Field of the invention
The present invention relates to a granular formulation of fungal and bacterial bio-control agents with enhanced shelf-life for the control of soil borne diseases. The present invention further provides a method for the preparation of the granular formulation that can be used for both fungal and bacterial bio-control agents and other beneficial microbes for bio-fertility.
Background of the invention and prior arts
Traditional methods used to protect crops from diseases have been largely based on the use of chemical pesticides. These chemical pesticides have several limitations in control of diseases and pests and also have negative environmental impacts. Biological control agents for disease control have emerged as potential agents to control plant pathogens resulting in minimal impact on the environment.
Although the research on use of beneficial microbes for plant disease suppression is going on for more than 50 years, the commercialization and use of these biocontrol agents is nowhere near chemical pesticide use, to till date the use of bio-control agents has not even crossed 3.0% of chemical pesticide use.
US4724147 relates to alginate gel pellets containing living fungal inoculums targeted to control selected soilborne plant diseases; said pellets comprising effective concentrations of living fungi which are selected to effectively control targeted soilborne plant diseases as the active ingredient incorporated into an alginate gel matrix carrier. '147 developed alginate gel pellets by mixing fungal propagules with sodium alginate and subsequently adding the homogenized mixture into a solution of calcium chloride/calcium gluconate dropwise. According to '147 four fungal bio-control agents viz., Gliocladium virens, Penicillium oxalicum, Talaromyces flavus and Trichoderma viride were used for alginate gel pellets preparation and were to control damping-off, Verticillium wilt. However the inventors of '147 failed to study the stability of their pellets beyond 12 weeks.

US2002/0015988 discloses the method of preparing granular formulation of beneficial bacteria Pseudomonas aurantiaca by using Vermiculite a water insoluble particulate substrate, a water absorbent polymer carragheenan and sodium alginate in different combinations. The application demonstrates that a granular formulation of Pseudomonas aurantiaca prepared with combination of 16% residual water, 1.5% microbial biomass, 81.9% vermiculite and 0.6% k-carragheenan to have stability up to 317 days at room temperature. However there is no report on stability of fungal biocontrol agents.
US6455036 described the preparation of granular formulation of entamopathogenic microorganisms and plant pathogens which are used as bioherbicides viz., Pseudomonas syringae, Fusarium oxysporum, Colletorichum gloeosporioides by using a combinations of starch graft polymer, disaccharides such as sucrose & trehalose, diatomaceous earth and vegetable oils. The formulations of Pseudomonas syringae, Fusarium oxysporum, Colletorichum gloeosporioides prepared by '036 demonstrate the shelf-life of 365 days, 114 days and 240 days respectively at storage temperature of 22°C. From the above formulation '036 could achieve only 114 days of stability of fungal bioagents and have not reported anything about formulation and stability of biocontrol agents used to control plant pathogens viz., Trichoderma harzianum, Bacillus subtilis, pseudomonas fluorescens etc.
Chumthong et. al. (2008) described a method for developing water-soluble granules from endospores of Bacillus megaterium using Lactose monohydrate, PVP and sodium alginate. They claimed to have achieved a shelf-life of 24 months with their formulation. But they have not included Fungi and non-spore forming bacteria such as Pseudomonas species in their formulation studies.
Thus there is a need to prepare a granular formulation of fungal and bacterial biocontrol agents with enhanced shelf-life for the control of soil borne diseases.
According to the present invention the bacterial and fungal beneficial microbes as Trichoderma species, Pseudomonas species, Bacillus species or mixtures thereof are used to control soil borne crop diseases (targeting disease causing pathogens/ plant pathogens) and enhance the plant growth. These biocontrol agents when applied colonizes the rhizosphere of the crops (by different mode of action e.g., competition, producing antibiotics etc) and does not allow the

harmful microbes that reside in the soil/rhizosphere to colonize. In turn, this helps to inhibit the growth of the harmful microbes that cause diseases on crops.
Object of the invention
It is an object of the present invention to overcome the drawbacks of the prior art.
It is another object of the present invention to provide a granular formulation of fungal and bacterial biocontrol agent with enhanced shelf-life for the control of soil borne diseases.
It is yet another object of the present invention to provide a method for the preparation of the granular formulation that can be used both for fungal and bacterial bio-control agents and other beneficial microbes for bio-fertility.
Summary of the invention
The present invention provides a granular bio-control composition having enhanced shelf-life comprising (i) at least one beneficial biocontrol agents, which colonizes the rhizosphere of the crops; (ii) at least one suitable membrane stabilizer; (iii) at least one slow release nutrient; (iv) and a suitable binder; and (v) at least one osmoprotectant.
The present invention further provides a process for the preparation of the granular biocontrol agent, comprising the steps of:
(i) Growing the biocontrol agent in suitable liquid medium;
(ii) Concentrating the microbial cell and spore suspension by centrifugation;
(iii) Suspending the concentrated suspension in 0.01 M phosphate buffer (K2HP04:KH2P04);
(iv) Mixing the fungal/bacterial suspension with combination of osmoprotectants (15-40% of total dry weight of formulation), PVP (0.5-5%), water-absorbent material (0.1-0.5%), sodium alginate (1-5%),

granulator (5-20%), slow release nutrients (30-60%) and mixed to form dough;
(v) Mixing the dough with vegetable oil (0.2-1.0% v/W);
(vi) Extruding the dough through a hand held granulator to form long strands and cutting into 3-5 mm length granules;
(vii) Air drying the granules in shade by passing stream of cool air.
Description of the accompanying figures
Figure 1 provides a graphical representation of survival of T. harzianum, T. viride, P. fluorescens, and B. subtilis in granular formulation-III at room temperature
Figure 2 represents a bar diagram of effect of biocontrol agents on the incidence soil borne diseases of tobacco at 60 days after planting
Figure 3 represents a bar diagram of bio-control agents on cured leaf yield of tobacco.
Figure 4 provides a graphical representation of the survival of Bacillus megaterium in granular formulation -03 at room temperature.
Figure 5 provides a graphical representation of the survival of Trichoderma harzianum ITC TH15 in different formulations at room temperature.
Figure 6 provides a graphical representation of the survival of Trichoderma viride ITC TV09 in different formulations at room temperature.
Figure 7 provides a graphical representation of the survival of Pseudomonas fluorescens PF1 in different formulations at room temperature.
Figure 8 provides a graphical representation of the survival of Bacillus subtilis BS1 in different formulations at room temperature.

Description of the invention
The present invention relates to a granular formulation of fungal and bacterial biocontrol agent with enhanced shelf-life for the control of soil borne diseases. The present invention further provides a method for the preparation of the granular formulation that can be used both for fungal and bacterial bio-control agents and other beneficial microbes for bio-fertility. The granular formulation of the present invention comprising, cell and spore suspension of microbe, water absorbent material, osmo-protectants, membrane stabilizers, binder, granulating agents, slow release nutrients, vegetable oil. The formulation uses commercially available low cost inputs and the process is easily scalable and commercially feasible.
The granular formulation for all fungal and bacterial biocontrol agents and biofertilizers have stability or shelf-life of at least 12 months for protecting plants from soil borne diseases and enhancing growth and yield parameters of agricultural crops. The efficacy of the stored product is proved by field trials. Further the technology developed is very simple, less expensive and can easily be adapted to industrial scale of production.
According to the present invention enhanced shelf life means, the survival of the beneficial microbes in the formulation, under room temperature, can be maintained for atleast 12 months. However, under refrigerated conditions (4°C - 10°C) the shelf life can be extended to about 10 years as well (preferably 120 months, more preferably 60 months and most preferably 36 months of shelf life).
According to the present invention the bacterial and fungal beneficial microbes as Trichoderma species, Pseudomonas species, Bacillus species or mixtures thereof are used to control soil borne crop diseases (targeting disease causing pathogens/ plant pathogens) and enhance the growth. These biocontrol agents when applied colonizes the rhizosphere of the crops (by different mode of action e.g., competition, producing antibiotics etc) and does not allow the harmful microbes that reside in the soil/rhizosphere to colonize. In turn, this helps to inhibit the growth of the harmful microbes that cause diseases on crops.
Further according to the present invention, the shelf-life of the formulation is increased by the following ways:

(i) The use of polyvinylpyrrolidone (PVP) of about 0.5-5% in the composition. PVP is a water-soluble polymer made from the monomer N-vinylpyrrolidone. PVP has high water-binding capacity, which reduces the toxic compounds in the medium and it is a very good membrane stabilizer known to protect cells, proteins and enzymes from adverse effects of temperature variations. These properties of PVP help in longer shelf-life of granular formulations.
(ii) By the use of slow release nutrients like rice flour (20% -40%) and wheat flour (20-40%) is known to supply microbes with required nutrients over a long period of time leading to longer survival of bio-control agents.
(iii) By the use of binder sodium alginate (1-5%) as an adhesive and gelling agent. Sodium alginate has the property of thickening the preparation and increasing the viscosity and binding the ingredients and microbes. This encapsulation of microbes by sodium alginates reduce direct exposure of microbe to environmental variations (like changes in temperature, humidity e.t.c.) and thus enhances the shelf-life.
The present invention has the following advantages over the prior art:
(1) The formulated product is effective in controlling soil borne plant pathogens and enhances plant growth and yield parameters.
(2) The present invention can be used for formulating all fungal and bacterial agents including biocontrol agents used for the control of soil borne plant pathogens.
(3) The shelf-life /viability of both fungal and bacterial agents in granular formulation is atleast 12 months.
(4) Uses slow release nutrients viz., rice flour, wheat flour and gluten, membrane stabilizing agent polyvinyl pyrrolidone and binder sodium alginate, which enhances the shelf-life of biocontrol agents.
The present invention relates to the method of preparing a stable granular formulation of both fungal and bacterial biocontrol agents and biofertilizers viz., Trichoderma harzianum, Trichoderma viride, Pseudomonas fluorescens, Bacillus subtilis and Bacillus megaterium. The

present invention uses the combination of water absorbent polymer, osmo-protectants to provide desiccation tolerance to living microbes, membrane stabilizers to stabilize the integrity of cell membrane from temperature fluctuations, combination of granulating agents and combination of slow release nutrients for longer nutrient supply to beneficial microbes to achieve easy granulation and longer shelf-life of the formulation.
According to the present invention, the formulation is prepared by the following process steps:
1. Biocontrol agent is grown in suitable liquid medium;
2. Concentrate the microbial cell and spore suspension by centrifugation;
3. Suspending the concentrated suspension in 0.01 M phosphate buffer (K2HP04:KH2P04);
4. The fungal/bacterial suspension is mixed with combination of osmoprotectants (15-40% of total dry weight of formulation), PVP (0.5-5%), water-absorbent material (0.1-0.5%), sodium alginate (1-5%), granulator (5-20%), slow release nutrients (30-60%) and mixed to form dough. The dough is mixed with vegetable oil (0.2-1.0% V/W). The dough is extruded through a hand held granulator to form long strands. The strands were cut into 3-5 mm length granules. The granules were air dried in shade by passing stream of cool air.



*rice flour also helps in granulation in addition to slow release nutrient
#gluten or wheat flour also helps in granulation in addition to slow release nutrient.
Process of granular formulation preparation:
Preparation of Fungal biocontrol agent's formulations:
Two sets of 2 X 500 ml of Potato Dextrose broth in 1000 ml capacity conical flasks were inoculated with 7-9 mm disc of 8-15 day old Trichoderma harzianum and Trichoderma viride grown of Potato dextrose agar plate. The inoculated flasks were incubated at room temperature for 10-15 days without agitation. After the incubation period the flasks were agitated well and centrifuged at 08-15°C to get concentrated biomass containing cells and spores of fungal bioagents. Subsequently the biomass of fungi is re-suspended in 300 ml of 0.01-0.03 M phosphate buffer (k2HP04:KH2P04, pH=7). The fungal suspension is poured in to clean tray (surface sterilized using 70% alcohol) and mixed with osmoprotectants mixture containing sucrose (10-30% of total dry weight of formulation), Lactose (3-15%), Dextrin (1-5%). subsequently water absorbent material i.e. aquakeep at 0.1-0.5%, Membrane stabilizer (Polyvinyl pyrolidone at 0.5-5%), were added and mixed well.
Thereafter, binder i.e. Sodium alginate at 1-5%, granulating agent (Diatamaceous earth at 5-20%), slow release nutrients (rice flour at 20-40%, wheat flour at 20-40%) and vegetable oil at 0.2-1.0% (v/w) were added and blended well to form a dough. The dough formed was extruded through a hand held granulator to form long strands and afterward the strands were cut into 3-5 mm length granules. The granules formed were air dried in shade or by passing stream of cool air in laminar air flow chamber.

Preparation of Bacterial biocontrol agent's formulations:
Two sets of 2 X 500 ml of Nutrient Broth in 1000 ml capacity conical flasks were inoculated with 1-5 ml of 24-48h old inoculum of Bacillus subtilis, Bacillus megaterium and Pseudomonas fluorescens. After inoculation the flasks were incubated at room temperature on a rotary shaker at 100 rpm for 5-7 days. After the incubation period the bacteria broth was centrifuged at 08-15°C to get pellet containing bacterial biomass. Subsequently the pellet is re-suspended in 300 ml of 0.01-0.03 M phosphate buffer (K2HP04:KH2P04, pH=7) and formulated exactly similar to fungal biocontrol agents formulation (as described above).


Table 3. Survival of Trichoderma harzianum ITCTH15, Trichoderma viride ITCTV09, Pseudomonas fluorescens ITCPF1, Bacillus subtilis ITCBS1 in Granular formulation-III at room temperature (Fig 1).
According to table 3, it is demonstrated that biocontrol agents T. harzianum, T. viride, P. fluorescens, B. subtilis demonstrate similar enhanced shelf-life in granular formulation III


Control of soil borne diseases of tobacco using granulated formulations of Biological control agents:
Table 4: Effect of Biocontrol agents on the incidence soil borne diseases of Tobacco at 60 days after planting (Fig 2).
Table 4 demonstrates that the biocontrol agents exhibit similar effect on disease incidence as compared to chemical control


Yield enhancement by biocontrol agents:
Table 5: Effect of Biocontrol agents on cured leaf yield of tobacco (Fig 3).
According to table 5, the biocontrol agents provides enhanced yield of cured leaf as compared to chemical controls.


Table 6. Survival of Bacillus megaterium in granular III formulation at room temperature (Fig 4).

The survival of Biocontrol agents in granular formulations I and II in comparison to Granular formulation III of the present invention:
Observations:
Tables 7-10 demonstrates the efficacy of granular III formulation in comparison with granular formulation I and II with respect to enhanced shelf-life of at least 12 months.
Table 7: Survival of Trichoderma harzianum ITC TH15 in different formulations at room temperature (Fig. 5)
According to table 7 it is demonstrated that T. harzianum exhibit enhanced survival (Logio CFU/g formulation) in granular formula III at 120 days, 180 days, 240 days and 300 days, 330 days and 360 days days as compared to granular formulation I and II



Table 8. Survival of Trichoderma viride ITC TV09 in different formulations at room temperature (Fig. 6)
According to table 8 it is demonstrated that Trichoderma viride ITC TV09 exhibit enhanced survival (Log10 CFU/g formulation) in granular formula III at 120 days, 180 days, 240 days, 300 days, 330 days and 360 days as compared to granular formulation I and II


Table 9. Survival of Pseudomonas fluoresceins PF1 in different formulations at room temperature (Fig. 7)
According to table 9 it is demonstrated that Pseudomonas fluorescens PF1 exhibit enhanced survival (Logio CFU/g formulation) in granular formula III at 240 days, 300 days, 330 days and 360 days as compared to granular formulation I and II


Table 10. Survival of Bacillus subtilis BS1 in different formulations at room temperature (Fig. 8)
According to table 10 it is demonstrated that Bacillus subtilis BS1 exhibit enhanced survival (Logio CFU/g formulation) in granular formula III at 240 days, 300 days, 330 days and 360 days as compared to granular formulation I and II

According to the present invention for the successful root colonization of any introduced microbe, it requires initial nutrition and favorable environment for establishment. The ingredients present in the formulation of the invention provides required nutrients for initial establishment and ultimately results in their better root colonization & efficacy in terms of disease suppression. Further the present invention resides in the select pick of ingredients (not only nutrients but also other elements for enhanced shelf-life of atleast 12 months.) and concentration range to achieve the desired benefit viz, (i) at least one beneficial biocontrol agents, which colonizes the rhizosphere of the crops; (ii) at least one suitable membrane stabilizer; (iii) at least one slow release nutrient; (iv) and a suitable binder; (v) atleast one osmoprotecant (e.g. Sucrose/lactose/dextrin/trehalose).
The present inventors have studied and optimized the best proportionate/concentration/combination of the ingredients which can give enhanced shelf-life of the granulated product. The mere addition of these ingredients may not provide the shelf-life of 12 months as observed in the prior art.

Further, it is surprisingly found that rice flour / wheat flour acts as "slow release ingredients" in the present invention although rice flour/wheat flour do not act as "slow release ingredients" in general. It has been designed (in terms of wt %) for the present invention in such a way to provide the desired benefits. Further the present invention uses polyvinylpyrrolidone (PVP) as membrane stabiliser and the same will provide the desired benefit only at a particular range
Hence the scope of the present invention resides in the combination of (i) at least one beneficial biocontrol agents, which colonizes the rhizosphere of the crops; (ii) at least one suitable membrane stabilizer; (iii) at least one slow release nutrient; (iv) and a suitable binder; (v) atleast one osmoprotecant (e.g. Sucrose/lactose/dextrin/trehalose).

We claim:
1. A granular bio-control composition having enhanced shelf-life comprising (i) at least one beneficial biocontrol agents, which colonizes the rhizosphere of the crops; (ii) at least one suitable membrane stabilizer; (iii) at least one slow release nutrient; (iv) a suitable binder; and (v) at least one osmoprotectant.
2. The granular biocontrol composition having enhanced shelf-life as claimed in claim 1 wherein the biocontrol agent is selected from Trichoderma species, Psendomonas species, Bacillus species or mixtures thereof.
3. The granular biocontrol composition having enhanced shelf-life as claimed in claim 1, wherein the said biocontrol agents colonizes the rhizosphere of the crops to inhibit the growth of harmful microbes.
4. The granular biocontrol composition having enhanced shelf-life as claimed in claim 1, wherein the membrane stabilizer is used in the range of 0.5-5 wt%.
5. The granular biocontrol composition having enhanced shelf-life as claimed in claim 1, wherein the membrane stabilizer is polyvinylpyrrolidone.
6. The granular biocontrol composition having enhanced shelf-life as claimed in claim 1, wherein the slow release nutrients are used in the range of 20-60%.
7. The granular biocontrol composition having enhanced shelf-life as claimed in claim 1, wherein the slow release nutrients are selected from rice flour, wheat flour or mixtures thereof.
8. The granular biocontrol composition having enhanced shelf-life as claimed in claim 1, wherein the binder is used in the range of 1-5%.
9. The granular biocontrol composition having enhanced shelf-life as claimed in claim 1, wherein the binder is sodium alginate.
10. A process for the preparation of the granular biocontrol agent as claimed in claim 1, comprising the steps of:
(i) Growing the biocontrol agent in suitable liquid medium;
(ii) Concentrating the microbial cell and spore suspension by centrifugation;
(iii) Suspending the concentrated suspension in 0.01 M phosphate buffer (K2HP04:KH2P04);

(iv) Mixing the fungal/bacterial suspension with combination of osmoprotectants (15-40% of total dry weight of formulation), PVP (0.5-5%), water-absorbent material (0.1-0.5%), sodium alginate (1-5%), granulator (5-20%), slow release nutrients (30-60%) and mixed to form dough;
(v) Mixing the dough with vegetable oil (0.2-1.0% v/W);
(vi) Extruding the dough through a hand held granulator to form long strands and cutting into 3-5 mm length granules;
(vii) Air drying the granules in shade by passing stream of cool air.

ABSTRACT

The invention relates to a granular bio-control composition having enhanced shelf-life comprising (i) at least one beneficial biocontrol agents, which colonizes the rhizosphere of the crops; (ii) at least one suitable membrane stabilizer; (iii) at least one slow release nutrient; (iv) a suitable binder; and (v) at least one osmoprotectant.
The invention provides a process for the preparation of the granular biocontrol agent comprising the steps of growing the biocontrol agent in suitable liquid medium and concentrating the cells and spore suspension by centrifugation; suspending the concentrated suspension in 0.01M phosphate buffer; mixing the cell suspension with combination of osmoprotectants (15-40% of total dry weight of formulation), PVP (0.5-5%), water-absorbent material (0.1-0.5%), sodium alginate (1-5%), granulator(5-20%) and slow release nutrients (30-60%) to form dough, mixing the dough with vegetable oil and extruding through hand-held granulator to form long strands cut into 3-5 mm lengths and air drying the granules by passing stream of cool air.