm depth ") f Cover with soil and irrigate the garden Repeat the application at least for one year at an intervals of 4 months Presently, no alternative bio formulation is known to mange the root knot disease (Meloidogyne incognita) in mulberry. The available methods are cultural and chemical i.e. soil application of neem oil cake and Furadan/Rugby 10 G. Moreover, chemical nematicides are having possible residual toxicity in mulberry for a longer period and also affect the beneficial microbes in soil while application of neem oil cake alone is not very effective in controlling the nematode disease. Hence a new ecofriendly technology was developed for effective management of the disease. The product Bionema has been developed using a native isolate of Verticillium chlamydosporium which was isolated from eggs of Meloidogyne incognita (nematode affected mulberry root knots). The process of preparation of Bionema comprises using V. chlamydosporium after mass multiplying on solid media (sorghum flour sand; 1:1), and after incubation of 10 days mixing of biomass with talc (2:1) to obtain the higher Colony Forming Unit/g in bioformulation (4.5 x 1011). Application method of Bionema after mixing with neem oil cake and Farm Yard Manure (1:24:200) is an ecofriendly strategy for effective management of root knot nematode in mulberry. EXAMPLE 4 Testing the technology at farmers field. Regional station of CSRTI and Department of Sericulture, Karnataka farms: The results obtained from field trials were on par with the experimental trials (Tables 3 & 4). Based on these demonstrations, it has been proved that bioformulation of the present invention is very effective in controlling the disease at farmer"s field. Regional Station and farm of department of Sericulture (DOS), Kamataka. At laboratory level, the technology reduced the disease by 85-90% with 23% improvement in leaf yield. Whereas at RSRS farms, DOS farms and farmers field it was reduced by 79-89, 86-90 and 81-90% respectively. By adoption of this technology, the net income of Rs. 7600/ha/yr with 1:1.7 cost benefit ratio can be achieved (Table-5). WE CLAIM; 1. A method for preparation of a bioformulation comprising selecting the effective bio-control agents 85 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 mixture, air drying the contents and packing them in polythene bags. 2. The method as claimed in claim 1, wherein the most effective biocontrol agents are V. chlamydosporium and T. harzianum. 3. The method as claimed in claim 1, wherein the ratio of sorghum flour and river sand is 1:1. 4. The method as claimed in claim 1, wherein said medium was sterilized at 15 lbs pressure for 30 minutes. 5. The method as claimed in claim 1, wherein the incubation was done at 28 JL20C for 10 days. 6. The method as claimed in claim 1, wherein the ratio of the biomass of biocontrol agent alongwith media and carrier is 2:1 w/w. 7. The method as claimed in claim 1, wherein said carrier is talc powder of aluminum silicate. 8. The method of applying the bioformulation into the said comprising mixing talc based bioformulation as claimed in claims to 1 to 8 with FYM in combination with neem oil cake applying the said mixture around the root zone of the plant at proper intervals. 9. The method as claimed in claim 1, wherein the ratio of talc based bioformulation and FYM and neem oil cake is 1:200:24. 10. The method as claimed in claim 1, wherein the mixture is applied at the rate of 200 gm/plant at 15 cm depth in 3 doses/year at an interval of 4 months. 11. The method for the preparation of a bioformulation substantially as herein described and illustrated.

Documents:

0163-mas-2002 abstract-duplicate.pdf

0163-mas-2002 abstract.pdf

0163-mas-2002 claims-duplicate.pdf

0163-mas-2002 claims.pdf

0163-mas-2002 correspondence-others.pdf

0163-mas-2002 correspondence-po.pdf

0163-mas-2002 description(complete)-duplicate.pdf

0163-mas-2002 description(complete).pdf

0163-mas-2002 form-1.pdf

0163-mas-2002 form-18.pdf

0163-mas-2002 form-26.pdf