DESC:Field of the invention
The invention relates to agricultural biotechnology and microbiology.
The invention relates to a novel endophytic Bacillus mojavensis with broad spectrum antipathogenic activity for crop protection. The invention also discloses optimized cultivation process of a novel endophytic Bacillus mojavensis with broad spectrum antipathogenic activity for crop protection. The invention further relates to a composition comprising a novel endophytic Bacillus mojavensis with broad spectrum antipathogenic activity for crop protection. The novel strain of Bacillus mojavensis and composition thereof also improves functional qualities of crop and increase crop productivity irrespective of climatic conditions.

Background of the invention
Chemical fertilizers have been used from decades in agriculture to provide essential macronutrients such as phosphorus and potassium. However, continuous use or overuse of chemical fertilizers have negative impact on soil and environment. Microbial based fertilizers are observed to be among prime agricultural components that improve crop productivity, and soil health which contribute to sustainable ecosystem. Application of fertilizers in minimum rational quantity combined with microbial enrichment will reduce need of chemical fertilizers, thereby preventing chemical leaching into soil.

Further exploration in terms of application of combined use of microbes and chemical fertilizer can improve the sustainability of soil in terms of improving soil microflora which in turn effects crop growth, defense against pest and crop yield. Microbes play important role in degrading the macronutrients, specifically P and K to a form which is easily available to the plants. Use of microbes affects the soil properties, reduces abiotic stress, and make it feasible for others beneficial plant associated microbes to flourish.

The world is looking for solutions to address the issue of food insecurity due to increase in population. Unscrupulous and exorbitant use of chemicals has led to pest resistance and negative impact of human health, animal health and environment. This has driven need to switch to biologically safe cultivation approach to get quality food for good health and sustainable environment.

Most of the agricultural microbial based products existing in market are rhizomicrobiome based products. In spite of their potential traits of performing well at laboratory scale, greenhouse and field conditions, they lack to perform well at varied agro-climatic conditions. This may be attributed to the fact that they face tough competition to assimilate with complex network of soil microbiome which end up in poor performance on plant growth enhancement.

To overcome these drawbacks of the rhizomicrobiome based products, endophytic microbes with multivariant traits would be the promising solution. But to date very limited endophytic microbial based products are available in the market. For successful commercial use of endophytes in agriculture, numerous factors have to be tested, viz. identification of new competent strains which are biotic and abiotic stress tolerant, development of new microbial inoculum for industrial application and their success at field level which is very challenging.

US2010092442 discloses methods of inducing systemic acquired resistance to infection in a plant by applying a composition comprising a Bacillus control agent to said plant wherein said plant is capable of producing defense proteins. The method comprising applying a Bacillus control agent comprising Bacillus mojavensis isolate 203-7 having accession number NRRL B-30893 and/or Bacillus mycoides isolate BmJ having accession number NRRL B-30890 to the plant. The document neither teaches the media composition to cultivate Bacillus control agents nor does it proposes application of the Bacillus mojavensis 203-7 isolate and the antibacterial agent together. Furthermore, the Bacillus mojavensis 203-7 isolate disclosed in the patent does not show fengycin hyper-producing properties and does not detect any of the bioactive metabolites at molecular level. No plant growth promoter (PGP) or abiotic assays screened.
WO9724433 discloses biocontrol methods and microorganisms, which are capable of controlling plant diseases of fungi origin. The microorganisms useful for biocontrol purposes are novel endophyte symbiotic Enterobacter cloacae strains. Said strains are suitable for passing useful genetic products into the plants. The used Enterobacter cloacae strains are rifampicin resistant. The Enterobacter cloacae strains ATCC55732 as analysed their 16S rRNA gene sequence was Bacillus mojavensis strains. The major shortfall of the isolate according to the patent is that no work in relation to detection of bioactive compounds, their biocontrol and PGP related enzymes determined nor composition and their application part in biotic/abiotic regions found therefore likely to be discouraged.

CN101163653B discloses an enhanced fertilizer comprising fertilizer particles, lactic acid bacteria and bacteria of the Baciliaceae family. The invention also relates to an enhancer for a fertilizer and a soil additive to enhance plant growth. The invention further describes the methods for increasing growth, development or yield of a plant and methods of enhancing a soil for increasing growth, development or yield of a plant.

WO2013148278A1 provides microbial composition comprising liquid fertilizer and processes for agricultural use. The compositions comprise a HYTa microbial composition and liquid fertilizer, preferably a liquid fertilizer that contains at least soluble nitrogen. The composition can be applied immediately to soil, foliage, seeds or seedlings. Alternatively, the composition may be activated by incubation for up to about seven days to form an activated composition. The incubation conditions influence the properties of the consortium after incubation. In one embodiment, the compositions comprise one or more lactic acid producing bacteria, one or more nitrogen fixing bacteria, and liquid fertilizer comprising soluble nitrogen. In preferred embodiments, there are multiple genera and/or species of lactic acid bacteria and nitrogen fixing bacteria.

WO2013034938A2 discloses a copper resistant, fengycin-producing Bacillus mojavensis strain for controlling vegetable pathogens, its use and compositions containing it. The Bacillus mojavensis R3 B mutant strain deposited under # NCAIM (P) B 001389 exerts the antagonism against the pathogens of green vegetables, preferably tomato, pepper, lettuce and/or cabbage. The document further discloses a pesticide composition, which contains a culture of the biological material and optionally contains a copper-containing pesticide, preferably copper-sulphate, copper-oxyquinolate, copper-oxide, copper-hydroxide and copper-oxy-chloride, and optionally an excipient. The composition is an aqueous suspension, suspension concentrate, capsulated concentrate, emulsion forming liquid spray, granule, granule dispersible in water, microgranule or water soluble powder dose formulation.
CN105831160A discloses composition utilizing Bacillus mojavensis KJS-3 strain or its culture to promote plant growth and prevent plant death. In addition, in order to promote plant growth or reduce mortality, the invention provides a root-endophytic method for the Bacillus mojavensis KJS-3 so as to promote plant growth and prevent plant death.
CN109266580A discloses a kind of drought-resistant rush seed of symbiosis sprouts endophyte and its application. The invention protects Bacillus mojavensis AER314-4, its deposit number is: CCTCC NO:M2018495. It was confirmed by experiments that Bacillus AER314-4 could increase the germination rate of Sophora japonica seeds by 54% under 25% PEG 6000 drought stress, and the starch was degraded to produce soluble sugar by activating the amylase in the seeds, which decreased the osmotic potential of the seeds and promoted the germination of water absorption. Bacillus has the characteristics of fast growth, strong resistance, no time and climate limitations, safety for human and animals, no pollution of the environment, no adverse effects on plants. The strain has the potential to be used as desert plant seeds to promote germination under drought stress, and can be applied to desert plant seeds to promote seed germination under drought stress to promote ecosystem restoration.
CN103931659A discloses application of bacillus mojavensis KJS-3 as biopesticide. The invention discloses the application of bacillus mojavensis KJS-3 as a microbial source biopesticide. The bacillus mojavensis KJS-3 (with the preservation number of KCCM 10961P) has very strong antibacterial and antifungal effects for the common pathogenic bacteria causing plant diseases, especially for pathogenic fungus, and is capable of remarkably promoting the growth of plants and protecting the health of the plants as a plant growth promoting rhizobacterium.
WO2021118412A2 relates the strain Bacillus mojavensis PS17 isolated from the seeds of the "Sadokat" variety of spring wheat, deposited in the National Bioresource Center - Russian National Collection of Industrial Microorganisms (NBC VKPM), NRC "Kurchatov Institute" - GosNIIgenetika, under registration number VKPM B-13415. The strain has a broad spectrum of activity against plant pathogenic fungi and improves the yield of agricultural crops. The strain is non-toxic to animals, exhibits growth-stimulating activity for plants and can be used to produce biopreparations on the basis thereof for the protection of plants from diseases caused by fungal pathogens.
RU2648163C1 relates to a Bacillus mojavensis Lhv-97 strain separated from rhizosphere soil of the Black Sea coast of the Caucasus, deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures "SCPM-Obolensk" under the number B-8101. Strain has a wide spectrum of action against pathogenic fungi and bacteria, including strains of the Candida genus, resistant to fluconazole, increases plant yield. Strain is nontoxic for plants and animals and can be used to create on its basis preparations for protection of humans, animals and plants from diseases caused by fungal and bacterial pathogens.
Most of the research is focussed towards the enhancement of biomass production (i.e., yield) and nutrition, increased tolerance to stress such as drought and pests. Despite of ongoing research, there is no commercially successful composition of Bacillus mojavensis available in the market. To date, a small number of endophyte based microbial studies have provided fitness benefits to plants against pathogens under field conditions. The endophytes have found to be ineffective or of limited efficacy in conferring benefits to a variety of crops. As such, they do not adequately address the need to provide broad spectrum disease control, enhanced novel bioactive compositions with multifunctional traits, improved yield and tolerance to environmental stresses present in many agricultural situations for such crops, particularly saline and drought.

In view of the above drawbacks in the existing state of the art, present invention is a ground breaking new approach to bring out the climate adaptive endophytic strains applicable at broad scale with enhanced bioactive composition.

Object of the invention
The main objective of the invention is to provide a composition of endophytic Bacillus mojavensis with plant growth promoting and broad spectrum antipathogenic activity for crop protection.

Yet another object of the present invention is to provide a media composition for endophytic Bacillus mojavensis with broad spectrum antipathogenic activity for crop protection activity and for improved quality, increase productivity of crop irrespective of climatic conditions.

Yet another object of the present invention is to provide a composition of endophytic Bacillus mojavensis capable of improving functional qualities of crop and increase productivity irrespective of climatic conditions.

Yet another object of the present invention is to provide a stable and industrially scalable composition of a novel endophytic Bacillus mojavensis with broad spectrum antipathogenic activity and for improved quality, increase productivity of crop irrespective of climatic conditions.

Summary of the invention
Accordingly, in an aspect of the present disclosure, the invention relates to a composition of endophytic Bacillus mojavensis with broad spectrum antipathogenic activity for crop protection and plant growth promoter activity.

In another aspect of the present disclosure, the invention relates to an optimized cultivation process of the endophytic Bacillus mojavensis with broad spectrum antipathogenic activity for crop protection and plant growth promoter activity.

In yet another aspect of the present disclosure, the invention relates to an optimized media composition for a novel endophytic Bacillus mojavensis with broad spectrum antipathogenic activity for crop protection and plant growth promoter activity.

In yet another aspect of the present disclosure, the invention relates to a composition comprising a novel endophytic Bacillus mojavensis with broad spectrum antipathogenic activity for crop protection and plant growth promoter activity.

In yet another aspect of the present disclosure, the invention relates to a composition comprising a novel endophytic Bacillus mojavensis capable of improving functional qualities of crop and increase productivity irrespective of climatic conditions.

In an aspect, the invention also relates to a method to obtain composition of the microbial formulation comprising novel strain of Bacillus mojavensis for use in agriculture.

In an embodiment, the endophytic gram positive bacterial strain of Bacillus mojavensis having a specific 16S ribosomal RNA (rRNA) sequence with NCBI Gene Bank accession No. OM110922 is isolated. from paddy crop (Oryza sativa), collected from Rohtak cultivated land, Haryana, India in the year 2021. The isolated strain possesses indole acetic acid (IAA) production capability along with phosphate solubilisation, zinc mobilization, thereby demonstrate a potential plant growth promoting activity. The B. mojavensis of the present invention produces specific and useful secondary metabolites such as enzymes, for example chitinase that helps in chitin degradation, cellulase for hydrolysing cellulose, siderophore and the presence of genes encoding antimicrobial peptides, viz., Iturin –A (lpa-14 & itu D1); sbo A (Subtilosin) and Iturin (itu C & itu D). These secondary metabolites have the ability to inhibit the growth of plant pathogens and provide tolerance to high salt concentration and drought.

In an embodiment, the invention provides novel endophytic bacterial strain B. mojavensis and/or antibiotic substances produced from the bacterial strain for use as an antifungal agent and inhibits plant pathogens such as pathogenic bacterial or pathogenic fungus. It is a potential biocontrol agent with broad spectrum antipathogenic activity.

In an embodiment, the invention provides novel endophytic bacterial strain B. mojavensis and/or antibiotic substances produced from the bacterial strain to inhibit pathogenic fungus selected from a group consisting of Botrytis, Rhizoctonia, Colletotricum, Fusarium, Alternaria, Aspergillus, Penicillium.

In an embodiment, the invention provides a non-toxic and biodegradable composition of novel endophytic bacterial strain B. mojavensis for use in agriculture and environmental protection, etc.

In an aspect, the invention provides a composition comprising live strain of novel endophytic bacteria B. mojavensis.

In an aspect, the invention provides a composition comprising live strain of novel endophytic bacteria B. mojavensis in combination with at least one other microorganism. The other microorganism may be selected from live bacteria, inactivated bacteria or extracts of the bacteria.

In an aspect, the composition of the invention further comprises a buffering agent and/or a wetting agent and/or a polysaccharide or a combination thereof.

In an aspect, the invention comprises an optimized cultivation process of B. mojavensis.

In an aspect, the invention comprises a composition comprising the B. mojavensis, a carrier, a surface active agent and can be applied under biotic/abiotic stress conditions.

In an aspect, the composition of the invention further comprises glycerol, polyethylene glycol, polyvinyl pyrollidone, mineral solutions or culture supports with enhanced lipopetides.

In an embodiment, the composition of the invention is used for crop protection and plant growth, wherein the application of composition may be on the ground, or on root part or it may be foliar treatment.

In an aspect, the composition may be in the form of suitable formulations including liquid formulation, powder formulations such as wettable powder, granules formulation, or suspension concentrate (flowable concentrate).

In an aspect, the formulation contains 106 to 1010 CFU /ml.

In an aspect, the formulation is packaged in packaging suitable to protect them from degradation and stored at room temperatures. The formulations are stable and can be stored at room temperature for at least 12 months to18 months.

The enhanced lipopeptide media composition of B.mojavensis acts as a highly efficient endophytic solution that help the crop to survive under any biotic/abiotic stress condition and improve fruit quality by supressing diseases that may cause economic loss. The composition of the present invention is eco-friendly, economical with better shelf life and replace the agri-based chemicals in safe sustainable manner.

In an aspect, the composition is a water-soluble formulation of augmented essential macronutrients that contains at least novel B.mojavensis, phosphorus and potassium together.

In an aspect, the composition is used in combination with a chemical fertilizer.

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.

Brief description of drawings
Fig.1 depicts screening of endophytic strain of Bacillus mojavensis for cellulase, chitinase, dual culture assay, plant growth promoter activity.
Fig. 2 depicts detection of iturin genes in isolates B005 and B193. Here, 1-2: lpa14 gene; 3-4: ituC gene; 5-6: ituD gene; 7-8: ituD1 gene and 9-10: sboA gene whereas M indicates 100 bp ladder.
Fig. 3 depicts poison assay of active compound extract at 0.5 mg/ml concentration (B.mojavensis - B005) against Botrytis sp. and Rhizoctonia solani.
Fig. 4 depicts B.mojavensis B005 was very effective against Sclerotium (85.5%), B.cinerea (74.82%), Curvularia sp.(71.42), it was found effective in controlling 21 pathogens out of 24 selected plant pathogens under invitro screening (dual culture method) method.
Fig. 5 depicts PGPR effects in B.mojavensis B005 with bioactive metabolite (SM) with varied concentration compared with control (SM9) in Egg Plant.
Fig.6 depicts estimation of chlorophyll and carotenoids content in presence of varied concentration of B. mojavensis SM in Egg plant.
Fig. 7 depicts shelf life analysis of bioactive enhanced liquid formulation of B. mojavensis.

Detailed description of the invention with non-limiting embodiments and examples

In the following detailed description of the invention, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be obvious to a person skilled in the art that the invention may be practiced with or without these specific details. In other instances, well known methods, procedures and components have not been described in details so as not to unnecessarily obscure aspects of the invention.

Furthermore, it will be clear that the invention is not limited to these alternatives only. Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art, without parting from the scope of the invention.

The B. mojavensis used in the present disclosure has been obtained from Rohtak cultivated paddy crop land, Haryana, India.

In an embodiment of the present disclosure, there is provided a fungicidal bioformulation comprises B.mojavensis with enhanced lipopeptides namely, iturin and fengycin in optimized culture media.

In an embodiment of the present disclosure, the fungicidal bioformulation of the present invention comprises B.mojavensis with enhanced lipopeptides namely, iturin and fengycin, buffering agent and/or a wetting agent and/or a polysaccharide or a combination thereof.
In an embodiment of the present disclosure, the bioformulation of the present invention is stable at room temperature for up to18 months.

In an embodiment, the bioformulation of the present invention is in the form of wettable powder, granules formulation, or suspension concentrate.

In an embodiment, the bioformulation of the present invention is a plant growth promoter and potential biocontrol agent with broad spectrum antagonistic activity.

In an embodiment of the present disclosure, the process of the bioformulation of the present invention comprises preparing an optimized culture media and sterilizing it.

In an embodiment of the present disclosure, the optimized culture media comprises of one or more components like water soluble polymers selected from glycerol, polyethylene glycol, polyvinyl pyrollidone; mineral solution; one or more protein source; one or more sugars and a surface active agent like SPAN 80.

In an embodiment of the present disclosure, the mineral solution is selected from MgS04, K2HPO4, NaCl or a combination thereof.

In an embodiment of the present disclosure, the protein source is selected from soy peptone, beef extract, casein hydrosylate or a combination thereof.

In an embodiment of the present disclosure, the one or more sugars is selected from sucrose, trehalose, maltose, lactose, glucose, dextrose in the range of 0.3-0.8 percentage.

In an embodiment of the present disclosure, the surface active agent is selected from like polysorbates/20/80/SPAN in the range of 0.05-0.2 percent.

The sterilized culture media is inoculated with starter culture of B. mojavensis and incubated at predetermined conditions. The predetermined conditions of incubation are 300C temperature for 48 hr to 72 hr at 150 rpm to180 rpm with a pH between 6 to 8.

The incubated culture media is centrifuged, and supernatant is separated. Vacuum evaporation of supernatant is done to obtain the B.mojavensis with enhanced lipopeptides namely, iturin and fengycin with viable cell count in the range of 106 to 109 CFU /ml. The B.mojavensis with enhanced lipopeptides is mixed with buffering agent and/or a wetting agent and/or a polysaccharide or a combination thereof to obtain the fungicidal bioformulation of the present invention.

The biofungicidal bioformulation of the present invention is an economical, eco-friendly, stable and industrially scalable composition with broad spectrum antagonistic activity and for improved quality, increase productivity of crop irrespective of climatic conditions.

In an embodiment of the present disclosure, there is provided an media composition for enhanced activity of the endophytic B.mojavensis with broad spectrum antagonistic activity. The media composition comprises soy peptone, beef extract, casein hydrosylate, MgSO4, K2HPO4, NaCl and dextrose.

In an embodiment of the present invention, isolated endophytic microorganism is classified as an endophytic gram positive bacterial strain of Bacillus mojavensis with a NCBI Gene Bank Accession No. OM110922. This novel endophytic bacterial strain produces specific and useful multivariate functions viz., phyto hormones such as auxin produce by PGPR, indole acetic acid (IAA) along with phosphate solubilisation and zinc mobilization, thereby demonstrate plant growth promoting activity. Siderophores are produced which supply iron to plants and promote their growth and aid in controlling the disease causing plant pathogens, producing mycolytic enzymes such as chitinase that helps in chitin degradation, cellulose for hydrolysing cellulose (Fig1). Fig. 1A shows isolation and purification of selected endophytes, Fig. 1B shows endophytes screening based on IAA, Zn and P solubilisation, and Fig. 1C shows endophytes screening based on cellulose, chitinase, dual culture assay, PGP.

In an embodiment, detection of lipopetide genes esp. molecules of iturin family in B.mojavensis using genomic DNA isolation kit and detection and amplification of Bacteriocin (SboA) and Iturin family (lpa14, ituC, ituD & ituD1) gene using degenerate PCR primers with amplification size ranges from 500-1400bp (Fig.2) is done. These genes play key role to inhibit growth of plant pathogens. In addition this strain provide tolerance to high salt concentration (upto2M) and drought tolerant (-12 Mpa).

In an embodiment, the present invention provides an optimized media composition. The media composition comprises
Ingredients %
Soy peptone 0.3-0.8
Beef extract 0.1-0.4
Casein Hydrosylate 0.1-0.4
MgS04 0.1-0.2
K2HPO4 0.1-0.2
NaCl 0.15-0.25
Dextrose 0.2-0.55

At flask mode of 1000 ml, 300 ml culture medium is kept at 1210C for 20 minutes for sterilization. The B. mojavensis ¬1%-2% starter culture was inoculated to activate the culture flask and kept at 300C temperature for 48-72 hr at 150-180 rpm with a pH between 6 to 8. Thereafter, the biomass and supernatant have been separated out using centrifugation followed by vacuum filtration method to remove the macromolecular cell particle debris from the supernatant and the remaining substance of some solubility have been evaporated with vacuum rotary evaporator system to obtain the B.mojavensis with enhanced lipopeptides namely, iturin and fengycin. The obtained B.mojavensis with enhanced lipopeptides is stored at 40C for further analysis.

Ultra High performance liquid chromatography (Waters-UHPLC) was used to detect and quantify iturin, used condition chromatographic column C18 10.7*250mm; Moving phase is 45%-70% acetonitrile: water: 0.5%-1% formic acid the 10mM ammonium acetate; Flow velocity 1mL/min; Detecting wavelength 280nm-480nm, is that standard is carried out assay determination with sigma company Iturin A standard substance; Rt 5-6 min.

The bioformulation of the present disclosure when tested under field conditions has been found to be very effective in controlling downy mildew of pea (70.33%) as compared to untreated control (52.87%). Similarly, the bioformulation has been found to be effective in controlling Cercospora leaf spot of Brinjal up to 76.00% when compared to untreated control (49.20%).
In an embodiment, the bioformulation of the invention is used for crop protection and plant growth, wherein the application of composition may be on the ground, or on root part or it may be foliar treatment.

In an aspect, the bioformulation may be in the form of liquid formulation, powder formulations such as wettable powder, granules formulation, or suspension concentrate (flowable concentrate).

In an embodiment, the invention provides a non-toxic and biodegradable composition of endophytic bacterial strain B. mojavensis for use in agriculture and environmental protection, etc.

In an aspect, the invention provides a bioformulation comprising live strain of endophytic bacteria B. mojavensis in combination with at least one other microorganism. The other microorganism may be selected from live bacteria, inactivated bacteria or extracts of the bacteria. In an aspect, the bioformulation of the invention further comprises a buffering agent and/or a wetting agent and/or a polysaccharide or a combination thereof.

In an aspect, the formulation contains 106 to 109 CFU /ml.

The bioformulation of the present disclosure is used as an antifungal agent and inhibits plant pathogens such as pathogenic bacterial or pathogenic fungus selected from a group consisting of downy mildew, Botrytis, Rhizoctonia solani Cercospora leaf spot, Red rot : Glomerella tucumanensis, Fusarium, Alternaria, Aspergillus, Penicillium etc. The broad spectrum antagonistic activity of the composition has been proven both under in vitro and in vivo conditions (Fig.3 to 6). Fig. 3 shows poison assay of active compound extract at 0.5 mg/ml concentration against Botrytis sp. and Rhizoctonia solani. Fig. 4 shows the effectiveness of the bioformulation against Sclerotium (85.5%), B.cinerea (74.82%), Curvularia sp.(71.42). The formulation has been found effective in controlling 21 pathogens out of 24 selected plant pathogens under in vitro screening (dual culture method) method. Fig. 5 shows PGPR effects of the bioformulation of the present disclosure with varied concentration compared with control (SM9) in Egg Plant. Fig. 6 shows estimation of chlorophyll and carotenoids content in presence of varied concentration of the bioformulation in Egg plant.

In an aspect, the bioformulation is packaged in packaging suitable to protect them from degradation and stored at room temperatures. The enhanced lipopeptide based bio formulation of the preset disclosure is stab7e and can be stored at room temperature for at least 12 months to18 months (Fig. 8).

The enhanced lipopeptide media composition of B.mojavensis acts as a highly efficient endophytic solution that help the crop to survive under any biotic/abiotic stress condition and improve fruit quality by supressing diseases that may cause economic loss. The composition of the present invention is eco-friendly, economical with better shelf life and replace the agri-based chemicals in safe sustainable manner.

The fungicidal bioformulation of the present disclosure is an economical, eco-friendly, stable and industrially scalable composition with broad spectrum antagonistic activity and for improved quality, increase productivity of crop irrespective of climatic conditions.

In an aspect, the composition is a water-soluble formulation of augmented essential macronutrients that contains at least B.mojavensis, phosphorus and potassium together. In an aspect, the composition is used in combination with a chemical fertilizer.

The above are preferred embodiments of the present application are based on value added research conducted on the premise to bring out a novel safe and sustainable delivery system for agricommunity with an intent to protect a complete application and are not intended to limit the present invention. It will nevertheless be understood that the embodiments and examples provided here are not departing from the design proposed during provisional application, all kinds of experimental research modifications and improvements made by skilled in the art to be constructed as being within the scope of the present invention.

,CLAIMS:1. A fungicidal bioformulation comprising B.mojavensis with enhanced lipopeptides namely, iturin and fengycin, buffering agent and/or a wetting agent and/or a polysaccharide or a combination thereof, said bioformulation is a plant growth promoter and potential biocontrol agent with broad spectrum antagonistic activity.
2. The bioformulation as claimed in claim 1, wherein said bioformulation is stable at room temperature for up to18 months.
3. The bioformulation as claimed in claim 1, wherein said bioformulation is in the form of wettable powder, granules formulation, or suspension concentrate.
4. A process of preparing bioformulation as claimed in claim 1, said process comprises
- Preparing an optimized culture media and sterilizing it,
- Inoculating said sterilized culture media with starter culture of B. mojavensis and incubating at predetermined conditions,
- Centrifuging the incubated culture media and separating the supernatant,
- Vacuum filtration of supernatant followed by vacuum evaporation to obtain the B.mojavensis with enhanced lipopeptides namely, iturin and fengycin with viable cell count in the range of 106 to 109 CFU /ml,
- Mixing said B.mojavensis with enhanced lipopeptides with buffering agent and/or a wetting agent and/or a polysaccharide or a combination thereof to obtain the fungicidal bioformulation.
5. The process of preparing bioformulation as claimed in claim 4, wherein said predetermined conditions of incubation are 300C temperature for 48 hr to 72 hr at 150 rpm to180 rpm with a pH between 6 to 8.
6. The process of preparing bioformulation as claimed in claim 4, wherein said optimized culture media comprises one or more water soluble polymers; one or more mineral solution; one or more protein source; one or more sugar and a surface active agent.
7. The process of preparing bioformulation as claimed in claim 4, wherein said one or more sugars in the range of 0.3-0.8 percent, one or more water soluble polymer in the range of 0.05-0.2 percent and a surface active agent in the range of 0.05-0.2 percent.
8. The bioformulation as claimed in claim 1, wherein said water soluble polymer is selected from glycerol, polyethylene glycol, polyvinyl pyrollidone.
9. The bioformulation as claimed in claim 1, wherein said protein source is selected from soy peptone, beef extract, casein hydrosylate or a combination thereof.
10. The bioformulation as claimed in claim 1, wherein said sugar is selected from sucrose, trehalose, maltose, lactose, glucose.
11. The bioformulation as claimed in claim 1, wherein said surface active agent is selected from polysorbates/20/80/SPAN.
12. A fungicidal bioformulation as claimed in claim 1, wherein said bioformulation is an economical, eco-friendly, stable and industrially scalable composition with broad spectrum antagonistic activity and for improved quality, increase productivity of crop irrespective of climatic conditions.