DESC:Field of the invention
The invention relates to agricultural biotechnology and microbiology.
The invention relates to a novel microbial consortium comprising two or more fungi belonging to two or more genus for insect and pest management in crops. The microbial consortium controls the soil, root, stem and foliar pest population in wide range of crops. The invention further relates to a process of preparing a formulation of microbial consortium of two or more fungi belonging to two or more genus for insect and pest management in crops.

Background of the invention
Annually billions of dollars of loss happen to the farmers in terms of productivity due to serious damage caused by the insect pests in the agricultural field. Recently, various chemical pesticides have been banned worldwide due to their adverse effect on human beings, animals, soil, water and overall environmental impact. People have become aware of dangerous side effects of chemical fertilizers and pesticides to the mankind and reducing biodiversity. With an increase in population, food demand is continuously increasing, and issues like changing environmental conditions, negative effect of chemicals usage on natural ecosystem has burden the agricultural workers. Microbial based pesticides are observed to provide effective solution to these problems and contribute to sustainable ecosystem. Thus, there is a growing demand to develop compositions and processes that not only effectively control pests and increase food production but also leads to decrease the use of harmful chemical insecticides.

Commonly used microorganisms used in development of biopesticides for biocontrol of hard body insects belong to order Orthoptera, Diptera, Cleoptera and Lepidoptera. These involve single strains of Metarhizium, Beauveria, Verticillium, Lecanicillium lecanii, Hirsutella thompsonii and Isaria fumosorosea etc.

WO2016135700A1 relates to development of microbial consortium containing five or more microbial species along with additional components like chitin, chitosan, amino acids etc. This consortium is used for degrading chitin containing biological material. The introduction of beneficial microbial consortium to soil leads to an increase in crop vigor and yield.

US20120015806A1 invention comprises microbial formulation for plant growth with customized solution to improve soil, plant growth promoting activities and increase soil micro and macro nutrients availability, the formulation comprises at least seven beneficial bacteria; at least two beneficial fungi; at least one yeast; and at least one compound which extends the effective life time of said formulation. The composition is in powder formulation that can be applied directly to the soil to enhance crop production and increase plant defence mechanism.

US7097830 discloses synergistic bioinoculant composition comprising Bacillus strains isolated from cows, either individually or in all possible combinations, and optionally a carrier, with each of the strains showing plant growth-promoting activity.

Several microbial consortia based bioproducts are commercially available and being used in the agriculture but limited only to improve the plant growth by PGPR (plant growth promoting) activity, nitrogen fixing, potash mobilizing and phosphate solubilizing activity.

There is urgent need develop strategies using two or more different microbes to control major pest population. The approach requires to develop two or more strains consortium to cover wide variety of pest population with one formulation. Use of single product to cover wide variety of insect is considered to be an economical and effective alternate to conventional pest control techniques in agriculture.

In view of the above background, the present invention provides a formulation of microbial consortium for control of wide range of insect pest in wide range of crops.

Object of the invention
The main objective of present invention is to provide a microbial consortium comprising two or more fungi belonging to two or more genus for insect and pest management in crops.

Yet another object of the present invention is to provide a formulation of microbial consortium comprising two or more fungi belonging to two or more genus with high viability and extended shelf life of microbes of at least 18 months to 24 months for powder and 18 months to 36 months for liquid formulation.

Yet another object of the present invention is to provide a formulation containing high colony forming units of fungal microbial cultures.

Yet another object of the present invention is to provide a process of preparing a microbial consortium comprising two or more fungi belonging to two or more genus for insect and pest management in crops.

Yet another object of the present invention is to provide a process of preparing a formulation of microbial consortium comprising two or more fungi belonging to two or more genus in combination with adjuvants for insect and pest management in crops.

Yet another object of the present invention is to provide a method of application of the formulation of microbial consortium comprising two or more fungi belonging to two or more genus in combination with adjuvants for insect and pest management in crops.

Summary of the invention
Accordingly, in an aspect of the present disclosure, the microbial consortium comprises two or more fungi belonging to two or more genus for insect and pest management in crops.

Present invention involves the method of development and application process of microbial consortium to cover wide range of insects and pest is an economical, cost-effective and user friendly way.

In an aspect of the present disclosure, the formulation comprises microbial consortium comprising two or more fungi belonging to two or more genus with high viability and extended shelf life of microbes of at least 18 months to 36 months. The formulation is stable at low temperature, high temperature and room temperature for at least 18 months.

In an aspect of the present disclosure, the microbes in the consortium demonstrate high colony forming units of microbial cultures in the formulation. The formulation comprises live viable cells which includes both the spores and mycelia that contributes to the activity of formulation.

In an aspect of the present disclosure, the microbes of the microbial consortium are selected from, but not limited to, Metarhizium anisopliae, Beauveria bassiana.

The formulation of the present disclosure is capable of being used to control wide range of pest belonging to order Orthoptera and/or Diptera and/or Cleoptera and/or Lepidoptera.

In an aspect of the present disclosure, the process provides a microbial consortium comprising two or more fungi belonging to two or more genus for insect and pest management in crops. The process of development of formulation of microbial consortia provides optimum conditions and process parameters to obtain microbial consortium with high viable cell count and longer shelf life. The process more particularly provides a liquid and wettable powder formulation of potent combination of microbial agents for sustainable insect pest and management. The microbial culture used in present invention are provided in suspension concentrate formulation with high spore density using optimized media. The process parameters include, but not limited to, standardization of carbon, nitrogen, macro elements, microelements and vitamins etc. to achieve desirable formulation.

In an aspect of the present disclosure, the process provides a formulation of microbial consortium comprising two or more fungi belonging to two or more genus in combination with adjuvants for insect pest management in crops. The adjuvants are selected from stickers, spreaders or surfactants for better efficacy of the formulation against insect/pest control.

In an aspect of the present disclosure, the invention also provides a method of application of the formulation of microbial consortium comprising two or more fungi belonging to two or more genus in combination with adjuvants for insect/pest management in crops.

In a non-limiting embodiment, the formulation comprises Metarhizium anisopliae and Beauveria bassiana. The formulation is provided in suspension concentrate formulation with shelf life of at least 18 months to 36 months and wettable powder form with at least 18 months to 24 months. The formulation has shown good efficacy in terms of pot and field trials on different crops. The formulation of microbial consortia of Metarhizium anisopliae and Beauveria bassiana effectively controls wide range of insect and pest population. The formulation of the present invention controls the soil, root, stem and foliar pest population in wide range of agricultural crops.

It will nevertheless be understood that the embodiments and examples provided herein are illustrative only with an intent to file a provisional application and not intended to be limiting. The alterations and further modifications in the present invention, and such further applications of the principles of the invention as would normally occur to those skilled in the art are to be construed as being within the scope of the present invention.

Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a nonexclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method.

Unless otherwise defined, all terms, and especially any technical and/or scientific terms, used herein may be taken to have the same meaning as commonly understood by one having an ordinary skill in the art.

The terminology and structure employed herein is for describing, teaching and illuminating some embodiments and their specific features and elements and does not limit, restrict or reduce the spirit and scope of the claims or their equivalents.

It will be understood by those skilled in the art that the summary of the invention provided herein is exemplary and explanatory of the invention and are not intended to be restrictive thereof. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The composition, methods, and examples provided herein are only illustrative and not intended to be limiting.

Statement of the invention
The present invention involves the development of consortium of two fungal strains belongs to the two different genus Beauveria bassiana and Metarhizium anisopliae. Both of the fungal strains are non-hazardous and non-toxic. Media constituents for the growth of Beauveria bassiana and Metarhizium anisopliae have been optimized under submerged condition in both lab and commercial scale. Product developed in two different formulations with optimum adjuvants to obtained shelf life upto 2 years. Effect of growth temperature and shelf life temperature have been studied. The sustainable consortium obtained is very effective in controlling all major sucking pest population and pest belonging to order Orthoptera and/or Diptera and/or Cleoptera and/or Lepidoptera in lab and open field.

Detail description of the Invention
The present invention deals with the development of consortium of two fungal strains effective in controlling different types of pest population and available in both liquid and powder formulation. The formulation is developed firstly identifying and evaluating the compatibility of two different strains from different genus to obtain wide range of pest control with minimum dosage.

Formulation development involves firstly, standardizing compatibility of two different strains chosen from different efficient fungal strains against different crop pests. Secondly, optimizing growth parameters for both of the fungal strains including nutritional parameters viz. carbon, nitrogen and mineral source; physical parameters like pH, aeration and temperature. The entire process involves mass multiplication of two fungal strains under submerged fermentation followed by adjuvant addition and final product formulation. The lab scale optimization studies were carried out in 1 L capacity Erlenmeyer flasks under shake flask condition followed by optimization of parameters in Lab scale fermenter up to 50 L capacity and final product preparation at commercial scale fermenters.

Strains for the study are selected from different genus belonging to Verticillium, Beauveria, Purpureocillium and Metarhizium for their compatibility of working together in the formulation against different pest of agriculture.

In an aspect of the present invention, two genus with different strains have been selected on the basis of compatibility and studied further. The formulation is developed using indigenous and wild type strains of Beauveria bassiana and Metarhizium anisopliae isolated from agricultural Mantori, Tissang, Kahutauli, Jansat, Muzzafarnagar, Uttar Pradesh, India. The strains are dully deposited IMTECH, Chandigarh, India with accession number of MTCC 25053 dated 11th January 2016 and MTCC 25014 dated 11th January 2016 for Beauveria bassiana and Metarhizium anisopliae strains, respectively.

Growth parameters optimization involves the use of various organic and inorganic source of carbon and nitrogen, simple or complex compounds for the growth of Beauveria bassiana and Metarhizium anisopliae strains. Preparation of high quality pre-inoculum is very important criteria for mass multiplication of the fungal strains under optimized aeration using flask volume and orbital shaker RPM adjustment from 150-200 RPM, initial pH from 4.5-6.5 and temperature from 25°C to 35°C to obtain desired viable count of the fungal strains. The optimum growth to achieve high CFU in the range of 2x109 CFU/ml to 2x1010 CFU/ml was attained in incubation time from 48-72 hrs.

The media components used for mass multiplication of Beauveria bassiana and Metarhizium anisopliae comprises of carbon source, nitrogen source and other micro and macro elements sources. The carbon sources are preferably simple or complex, single or two sources chosen from glucose, glycerol, sucrose, lactose, fructose, maltose, jaggery, molasses etc. The nitrogen source comprises of organic or inorganic sources like ammonium sulphate, ammonium nitrate, potassium nitrate, fish meal, yeast extract, corn steep liquor, soya flour, peptone, cotton seed flour etc. individual or mixture thereof.

The essential macro and microelements involve in mass production of the strains are from organic or inorganic sources viz. corn steep liquor, molasses, gram flour, potassium nitrate, potassium dihydrogen phosphate, manganese sulphate, magnesium chloride, calcium chloride, ferrous sulphate, ferric citrate, zinc sulphate, cobalt chloride, potassium molybdate etc. single or mixture thereof.

The pre-inoculum is added to the sterilized media in the fermenter.

Submerged fermentation is done in fermenter with optimized parameters as follows:
Process Time: 24-96 h
Temperature: 25 to 35° C
Agitation: 25-100 RPM (Rotation per minute)
Aeration: 0.2-1.0 VVM (Vessel Volume per minute)

After attaining optimum growth, the fermented broth is mixed with preservatives before harvesting.

The preservative comprises Polyethylene glycol, Potassium hydroxide and Sodium chloride at different concentration of:
Polyethylene glycol: 5- 10 %
Potassium chloride: 1.0-10.0 %
Sodium chloride: 0.5-2.0%

The Fermented broth is harvested after mixing with the preservatives and bottled in different capacity of 100 ml to 5 litres HDPE container.
Thus, the process to obtain the microbial formulation comprises selecting two compatible fungal strains belonging to two genus and preparing pre-inoculum of each of the fungal strain in optimized growth media in flask, under optimized aeration from 150-200 RPM, initial pH from 4.5-6.5 and temperature from 25°C to 35°C to obtain optimum growth to achieve high CFU in the range of 1x109 CFU/ml to 1x1010 CFU/ml in incubation time from 48-72 hrs. Thereafter the fermenter media is inoculated with the pre-inoculum of each of the fungal strains for submerged fermentation under pre-determined process parameters to obtain fermenter broth having optimum viable cell count of the microbial consortium of the fungal strains. The preservatives comprising Polyethylene glycol, Potassium hydroxide and Sodium chloride at different concentration of: Polyethylene glycol: 5- 10 %, Potassium chloride: 1.0-10.0 %, Sodium chloride: 0.5-2.0% are added in the fermenter broth followed by harvesting to obtain harvested broth or formulation of the microbial consortium.

The optimized growth media and fermenter media comprises carbon source selected from glucose, glycerol, sucrose, lactose, fructose, maltose, jaggery, molasses; nitrogen source ammonium sulphate, ammonium nitrate, potassium nitrate, fish meal, yeast extract, corn steep liquor, soya flour, peptone, cotton seed flour; and other essential micro and macro elements sources are selected from corn steep liquor, molasses, gram flour, potassium nitrate, potassium dihydrogen phosphate, manganese sulphate, magnesium chloride, calcium chloride, ferrous sulphate, ferric citrate, zinc sulphate, cobalt chloride, potassium molybdate.

The pre-determined process parameters for submerged fermentation comprises Process Time: 24 h to 96 h, Temperature: 25 °C to 35 °C, Agitation: 25 RPM to 100 RPM (Rotation per minute); Aeration: 0.VVM to-1.0 VVM (Vessel Volume per minute). The optimum viable cell count of the microbial consortium in the formulation is in the range of 2×109 CFU/ml to 2 × 1010 CFU/ml.

The formulation can be formulated into powder as well as liquid formulation using following components and mass percentage thereof:
Liquid formulation
Harvested broth 90-95%
Adhesive 0.05-3.0%
Adjuvant 0.02-0.5 %

The adhesive used in the liquid formulation is Polyvinylpyrrolidone.

The adjuvant used in the formulation is Tween 80.

Powder formulation
Harvested broth 15-30%
Adhesive 0.05-3.0%
Adjuvant 0.05-1.5 %
Carrier 60-80%

The adhesive used in the powder formulation is Polyvinylpyrrolidone

The adjuvant used in the powder formulation is Tween 20.

The carrier used in the powder formulation is talc.

The optimum viable cell count of the microbial consortium in the powder formulation is in the range of 2×108 CFU/ml to 2 × 109 CFU/gm.

The formulations obtained are very effective in controlling all major sucking pest population viz. jassids, aphids, thrips, whitefly and mealy bugs pest belong to order Lepidoptera fruit and shoot borers, spodoptera, Helicoverpa armigera etc in lab and open field. These are the major pest of the Agriculture not only in India but also all over the world for about 15-30% losses in the yield. Metarhzium anisopliae fungus and Beauveria bassina colonize on the insect body leads to double benefit in the control of pest population by entering into the insect through feeding on the plant and by entering through the skin by direct contact with the product. Microscopic and microbiological identification techniques proves that Metarhzium anisopliae established its presence in the plant and both of the strain growth on insect body.

The microbial consortium of the present invention more than 95% efficacy as compared to individual microorganism which shows bioefficacy between 66 % to 77% as shown in Table 1 below.
Table 1: Bio efficacy data

Organism CFU/ml Dosage Total no. of Aphid nymph Total no. dead Aphid nymph Percent Mortality
7 DAT
Consortium of present invention 2*1010 5 ml/lit 10 9.54 95.40
M. anisopliae L MTCC 25014 1*1010 5 ml/lit 10 7.65 76.50
B. bassiana L MTCC 25053 1*1010 5 ml/lit 10 6.66 66.60
Control - - 10 0.00 0.00

The shelf life studies shows that the shelf life of the liquid formulation is more than 36 months and for powder formulation is 24 months. The shelf life of the liquid was tested at every 3 months interval for 36 months. While the shelf life of the powder formulation was tested at every 3 months interval for 24 months. The shelf life data for liquid and powder formulation are presented in table 2 and 3 below, respectively.
Table 2: Shelf life data of liquid formulation
S.no Months of study Optimized Media PDB (Broth)
1 3 Month 2 x 1010 4 x 108
2 6 Month 2 x 1010 3 x 105
3 9 Month 1 x 1010 2 x 105
4 12 Month 1 x 1010 2 x 104
5 15 Month 8 x 109 2 x 103
6 18 Month 6x 109 2 x 103
7 21 Month 6 x 109 Nil
8 24 Month 5 x 109 Nil
9 27 Month 4 x 109 Nil
10 30 Month 3 x 109 Nil
11 33 Month 2 x 109 Nil
12 36 Month 2 x 109 Nil

Table 3: Shelf life data of powder formulation
S.no Months of study Optimized Media +Talc PDB (Broth)+ Talc
1 3 Month 2 x 109 1 x 108
2 6 Month 2 x 109 2 x 106
3 9 Month 1 x 109 3 x 105
4 12 Month 1 x 109 1 x 104
5 15 Month 9 x 108 1 x 103
6 18 Month 7 x 108 Nil
7 21 Month 3 x 108 Nil
8 24 Month 2 x 108 Nil

Uniqueness in the formulation and process of manufacturing:
1. The consortium of Beauveria bassiana and Metarhizium anisopliae in liquid formulation having viable cell count of minimum of 2×109 CFU/ml and maximum of 2 × 1010 CFU/ml.
2. The production and mass multiplication to attain the high CFU count is only 24-96 hrs of batch run.
3. The microbial formulation comprising Beauveria bassiana MTCC 25053 and Metarhizium anisopliae MTCC 25014 having high viable cell count in the range of 2×108 CFU/ml to 2 × 1010 CFU/ml, and preservatives comprising Polyethylene glycol, Potassium hydroxide and Sodium chloride at specific concentrations, in liquid and powder form.
4. The formulation has shelf life of more than 36 months.
5. The formulation shows bioefficacy of more than 95% as compared to individual microorganism which shows bioefficacy between 66% to 77%.
,CLAIMS:1. A microbial consortium comprising two fungal strains belonging to two genus for broad spectrum insect and pest management in crops, wherein said fungal strains are Beauveria bassiana MTCC 25053 and Metarhizium anisopliae MTCC 25014 having high viable cell count in the range of 2×109 CFU/ml to 2 × 1010 CFU/ml.

2. A microbial formulation for broad spectrum insect and pest management in crops, said formulation comprises fungal strains from two different genus, said fungal strains are Beauveria bassiana MTCC 25053 and Metarhizium anisopliae MTCC 25014 having high viable cell count in the range of 2×108 CFU/ml to 2 × 1010 CFU/ml, and preservatives comprising Polyethylene glycol, Potassium hydroxide and Sodium chloride at specific concentrations.

3. The formulation as claimed in claim 2, wherein the specific concentration of preservatives is Polyethylene glycol: 5 % to 10 %, Potassium chloride: 1.0 % to10.0 % and Sodium chloride: 0.5 % to 2.0%.

4. The formulation as claimed in claim 2, wherein said formulation has shelf life of 18 moths to 36 months.

5. The formulation as claimed in claim 2, wherein said formulation is a liquid formulation comprising the formulation in the range of 90-95%, Adhesive in the range of 0.05-3.0%, Adjuvant in the range of 0.02-0.5 % having viable cell count in the range of 2×109 CFU/ml to 2 × 1010 CFU/ml.

6. The formulation as claimed in claim 2, wherein said formulation is a powder formulation comprising the formulation in the range of 15-30%, Adhesive in the range of 0.05-3.0%, Adjuvant in the range of 0.05-1.5 %, Carrier in the range of 60-80% and Dispersant in the range of 0.5-3.0% having viable cell count in the range of 2×108 CFU/ml to 2 × 109 CFU/ml.
7. A process to obtain the microbial formulation as claimed in claim 2, wherein said process comprises
- Selecting two compatible fungal strains belonging to two genus,
- Preparing pre-inoculum of each of the fungal strain in optimized growth media in flask, under optimized aeration from 150-200 RPM, initial pH from 4.5-6.5 and temperature from 25°C to 35°C to obtain optimum growth to achieve high CFU in the range of 1x109 CFU/ml to 1x1010 CFU/ml in incubation time from 48-72 hrs,
- Inoculating sterilized fermenter media with pre-inoculum of each of the fungal strains for submerged fermentation under pre-determined process parameters to obtain fermenter broth having optimum viable cell count of the microbial consortium of the fungal strains,
- Adding preservatives comprising Polyethylene glycol, Potassium hydroxide and Sodium chloride at different concentration of: Polyethylene glycol: 5- 10 %, Potassium chloride: 1.0-10.0 %, Sodium chloride: 0.5-2.0% in the fermenter broth followed by mixing the fermented broth with the preservatives and harvesting and bottling in different capacity of HDPE container.

8. The process as claimed in claim 8, wherein the optimized growth media and fermenter media comprises carbon source selected from glucose, glycerol, sucrose, lactose, fructose, maltose, jaggery, molasses; nitrogen source ammonium sulphate, ammonium nitrate, potassium nitrate, fish meal, yeast extract, corn steep liquor, soya flour, peptone, cotton seed flour; and other essential micro and macro elements sources are selected from corn steep liquor, molasses, gram flour, potassium nitrate, potassium dihydrogen phosphate, manganese sulphate, magnesium chloride, calcium chloride, ferrous sulphate, ferric citrate, zinc sulphate, cobalt chloride, potassium molybdate.

9. The process as claimed in claim 8, wherein the pre-determined process parameters are Process Time: 24 h to 96 h, Temperature: 25 °C to 35 °C, Agitation: 25 RPM to100 RPM (Rotation per minute); Aeration: 0.VVM to-1.0 VVM (Vessel Volume per minute).

10. The process as claimed in claim 8, wherein said optimum viable cell count of the microbial consortium is in the range of 2×109 CFU/ml to 2 × 1010 CFU/ml.