DESC:Field of the invention:
The present invention relates to microbial formulation capable of being used as plant growth promoter to stimulate plant growth in an eco-friendly manner. More particularly, the present invention relates to a bio-formulation of Bacillus aerius for enhancing soil fertility and facilitating plant growth and development by solubilizing phosphate and zinc, and mobilizing potash.

Background of the invention:
Agricultural production currently depends on large scale use of chemical fertilizers to provide essential plant nutrients nitrogen, phosphorus and potassium. However, the continuous use or overuse of fertilizers has caused unanticipated environmental impacts.

Plant microbe interactions contributes significantly to soil fertility and growth, health and productivity of plants. Plant growth promoting (PGP) bacteria enhance plant growth and protect plants from abiotic stress. The beneficial plant growth promoting properties of PGP bacteria are nitrogen fixation, iron chelation, phosphate solubilization, potash mobilization, inhibition of non-beneficial microorganisms, resistance to pest, and decomposition of plant material in soil to increase soil organic matter.

Plant rhizosphere contains billions of microorganisms known to have beneficial effects on plant growth. Therefore, the plant growth promoter based technology is an effective substitute of chemical fertilizers for sustainable development. This technology depends on developing efficient inoculants of PGP bacteria. Bacterial inoculants can contribute significantly to improve plant growth and productivity. Therefore, it is of utmost importance to increase the efficiency of inoculation.

US 2010/0300166 A1 discloses a biofertilizer composition wherein strain of Tsukamurella paurometabola is combined or mixed with other biofertilizer micro-organisms, such as Bacillus subtilis, Rhizobium leguminosarum, Azotobacter chroococcum, Pseudomonas fluorescens, Glomus fasciculatum and Glomus clarum, or a mutant derived from Such organisms, as well as any active principle or metabolite obtained from said strains by a natural, recombinant or synthetic way, in an appropriate carrier.

IN225918 discloses composition of the microbial plant growth promoter comprising Bacillus polymyxa, Lactobacillus acidophilus, Propionibacterium freudenreichii, Candida tropicallis, Azotobacter vinelendi, Nitrobacter and Bacillus licheniforrnis bioenhancers; (sugar base) khandsari and zeolite.

Genet Mol Biol 2015 Dec; 38(4):401-19 provides that at a global scale, the effects of continuous agricultural practices such as fertilizer application can cause serious damage to the environment. Inoculation of microbes in soil in form of bio fertilizer is one of the most important sustainable practices in agriculture, because microorganisms establish associations with plants and promote plant growth by means of several beneficial characteristics. The combination of different methodologies with these bacteria, such as identification of plant growth promoting characteristics, the identification of bacterial strains, as well as assays of seed inoculation in laboratory conditions and cultivation experiments in the field, are part of the search for new technologies for agricultural crops. Thus, when this search shows a potential bacterial inoculant, adequate for reintroduction in the environment, many genera such as Azospirillum, Bacillus and Rhizobium may be primary candidates. Finally, the research for beneficial bacteria is important for the development of new and efficient inoculants for agriculture. Also important are investments in technologies that can contribute to increase the inoculum efficiency and the survival rate of bacteria adherent to the seeds, which are other essential factors for successful inoculation. Thus, the introduction of beneficial bacteria in the soil tends to be less aggressive and cause less impact to the environment than chemical fertilizer, which makes it a sustainable agronomic practice and a way of reducing the production costs.
Phosphate Solubilizing Microbes (PSMs) has the natural ability to liberate unavailable phosphorus from soil, rocks, and mines for plants. The main mechanism behind this is production of mineral dissolving compounds such as organic acids, protons and hydroxyl ions and (Rodríguez and Fraga, 1999; Sharma et al., 2013).

The present invention provides a novel microbial plant growth promoter formulation of Bacillus aerius. The formulation provides highly efficient bacterial inoculant showing phosphate solubilizing, zinc solubilizing and potash mobilizing properties. The invention aims at Bacillus aerius formulation with multiple PGP properties as potential ingredient for biofertilizer to improve the plant growth and develop cost effective agriculture practices.

Object of the invention:
The main object of the present invention is to provide a novel plant growth promoter formulations of B. aerius.

Another object of the present invention is to provide an optimized media composition to obtain highly efficient bacterial inoculant of Bacillus aerius.

Yet another object of the present invention is to provide novel plant growth promoter formulation of Bacillus aerius having highly efficient bacterial inoculant with phosphate solubilizing, zinc solubilizing and potash mobilizing properties.

Yet another object of the present invention is to provide novel water soluble formulation of Bacillus aerius.

Yet another object of the present invention is to provide powdered formulation of B. aerius having highly efficient bacterial inoculant.

Yet another object of the present invention is to provide granular formulation of B. aerius having highly efficient bacterial inoculant with phosphate solubilizing, zinc solubilizing and potash mobilizing properties.

Yet another object of the present invention is to provide an optimized media composition to obtain highly efficient bacterial inoculant of Bacillus aerius.

Yet another object of present invention is to provide a novel plant growth promoter formulation of Bacillus aerius of high shelf life of more than 12 months.

Yet another object of the present invention is to provide a process to obtain a formulation of Bacillus aerius of highly efficient bacterial inoculant for plant growth and development by phosphate solubilizing, zinc solubilizing and potash mobilizing properties.

Summary of the present invention:
It will nevertheless be understood that no limitation of the scope of the invention is thereby intended by way of embodiments and examples. Such 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 constructed as being within the scope of the present invention.

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.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive 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 a process or method. Similarly, one or more steps of method or components preceded by "comprises... a" does not, without more constraints, preclude the existence of other, steps or components. Appearances of the phrase "in a preferred embodiment”, “in an embodiment", “in another embodiment” and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.

Accordingly, the present invention provides a microbial plant growth promoter formulation to stimulate plant growth in eco-friendly manner. The formulation comprises phosphate solubilizing soil bacteria for plant growth. The bacteria is Bacillus aerius contributing to enhance soil fertility and facilitating plant growth and development by solubilizing phosphate and zinc, and mobilizing potash.

In one of the basic embodiments, the formulation helps plants to optimize the acquisition of organic matter, favouring the acquisition of macro- and micro- nutrients by the activity of Bacillus aerius. The formulation is applied independently or in combination with other biofertilizers and bio-stimulators.

In another embodiment, the bacteria B. aerius is combined or mixed with other biofertilizing microorganisms.

In another non-limiting embodiment, the formulation enhances the growth of Zea mays (maize) plants.

The present invention also provides a process for optimization growth parameters of bacteria B. aerius to obtain highly efficient bacterial inoculant both in fermenter and shake flask. The process parameters optimized for optimized growth of B. aerius are aeration, agitation, temperature and pH, along with media optimization through carbon and nitrogen sources to achieve a high cell density growth of more than 1x1010 CFU/ml.
The formulation is provided in different formulation such as wettable powder, liquid, granule and water-soluble formulation. The shelf life in all the formulation is more than 12 months. The product has established good efficacy in terms of pot culture and field trials.

Example:
Out of total 137 bacterial isolates, one potential bacteria having multiple plant growth promoting (PGP) properties was isolated and studied for green house experiments in pots. Its phosphate solubilization ability was shown by the clear zone formation on Pikovskaya's agar, Phosphate medium with solubility index (PSI) of 5.153333. The potential isolate was found to solubilize tri calcium phosphate (Ca3(PO4)2) at a rate of 790.67 mg L-1. The bacterial isolate was further screened for other plant growth promoting activity and was found to be positive for potash solubilization (KSI value-4.016), zinc solubilzation (ZSI value-7.875) and siderophore production (SI-3.5). On the basis of morphological and molecular characterization, isolate was identified as Gram positive bacterium, Bacillus aerius. LabID-B197 was assigned to Bacillus aerius for further pot experiments.

Greenhouse experiments demonstrated that maize plants inoculated with B197 incorporated more phosphate which was observed as increased shoot and root length and much higher biomass weights than those plants without any added bacteria or controls. Further the pot culture experiment was carried out to evaluate the effect of phosphate solubilizing B197 on the growth of maize. Germination percentage of 93.3 per cent was observed in the maize when it was treated with only B197 and no other fertilizers. The isolate was found to solubilize phosphate, zinc and mobilize potash.

The effective amount of formulation is applied on soil or substrate in aqueous suspension. The substrate can be seedling, seed or set of crops.

DETAILED DESCRIPTION OF INVENTION WITH ILLUSTRATIVE EXAMPLES AND NON-LIMITING EMBODIMENTS:
It should be noted that the particular description and embodiments set forth in the specification below are merely exemplary of the wide variety and arrangement of instructions which can be employed with the present invention. The present inventions may be embodied in the other specific forms without departing from the spirit or essentials characteristics thereof. All the features disclosed in this specification may be replaced by similar other or alternative features performing similar same or equivalent purposes. Thus, unless expressly stated otherwise, they all are within the scope of present invention. Various modifications or substitutions are also possible without departing from the scope or spirit of the present invention. Therefore, it is to be understood that this specification has been described by way of most preferred embodiment and for the purpose of illustration and not limitation.

The present invention provides a microbial plant growth promoter formulation of
Bacillus aerius exhibiting phosphate solubilizing, zinc solubilizing and potash mobilizing properties. The formulation of the present invention is stable at room temperature and has a shelf life of more than 12 months.

The microbe Bacillus aerius was isolated from rock phosphate mine of Jhamarkotra, Udaipur Rajasthan. Samples were collected in sterile containers. The organism is deposited at IMTECH (Institute of Microbial Technology), Chandigarh, India bearing accession no. MTCC 25483 dated December 03, 2021.

The isolated bacteria is purified and cultured on an optimized culture medium in shake flask. The optimized culture medium comprises a carbon source, a nitrogen source and other nutrient source in pre-determined amount.

The carbon source is selected from mannitol, fructose, glucose, sucrose and glycerol.
The nitrogen source is selected from soyameal, urea, beef extract, and ammonium sulphate.

The other nutrient source is selected from ammonium chloride (NH4Cl), potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), either alone or in combination.

In a preferred embodiment, the optimized culture medium comprises mannitol, soyameal, NH4Cl, KH2PO4 and K2HPO4 in 100 ml distilled water at pH 7.2-7.5. The optimized culture medium is diluted in water, wherein the medium and water ratio is 1:5.

In a most preferred embodiment, the optimized culture medium comprises NH4Cl 0.5%, KH2PO4 0.5%, K2HPO4 0.5%, Mannitol 0.3%, Soyameal 0.5% in 100 ml distilled water at pH 7.2-7.5. The optimized culture medium is diluted in water, wherein medium and water ratio is 1:5.

The optimized culture medium is inoculated with 0.5% (v/v) the bacterial culture and incubated for 48 hr, with agitation rate of 420 RPM and aeration of 0.9 VVM to 1VVM. Fermentation is carried out in shake flask and yields fermented broth containing bacterial inoculant with optimized bacterial count (spore, etc) of 1X1010 cfu/ml. The bacterial cells in fermented broth are motile rods. The fermented broth is brownish yellow in colour and pH was 7.5-8.5.

The fermentation is also carried out in fermenters with the same optimized fermenter medium, using 0.5% pre-inoculum from the shake flask. The process parameters for incubation and fermentation in the fermenters are same as in case of shake flask, thereby making the process scalable.

The fermented broth is harvested, and formulation is prepared as liquid, wettable powder, granule and water soluble formulation. Preservative is added to the formulation in specific amount to obtain a highly stable formulation. The preservative is selected from PVP (Polyvinylpyrrolidone), PEG (Polyethylene glycol) or a combination thereof in a range of 1% to 5%. The pH of the formulation is in the range of 7.0 to 8.5.

The shelf life of the formulation is more than 12 months. In a preferred embodiment, the shelf life of the formulation is in the range of 12 months to 24 months. The bacterial CFU count per ml of the formulation is in the range of 1 X 109 to 1 X 1010.

In another embodiment, the granule formulation is prepared by mixing harvested broth with 5% PEG. The ratio of fermented broth and PEG in the formulation is 1:4.5. To obtain granule formulation, PEG is added in the flask or fermenter during incubation. The moisture content in the granule formulation is 10% to 15%.

In yet another embodiment, the powder formulation is obtained by mixing harvested broth with aluminium silicate and rock phosphate in a ratio of 1:3:1. 5% PVP as a preservative is added during the mixing. The moisture content in the powder formulation is 10% to 15%.

The water soluble powder formulation is obtained by adding dextrose as a carrier. The harvested broth and carrier are mixed in a ratio of 1:20. 3-5% preservative is added during mixing. The moisture content in the water soluble powder formulation is 5%.

The field experiments have been conducted to evaluate the efficiency of the bacterial inoculum in the formulation in greenhouse and fields.

During greenhouse experiment, field soil has been sterilized thrice by autoclaving for 20 min at 120°C within 24 hr interval. Seeds have been sterilized with 0.1% mercuric chloride for 30 sec and then washed with sterilized distilled water thrice, then sterilization with 70% ethanol for 1 min has been done and then washed twice with autoclaved distilled water. Ten seeds have been sown at 4 to 5 cm depth of sterile soil in each pot, in triplicates. Total 6 pots have been used for experiment, 3 replicate pot for uninoculated seed which have been used as control (C) and 3 replicates pot having seed treated with bacterial inoculum of the present invention have been used as test (T). Treatments have been arranged in a factorial experiment based on completely randomized design. Pots have been watered daily and no artificial fertilizer was used. After 30 days, fresh weight of root and shoot have been determined and dry weight of root and shoot have been calculated by drying plants in an oven at 75°C until the weight remained constant. Besides this, % germination have also been calculated.
Germination %= (No of germinated seed/ Total number of seed) X 100

After 30 days of application of bacterial inoculum, the plants showed significant increase of up to 155% in mean shoot length, up to 45% in mean root length in comparison to control. Similarly, significant increase in fresh shoot weight of up to 165%, dry shoot weight of up to 124%, and fresh root weight of up to 75.95%, while a decrease in dry root weight of up to 14.18% was observed in comparison to control after 30 days. Germination percentage recorded in the maize seeds was 93.3% as compared to control which is 66%. The results are presented in Table 3.

Table 3. Effect of bacterial inoculum of B. aerius of the present invention on growth of maize during Greenhouse experiments.
Type Mean Shoot length (cm) Mean Root Length (cm) Shoot fresh weight (gm)
Root fresh weight (gm) Shoot dry weight (gm)
Root dry weight (gm)
Germination %
Control (C) 2.833 24 1.485 0.84 0.200333 0.228 66%

Test (T) 18.67
61.33333 3.935667
1.478
0.448333
0.195667
93.3%
During field experiment, maize seeds were treated with powder formulation of the invention and sown. The field of study was conducted in Bijnor, UP, India. The study was divided into segments. Each segment was demarcated and divided into grids. The grids denoted the Control field (C), and the test field (T). The experiment was done in random block design (RBD) in triplicates for each treatment. The size of each plot was 100 square meter. The maize seeds were planted at a distance of 20-30 cm apart from each other. The seeds were regularly watered and growth was measured in terms of plant height, shoot length, root length, fresh biomass and dry biomass of maize plant. Growth parameters were studied on 60th days after sowing.

The results showed that the application of formulation of the present invention had significant effect on plant height, fresh and dry shoot and root weight in comparison to control (Table 3).

The plants from the treated seeds showed increase in mean shoot length of up to 17.39% and mean root length of up to 27.27% as comparison to control. Similarly, significant increase in fresh shoot weight of up to 10.91%, dry shoot weight of up to 26.41 %, and fresh root weight of up to 7.96 %, while a decrease in dry root weight of up to 16.70% was observed in comparison to control after 60 days. Application of formulation of the present invention on maize seeds significantly influenced the germination percentage due to its phosphate solubilizing property. Germination percentage recorded in the maize seeds was 80 per cent. Besides effect on germination percentage, the formulation had significant effect on shoot and root length in comparison to control (Table 4).
Table 4. Effect of powder formulation of the present invention on growth of maize in field experiments.
Type Mean Shoot length (cm) Mean Root Length (cm) Shoot fresh weight (gm) Root fresh weight (gm) Shoot dry weight (gm)
Root dry weight (gm) % germi
nation
Control (C) 230 55 33.3 28.23 12.22 10.45 65%
Test (T) 270 70 36.935667
30.478
15.448333
12.195667
80%

CLAIMS:We Claim:
1. A microbial plant growth promoter formulation comprising highly efficient bacterial inoculant of Bacillus aerius for enhancing soil fertility and facilitating plant growth and development by solubilizing phosphate and zinc, and mobilizing potash, wherein said bacterial inoculant has high cell density growth in the range of 1 X 109 to 1 X 1010.

2. The formulation as claimed in claim 1, wherein said formulation has a shelf life of 12 months to 24 months.

3. The formulation as claimed in claim 1, wherein said formulation is in the form of liquid, wettable powder, granule and water soluble formulation.

4. The formulation as claimed in claim 1, wherein preservative is added to the formulation, said preservative is selected from PVP (Polyvinylpyrrolidone), PEG (Polyethylene glycol) or a combination thereof in a range of 1% to 5%.

5. The formulation as claimed in claim 1, wherein pH of the formulation is in the range of 7.0 to 8.5.

6. The formulation as claimed in claim 1, wherein said highly efficient bacterial inoculant is obtained by culturing the Bacillus aerius on an optimized culture medium.

7. The formulation as claimed in claim 6, wherein said optimized culture medium comprises a carbon source, a nitrogen source and other nutrients source in pre-determined amount.

8. The formulation as claimed in claim 6, wherein said carbon source is selected from mannitol, fructose, glucose, sucrose and glycerol.
9. The formulation as claimed in claim 6, wherein said nitrogen source is selected from soyameal, urea, beef extract, and ammonium sulphate.

10. The formulation as claimed in claim 6, wherein said other nutrients source is selected from ammonium chloride (NH4Cl), potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), either alone or in combination.

11. The formulation as claimed in claim 6, wherein said optimized culture medium comprises NH4Cl 0.5%, KH2PO4 0.5%, K2HPO4 0.5%, Mannitol 0.3%, Soyameal 0.5%.

12. The formulation as claimed in claim 6, wherein said optimized culture medium is diluted in water in a ratio of medium and water ratio being 1:5 at pH 7.2-7.5.

13. A process to obtain the formulation as claimed in claim 1, wherein said process comprises:
- preparing pure culture of isolated Bacillus aerius,
- preparing the optimized culture medium,
- inoculating the optimized culture medium with the 0.5% (v/v) bacterial culture under specific process parameters to obtain fermented broth having bacterial inoculant with optimized bacterial count in the range of 1X109 to 1X1010 cfu/ml,
- harvesting said fermented broth to obtain the formulation,
- mixing the formulation with a preservative.

14. The process as claimed in claim 13, wherein said specific process parameters are incubation time of 48 hr at agitation rate of 420 RPM and aeration of 0.9 VVM to 1VVM.
15. The process as claimed in claim 13, wherein said fermented broth is brownish yellow in colour with pH in the range of 7.5 to 8.5.

16. The process as claimed in claim 13, wherein said preservative is selected from PVP (Polyvinylpyrrolidone), PEG (Polyethylene glycol) or a combination thereof in a range of 1% to 5%.

17. The formulation as claimed in claim 1, wherein said formulation helps plants to optimize the acquisition of organic matter and favouring the appropriation of macro- and micro- nutrients.

18. The formulation as claimed in claim 1, wherein said formulation can be applied in combination with other biofertilizers and bio-stimulators, or independently or with other biofertilizing microorganisms.