DESC:FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003

COMPLETE SPECIFICATION
(See Section 10 and Rule 13)

Title of invention:
AGROBIOLOGICAL FORMULATION FOR PLANT GROWTH

Applicant:
1000 Farms Agritech Private Limited
An Indian entity having address as
SY NO. 155/3A, Tirupati Road, Venkatapuram, Karkambadi, Tirupati, Chittoor, Andhra Pradesh, India, 517520

The following specification particularly describes the invention and the manner in which it is to be performed

CROSS REFERENCE TO RELATED APPLICATIONS AND PRIORITY
[0001] The present application claims priority from Indian provisional patent application no. 202441013730 filed on the 26th day of February 2024, the details of which are incorporated herein by reference.

TECHNICAL FIELD
[0002] The present subject matter, in general, is directed to the field of agricultural science. More particularly, the present subject matter relates to an agrobiological formulation for plant growth and a seed coating composition, an artificial nodule stimulant, and a root dipping composition comprising thereof.

BACKGROUND
[0003] The revolutionization in agricultural practices has brought forth newer robust varieties of crops and fertilizers that are aimed towards obtaining higher yields of crops. It is known that proper nutrient management not only results in higher crop yields, increasing the potential for increased profits, but also allows for maximum yield from limited land resources. For steady soil replenishment, particularly in an agricultural ecosystem, numerous chemically derived fertilizers or agrochemicals have been developed. However, many of these agrochemicals have demonstrated drawbacks such as increased acidity level, water contamination, soil quality depletion, extremes such as overgrowth or barrenness, acceleration in climate change, and damage to living beings, providing an impetus for developing natural means for sustainability.

[0004] Natural means, such as microorganisms, have presently transpired as a reliable alternative for providing productive and sustainable solutions for addressing increased demands on the global food chain. Microorganisms offer an excellent alternative technology for fixing, solubilizing, mobilizing, and recycling nutrients, and provide an excellent substitute to agrochemicals. Various microbial taxa are explored as biofertilizers, based on their capacity to access nutrients from fertilizers and soil stocks, to fix atmospheric nitrogen, to improve water uptake or to act as biocontrol agents.

[0005] In the soil, the nitrogen-fixing organisms (nitrogen fixer(s)) live in close association with the roots of the plants and implement organic acids and sugars from root exudates as carbon sources to fix dinitrogen into ammonia, which can be utilized by the plants as a nitrogen source. Nitrogen fixation is carried out by nitrogenase, a complex enzyme, that is sensitive to oxygen and consists of two activity subunits/components – dinitrogenase reductase and dinitrogenase. Since the biological nitrogen fixation process is excessively energy-intensive, these organisms struggle to find their niche in the soil where they can respire and fix nitrogen while protecting the nitrogenase enzyme complex from oxygen. Further, there is a high requirement for organic carbon during nitrogen fixation, which, when lacking, can create a highly stressful environment by generating reactive oxygen species. This rapidly declines not only the nitrogen fixing capacity of the nitrogen fixer(s), but also their shelf-life.

[0006] It is further known that free-living nitrogen fixers are less efficient compared to their symbiotic counterparts. In a free-living nitrogen fixer, the energy consumption for nitrogen fixation is significantly higher, as these organisms are not symbiotically associated with plants, therefore making them more susceptible to oxidative stress due to higher respiration, rendering the nitrogenase enzyme further sensitive to damage. Hence, the nitrogen fixed by free-living nitrogen fixers is fundamentally reported to be low. There is therefore a requirement to identify anti-stressor(s) that can increase microbial survivability and preserve nitrogenase activity of both symbiotic as well as free-living nitrogen fixers.

[0007] Further, the establishment of the potential assistance of these anti-stressors in enhancing nitrogen fixation and promoting plant growth is also desirable. There is therefore a requirement to devise agrobiological formulations that increase nutrient availability to agricultural plants, and promote plant development, without affecting soil structure.

[0008] Prior arts demonstrate the implementation of conductive polymer-based photosynthetic biohybrid, and genetic engineering for improving nitrogen fixation. However, these methodologies are reported to be time-consuming and uneconomical.

[0009] The present study addresses existing limitations in sustainable agriculture by utilizing an agrobiological formulation designed to elevate soil ammoniacal nitrogen concentrations and enhance nitrogenase enzymatic activity. This formulation also facilitates microbial aggregation and proliferation, while improving critical agronomic parameters such as seed surface colonization, seedling biomass, root and shoot development, and overall microbial population density. It is intended as a crop-agnostic solution, applicable to both symbiotic and non-symbiotic associations in leguminous and non-leguminous plant species, with the goal of reducing or replacing conventional nitrogenous chemical fertilizers.

[0010] Furthermore, given that the efficacy of microbial biofertilizers is strongly influenced by their viability post-storage and their ability to rapidly establish in the rhizosphere, the inventors have also focused on the development of an optimized carrier system. This carrier is engineered to provide a protective microenvironment, ensure stability during packaging and storage, and enable efficient delivery and establishment of the microbial consortia in the target soil ecosystem

SUMMARY
[0011] Before the present composition and method and its components are summarized, it is to be understood that this disclosure is not limited to the formulation/composition as described, as there can be multiple possible embodiments that are not expressly illustrated in the present disclosure. The present application overcomes one or more shortcomings of the prior art and provides additional advantages discussed throughout the present disclosure. Additional features and advantages are realized through the techniques of the present disclosure. It is also to be understood that the terminology used in the description is for the purpose of describing the versions or embodiments only and is not intended to limit the scope of the present disclosure. This summary is not intended to identify essential features of the claimed subject matter nor is it intended for use in detecting or limiting the scope of the claimed subject matter.

[0012] An embodiment of the instant disclosure relates to an agrobiological formulation for plant growth comprising a free-living nitrogen-fixing bacterium; at least one anti-stress agent; a carbohydrate-binding lectin; and a carrier, wherein the microbial formulation improves ammoniacal nitrogen in soil and nitrogenase activity.

[0013] A related embodiment of the instant disclosure relates to a seed coating composition for plant growth comprising the agrobiological composition

[0014] Another related embodiment of the instant disclosure relates to an artificial nodule stimulant for plant growth comprising the agrobiological composition.
In a related embodiment, the artificial nodule stimulant functions by simulating the effects of nodulation in non nodulating plants, while in nodulating plants, it acts as a stimulant to enhance natural nodule formation and activity.

[0015] Yet another related embodiment of the instant disclosure relates to a root dipping composition for plant growth comprising the agrobiological composition.

BRIEF DESCRIPTION OF FIGURES
[0016] Having thus described the disclosure in general terms, references will now be made to the accompanying figures, wherein:

[0017] Figure 1 illustrates the growth kinetics of Azospirillum lipoferum in Dobereiner medium (DOB).

[0018] Figure 2 illustrates effect of different concentrations of malic acid as a carbon source on the growth kinetics of a free-living nitrogen fixing bacterium.

[0019] Figure 3 illustrates effect of anti-stress agent(s) on the growth kinetics of a free-living nitrogen fixing bacterium

[0020] Figure 4 (A-B) illustrates estimation of ammoniacal nitrogen and nitrogenase activity.

[0021] Figure 5 (A-D) illustrates the effect of carbohydrate-binding lectin and carrier on the growth kinetics of a free-living nitrogen fixing bacterium: aggregation and Biofilm Behavior of the free-living nitrogen fixing bacterium Treated with WGA and Succinoglycan.

[0022] Figure 6 (A-B) illustrates estimation of ammoniacal nitrogen and nitrogenase activity – cultures subjected to aggregation treatments.

[0023] Figure 7 (A-F) illustrates SEM images to evaluate microbial colonization and surface modification - Figs. 7(A, C-F), and SEM images to evaluate Azos + NAC (without seed) – Fig. 7B.

[0024] Figure 8 (A-E) illustrates plant growth performance.

[0025] Figure 9 represents root and shoot development.

[0026] Figure 10 (A-C) illustrates root length, shoot length, and plant biomass estimation.

[0027] Figure 11 illustrates nitrogenase activity.

[0028] It should be noted that the accompanying figures are intended to present illustrations of exemplary embodiments of the present disclosure. These figures are not intended to limit the scope of the present disclosure. It should also be noted that accompanying figures are not necessarily drawn to scale.

DETAILED DESCRIPTION
[0029] Reference will now be made in more detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein reference numerals refer to like elements throughout the specification. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of embodiments of the present description. As used herein, the term ‘and/or’ includes any and all combinations of one or more of the associated listed items. Throughout the present disclosure, the expression ‘at least one of a, b and c’ indicates only a, only b, only c, both a and b, both a and c, both b and c, all of a, b, and c, or variations thereof.

[0030] The subject matter of the present application may include various modifications and various embodiments, and example embodiments will be illustrated in the drawings and described in more detail in the detailed description. Effects and features of the subject matter of the present disclosure, and implementation methods therefore will become clear with reference to the embodiments described herein below together with the drawings. The subject matter of the present application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0031] Hereinafter, embodiments of the present application will be described in more detail with reference to the accompanying drawings. The same or corresponding elements will be denoted by the same reference numerals, and thus, redundant description thereof will not be repeated.

[0032] It will be understood that although the terms ‘first,’ second,’ etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another.

[0033] An expression used in the singular may also encompass the expression of the plural unless it has a clearly different meaning in the context.

[0034] In the following embodiments, it is to be understood that terms such as ‘including,’ includes,’ having,’ having,’ comprises,’ and ‘comprising,’ are intended to indicate the existence of the features or elements disclosed in the specification and are not intended to preclude the possibility that one or more other features or elements may exist or may be added. Further, it is to be understood that the terms “component”, “component”, “agent”, and “agents” pertain to any of the features of instant invention disclosed herein.

[0035] In order to facilitate an understanding of the composition and/or formulation and/or product discussed herein, a number of terms are defined below. The terms defined below, as well as other terms used herein, should be construed to include the provided definitions, the ordinary and customary meaning of the terms, and any other implied meaning for the respective terms. Thus, the definitions below do not limit the meaning of these terms but only provide exemplary definitions.

[0036] The present disclosure, in general, relates to the field of agricultural science and particularly relates to an agrobiological formulation for plant growth.

[0037] An aspect of the instant disclosure relates to an agrobiological formulation.
Now, for the purpose of instant disclosure and as is perceivable to a person skilled in the art, the term “agrobiological” pertains to the application of biological principles, organisms, and processes. Further, the term “formulation” or “formulations” pertains to a composition.
In a related embodiment, the instant disclosure relates to an agrobiological formulation for plant growth.
For the purpose of instant disclosure and as is perceivable to a person skilled in the art, the term “plant growth” pertains to the increase in plant volume and/or mass with or without formation of new structures such as organs, tissues, cells, or cell organelles. Growth is usually associated with development (cell and tissue specialization) and reproduction (production of new individuals).
In an embodiment, the plant is selected from legumes (Fabaceae), monocots, dicots such as soybean, and pea, Actinorhizal plants, Parasponia (non-legume), aquatic legumes, graminaceous crops such as barley, rice, maize, wheat, sugarcane, grasses, monocots, cereal crops, sugarcane, and vegetables such as tomato, carrot, and lettuce, native trees, and shrubs, as is perceivable to a person skilled in the art.

[0038] In an embodiment, the agrobiological formulation comprises at least one nitrogen fixer.
For the purpose of instant disclosure, the term “nitrogen fixer”, or “nitrogen fixers” pertains to all the conventionally known symbiotic as well as non-symbiotic (free-living) nitrogen-fixing microorganisms. Further, the term “symbiotic” pertains to mutualistic microorganisms. In addition to this, the term “non-symbiotic” or “free-living” pertains to non-mutualistic microorganisms. Furthermore, the terms “organism” or “organisms” for the purpose of instant disclosure pertains to microorganism(s), as is perceivable to a person skilled in the art and/or as is fundamentally defined.
In a related embodiment, the nitrogen fixer is a free-living nitrogen-fixer, and preferably, a free-living nitrogen-fixing bacterium.
In another related embodiment, the free-living nitrogen-fixing bacterium is selected from cyanobacteria (or blue-green algae) Anabaena and Nostoc, genera such as Azotobacter, Beijerinckia, Clostridium, Rhizobium, Frankia, Azospirillum, Mesorhizobium, Sinorhizobium, and/or any other nitrogen fixers and combinations thereof, perceivable to a person skilled in the art; and preferably selected from Azospirillum lipoferum, Azospirillum brasilense, Azotobacter chrrococcum, Azotobacter beijerinckii, Azotobacter insignis, Azolla, Anabaena, Herbaspirillum seropedicae, Gluconacetobacter diazotrophicus, and Pseudomonas sp.
In yet another related embodiment, the nitrogen fixer; and preferably, the free-living nitrogen-fixing bacterium is present in a specific ratio and/or concentration as per requirements.
In an embodiment, the nitrogen fixer; and preferably, the free-living nitrogen-fixing bacterium, is present in 0.001% to 10% (w/v) concentration of the total agrobiological formulation.
For the purpose of instant disclosure and as is perceivable to a person skilled in the art, the term “total agrobiological formulation” pertains to the complete or holistic agrobiological formulation described herein.

In an embodiment, the nitrogen fixers; and preferably, the free-living nitrogen-fixing bacterium, described above, either individually or in any combination, may be combined with any plant, active molecule (synthetic, organic, etc.), adjuvant, carrier, supplement, or biological component known to a skilled person in the art.
In a related embodiment, the nitrogen fixers may be combined with emulsifiers; and preferably, non-ionic emulsifiers such as Tween 20, and Tween 80, and anionic emulsifiers.
In another related embodiment, the nitrogen fixers may be combined with carbon sources such as but not limited to malic acid, and mannitol.

[0039] In another embodiment, the agrobiological formulation comprises at least one anti-stress agent.
For the purpose of instant disclosure and as is perceivable to a person skilled in the art, the term “anti-stress agent”, “anti-stress agents”, “anti-stressor” or “anti-stressors” pertains to any molecule that protects the microorganism from the effects of stresses, in this case, oxidative stress. Bacteria are known to produce anti-stressors such as glutamate, trehalose, glycine betaine, and proline under osmotic stress.
In a related embodiment, the at least one anti-stress agent is a water-soluble component, an endogenous tripeptide, a lipid-soluble component, or an antioxidant.
In another related embodiment, the antioxidant is selected from N-acetyl cysteine (NAC), vitamin C (Ascorbic acid), glutathione (GSH), vitamin E (tocopherol), melatonin and/or polyphenols.
In yet another related embodiment, the polyphenols are at least resveratrol, or quercetin.
The anti-stress agent of the instant disclosure acts as protectors against oxidative stress as it can scavenge free radicals and reduce reactive oxygen species (ROS). The anti-stress agent may also protect from other environmental stresses, such as saline stress, heat stress, osmotic stress, and drought.
In yet another related embodiment, the at least one anti-stress agent is present in a specific ratio and/or concentration as per requirements.
In an embodiment, the at least one anti-stress agent is present in 0.001% to 0.003% (w/v) concentration of the total agrobiological formulation described above.

[0040] In yet another embodiment, the agrobiological formulation comprises a lectin; and preferably, a carbohydrate-binding lectin.
For the purpose of instant disclosure and as is perceivable to a person skilled in the art, a “carbohydrate-binding lectin” pertains to protein that recognizes and binds to specific carbohydrate structures, often sugars, in a reversible and non-immune manner. These proteins are found in a wide range of organisms and play various roles in cell recognition, cell adhesion, and other biological processes.
In a related embodiment, the carbohydrate-binding lectin is selected from Concanavalin A (Con A), Snodrop Lectin, Ricinus communis Agglutin, Peanut Agglutin (PNA), Wheat Germ Agglutin (WGA), Aleuria europaeus Agglutin, Soybean Agglutinin (SBA), Jacalin- related lectins, ZmLEC1 – like lectins, PSA (Pea seed agglutinin), Dolichos biflorus Agglutinin (DBA), Ulex europaeus Agglutinin I (UEA-I), MLL ( Morus alba lectin), CsinLec ( Citrus sinensis lectin), VvML (Vitis vinifera mannose lectin), MsLec (Medicago sativa lectin), CaLec (Coffea arabica lectin), OliLec (Olea europaea lectin), PsL (Prunus species lectin), and AlLec (Almond Lectin).
In yet another related embodiment, the carbohydrate-binding lectin is present in a specific ratio and/or concentration as per requirements.
In an embodiment, the carbohydrate-binding lectin is present in 0.004 to 0.02% (w/v) concentration of the total agrobiological formulation described above.

[0041] In a further embodiment, the agrobiological formulation comprises a carrier.
For the purpose of instant disclosure and as is perceivable to a person skilled in the art, the term “carrier” or “carriers” pertain to a diluent, adjuvant, excipient, or vehicle with which the agrobiological formulation described above can be administered to its target, which does not detrimentally affect the composition/formulation, and which is an agriculturally acceptable in nature. Non-limiting examples of agriculturally acceptable carriers are exopolysaccharides, lipopolysaccharides, mucopolysaccharides, polypeptides, lipids, modified polysaccharides, Glycoproteins, and derived polysaccharides, such as PVA, xanthan gum, starch, chitin, chitosan, Inulin, carboxy methyl cellulose, alginate, pectin, starch derivatives, guar gum, Gum arabic, polyvinyl alcohol (PVA), poly (ethylene glycol) (PEG), polyvinyl pyrrolidine (PVP), polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyacrylamides, N-(2-Hydroxypropyl) methacrylamide (HPMA), divinyl ether-maleic anhydride (DIVEMA), polyphosphates, polyphosphazene, glycerol, succinoglycan, succinylated alginate, levan, glucomannan, and/or any other agriculturally acceptable carriers perceivable to a person skilled in the art.
Additional examples of agriculturally acceptable carriers are known in the art. The carrier can be a solid carrier or liquid carrier, and in various forms including microspheres, powders, emulsions, and the like. The carrier may be any one or more of a number of carriers that confer a variety of properties, such as increased stability, wettability, or dispersibility. Wetting agents such as natural or synthetic surfactants, which can be non-ionic or ionic surfactants, or a combination thereof may be included in the agrobiological formulation. Suitable formulations that may be prepared include wettable powders, granules, gels, agar strips or pellets, thickeners, and the like, microencapsulated particles, and the like, liquids such as aqueous flowable, aqueous suspensions, water-in-oil emulsions, etc. In some embodiments, the agricultural carrier is a soil or a plant growth medium. Other agricultural carriers that may be used include water, fertilizers, plant-based oils, humectants, or combinations thereof. Alternatively, the agricultural carrier may be a solid, such as diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, seed cases, other plant and animal products, or combinations, including granules, pellets, or suspensions. Mixtures of any of the aforementioned ingredients are also contemplated as carriers, such as but not limited to, pesta (flour and kaolin clay), agar or flour-based pellets in loam, sand, or clay, etc.
The carrier described herein may act as a signaling molecule modulator and contribute to enhanced bacterial adhesion, proliferation, and intercellular communication. By facilitating quorum sensing and other signaling pathways, the carriers support the establishment and sustained activity of beneficial microbial populations on plant surfaces and in the rhizosphere.
In a related embodiment, the carrier is a gel-based carrier.
In another related embodiment, the carrier is polymer-based; preferably water-soluble, and more preferably, biodegradable.
In yet another related embodiment, the carrier is present in a specific ratio and/or concentration as per requirements.
In an embodiment, the carrier is present in 0.01 to 10 % (w/v) concentration of the total agrobiological formulation described above.

[0042] In yet another further embodiment, the agrobiological formulation described above may have consistency of liquid, solgel, or gel; and more preferably, the agrobiological formulation is gel-based.
As is known to a skilled person in the art, gel-based formulations demonstrate advantages such as being easy to use, simple application procedure on any surface, and being washable. Gel composition requires less volume than any liquid composition. Such compositions have easy spreadability and higher stability for a longer time than liquid compositions.

[0043] Another aspect of the instant disclosure pertains to a process for preparing the agrobiological formulation described above.
In an exemplary embodiment, the concentration of the free-living nitrogen-fixing bacterium; the at least one anti-stress agent; the carbohydrate-binding lectin; and the carrier described above is 0.01% to 10%, 0.01% to 0.03%, 0.004% to 0.02%, and 0.01% to 10% (w/v), respectively of the total agrobiological formulation described above.
In a related embodiment, the agrobiological formulation further comprises water; preferably, in a concentration ranging from 60% to 80% (w/v) of the total agrobiological formulation described above.
In another related embodiment, the agrobiological formulation comprises buffer components perceivable to a skilled person in the art.
In an exemplary embodiment, the agrobiological formulation comprises 0.1M Phosphate buffer 7.2 (sodium phosphate dibasic and monobasic) and water.

[0044] Yet another aspect of the instant disclosure pertains to the application of the agrobiological formulation described above; preferably, to at least one part of the plant, described above, including but not limited to roots, nodule(s), seed, internode, bud, stem, blade, petiole, veins, leaves, stomata, sepals, petals, fruit(s), etc. perceivable to a skilled person in the art.
In a related embodiment, the application of the agrobiological formulation is carried out by at least one of dipping, spraying, seed coating, soil/plant drenching, soil briquettes, hydroponic-system based methods perceivable to a skilled person in the art.
In another related embodiment, the application of the agrobiological formulation is carried out in a range of 100 mL to 1 L per acre.
In an exemplary embodiment, the agrobiological formulation described above may be applied as at least one of a seed coating composition, artificial nodule stimulant, and a root dipping composition.

[0045] An embodiment of the instant disclosure pertains to a seed coating composition for plant growth comprising the agrobiological formulation described above.
For the purpose of instant disclosure and as is perceivable to a person skilled in the art, the term “seed coating composition” pertains to a composition/formulation applied around a seed to improve its handling, protection, germination, and early-stage growth without significantly altering its shape or size.
In a related embodiment, the seed coating composition comprises the agrobiological formulation described above; and specifically, the components of the agrobiological formulation as described above.
In another related embodiment, the seed coating composition is applied using methods described above.
In a further related embodiment, the seed coating composition is applied by coating about 1 g of the seed with 2 mL to 10 mL of the seed coating composition.

[0046] Another embodiment of the instant disclosure pertains to an artificial nodule stimulant for plant growth comprising the agrobiological formulation described above.
In a related embodiment, the artificial nodule stimulant functions by simulating the effects of nodulation in non nodulating plants, while in nodulating plants, it acts as a stimulant to enhance natural nodule formation and activity.
For the purpose of instant disclosure and as is perceivable to a person skilled in the art, the phrase “the artificial nodule stimulant functions by simulating the effects of nodulation in non nodulating plants” pertains to the process of stimulating the formation of root nodules in non nodulating plants by mimicking or enhancing the natural signaling processes in the non nodulating plants (as is perceivable to a person skilled in the art, and as is described above).
Further, the phrase “while in nodulating plants, it acts as a stimulant to enhance natural nodule formation and activity” pertains to the process of further enhancing/encouraging formation of root nodules in nodulating plants by providing better rhizospheric environment in a nodulating plant (as is perceivable to a skilled person in the art, and as is described above).
In a related embodiment, the artificial nodule stimulant comprises the agrobiological formulation described above; and specifically, the components of the agrobiological formulation as described above.
In another related embodiment, the artificial nodule stimulant is applied using methods described above.
In an embodiment, the artificial nodule stimulant may be applied as seed coating on the seed surface of the plants described above.
In another embodiment, the artificial nodule stimulant may be applied on root surface of the plants described above by dipping before transplant

[0047] Yet another embodiment of the instant disclosure pertains to a root dipping composition for plant growth comprising the agrobiological formulation described above.
For the purpose of instant disclosure and as is perceivable to a person skilled in the art, the term “root dipping composition” pertains to a formulation intended to enhance root health, microbial colonization, nutrient uptake, and transplant survival, and may also protect against soil-borne pathogens.
In a related embodiment, the root dipping composition comprises the agrobiological formulation described above; and specifically, the components of the agrobiological formulation as described above.
In another related embodiment, the root dipping composition is applied using methods described above.

[0048] In an embodiment, the agrobiological formulation improves ammoniacal nitrogen in soil. Consequently, the seed coating composition, the artificial nodule stimulant, and the root dipping composition improve ammoniacal nitrogen in soil.
For the purpose of instant disclosure and as is perceivable to a person skilled in the art, the term “ammoniacal nitrogen in soil” pertains to the form of nitrogen present in soil as ammonium ions (NH4?).
In an embodiment, the agrobiological formulation improves ammoniacal nitrogen in soil by 125%.

[0049] In another embodiment, the agrobiological formulation improves nitrogenase activity. Consequently, the seed coating composition, the artificial nodule stimulant, and the root dipping composition improve nitrogenase activity.
For the purpose of instant disclosure and as is perceivable to a person skilled in the art, the term “nitrogenase activity” pertains to biochemical process carried out by the nitrogenase enzyme complex, which catalyzes the reduction of atmospheric nitrogen gas (N2) into ammonia (NH3) under anaerobic conditions.
In an embodiment, the agrobiological formulation improves the nitrogenase activity by 140% - 150%.

[0050] In yet another embodiment, the agrobiological formulation improves at least one of bacterial aggregation and bacterial proliferation. Consequently, the seed coating composition, the artificial nodule stimulant, and the root dipping composition improve at least one of bacterial aggregation and bacterial proliferation.
For the purpose of instant disclosure and as is perceivable to a person skilled in the art, the term “bacterial aggregation” pertains to the process by which individual bacterial cells adhere to one another, forming multicellular clusters or clumps. Whereas the term “bacterial proliferation” pertains to refers to the increase in the number of bacterial cells through the process of cell division, most commonly by binary fission, resulting in population growth under favorable environmental conditions.
In an embodiment, the agrobiological formulation improves at least one of bacterial aggregation, and bacterial proliferation by 70-90%, and 70-90%, respectively.

[0051] In a further embodiment, the agrobiological formulation improves at least one of improves at least one of seed surface colonization, seedling biomass, root and shoot development, and microbial count. Consequently, the seed coating composition, the artificial nodule stimulant, and the root dipping composition improve at least one of seed surface colonization, seedling biomass, root and shoot development, and microbial count.
For the purpose of instant disclosure and as is perceivable to a person skilled in the art, the term “seed surface colonization” pertains to attachment and establishment of microorganisms (specifically, the free-living nitrogen-fixing bacterium described above) on the outer surface of a seed the plant described above. Further, the term “seedling biomass” pertains to total mass of living plant tissue of a seedling, typically measured after germination and early growth, which includes both above-ground (shoots, leaves, stems) and below-ground (roots) tissues. It is an important indicator of the health, growth rate, and nutritional status of a seedling during its early development stages. The term “root and shoot development” pertains to the coordinated growth and differentiation of the root system and shoot system of a plant from the seedling stage through maturity. It encompasses the processes by which plant organs—roots (below ground) and shoots (above ground, including stems and leaves)—develop, differentiate, and grow to perform their respective functions in the plant’s survival and reproduction. In addition to this, the term “microbial count” pertains to quantification of microorganisms in a specific sample, typically expressed as the number of colony-forming units (CFU) per unit of volume, mass, or area. It is a key metric used to assess the abundance, diversity, and activity of microbial populations in various environments such as soil, water, air, food, and clinical settings.
In an embodiment, the agrobiological formulation improves at least one of seed surface colonization, seedling biomass, root and shoot development, and microbial count by 70-90%, 100-120%, 80-90%, and 25-30%, respectively.

In an embodiment, the agrobiological formulation increases the microbial count described above. Consequently, the seed coating composition, the artificial nodule stimulant, and the root dipping composition increase the microbial count described above.
In a related embodiment, the agrobiological formulation increases microbial count by 100000 times.

[0052] The formulations and compositions described herein can be applied across various sectors of agricultural sciences, including but not limited to crop nutrition, seed treatment, soil health enhancement, biological nitrogen fixation, plant growth promotion, stress tolerance management, and integrated pest and disease control. These formulations/compositions may be developed and produced at different scales of operation, ranging from small-scale laboratory preparations to medium-scale pilot production and full-scale industrial manufacturing, depending on the intended application and commercial requirements.

[0053] Various modifications to the embodiment will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments. However, one of ordinary skill in the art will readily recognize that the present disclosure is not intended to be limited to the embodiments illustrated but is to be accorded with the widest scope consistent with the principles and features described herein.

[0054] The foregoing description shall be interpreted as illustrative and not in any limiting sense. A person of ordinary skill in the art would understand that certain modifications could come within the scope of this disclosure. The features and properties of the present disclosure are described in further detail below with reference to examples.

[0055] Example 1
Characterizing growth kinetics of a free-living nitrogen fixing bacterium
For this experiment, the growth kinetics of Azospirillum lipoferum (hereinafter referred to as “Azos”, “culture”, or “bacterium”) commercially sourced from Tamil Nadu Agriculture University (TNAU) [AZ 204] were studied using Dobereiner medium (DOB) medium with a carbon source (D-Mannitol) [of about 5g/L].
The average of triplicate measurements with ± 1%-10 % standard deviation is demonstrated in Fig. 1 (growth pattern of the microbial culture over time is illustrated through optical density (OD) measurements)
Observation:
Referring to Fig. 1, a rapid increase in OD is observed within the first 24 h., indicating the onset of the exponential (log) growth phase. The culture reaches its maximum OD at approximately 48 h., representing peak cell density. Thereafter, a slight decline or plateau is noted beyond 72 h., suggesting the depletion of available nutrients and/or the transition into the stationary phase. The inclusion of error bars, representing standard deviation within a range of ±1% to ±10%, confirms the reproducibility and consistency of the experimental results across replicates.

[0056] Example 2
Effect of different concentrations of malic acid as a carbon source on the growth kinetics of a free-living nitrogen fixing bacterium
For this experiment, DOB medium supplemented with varying concentrations of malic acid (about 2.5, 5, 7.5, and 10 g/L) as the sole carbon source were employed to evaluate the growth kinetics of Azospirillum lipoferum, as described in Example 1. A negative control (without malic acid) was also included to assess baseline growth. The average of triplicate measurements with ± 1%-10 % standard deviation is demonstrated in Fig. 2 (growth pattern of the microbial culture over time is illustrated through optical density (OD) measurements)
Observation:
No growth was observed in the negative control, confirming the necessity of malic acid. Minimal growth occurred at 2.5 g/L, moderate growth at 5 g/L, and a high growth rate at 7.5 g/L, indicating near-optimal conditions. The highest optical density was recorded at 10 g/L, establishing it as the most effective concentration. A dose-dependent increase in growth was observed across the tested concentrations. Growth plateaued after 72 h., indicating transition to the stationary phase

[0057] Example 3
Effect of anti-stress agent(s) on the growth kinetics of a free-living nitrogen fixing bacterium
To evaluate the growth response of Azospirillum lipoferum, described in Example 1 in the presence of anti-stress agents - NAC and Vitamin, individually and in combination, the cultures were incubated under the following treatment conditions and growth was evaluated (demonstrated in Figure 3) - Control (Azos without any anti-stress agent); Azos + NAC (in the range of 0.001 to 0.003% (w/v)) ; Azos + Vitamin C (in the range of 0.001 to 0.003% (w/v)); and Azos + NAC (in the range of 0.001 to 0.003% (w/v)) + Vitamin C (in the range of 0.001 to 0.003% (w/v)).
All treatments were maintained under identical growth conditions. Growth was monitored and compared at the end of about 48 h. period.
Observation:
The anti-stress agents NAC and Vitamin C, when applied individually in the range of 0.001 to 0.003% (w/v), promoted the growth of the bacterium compared to the untreated control. The combination of NAC and Vitamin C (in the range of 0.001 to 0.003% (w/v) each - concentrations) exhibited a synergistic effect, resulting in the highest optical density and thus the most enhanced growth. These observations confirm the efficacy of anti-stress treatments in improving microbial performance under standard conditions, wherein the concentration range of 0.001 to 0.003% (w/v) was identified as the optimized concentration for the anti-stress agents.

Estimation of Ammoniacal Nitrogen and Nitrogenase Activity:
Ammoniacal nitrogen in the culture supernatant and nitrogenase activity of Azos were quantified. Data represents the mean of triplicate measurements with a standard deviation ranging from ±1% to ±10% (see Figs. 4A & 4B)
Graph A: Y-axis: Ammonia concentration (mg/L)
Description: Quantification of ammoniacal nitrogen (mg/L) in culture supernatants under different treatment conditions.
Observations:
The bacterium without treatment showed the lowest ammonia concentration (~64 mg/L). Treatments with either NAC or Vitamin C individually increased ammonia levels to approximately 110–115 mg/L. The combination of NAC and Vitamin C resulted in the highest ammonia production, ranging from 120–130 mg/L.
Conclusion:
Anti-stress compounds NAC and Vitamin C enhance nitrogen fixation in the bacterium, as evidenced by the increased levels of ammoniacal nitrogen and nitrogenase activity.

Graph B: Nitrogenase Activity (nanomol/min)
Y-axis: Nitrogenase activity (nanomol/min)
X-axis: Treatment groups – Azos, Azos + NAC, Azos + Vitamin C, Azos + NAC + Vitamin C
Observations:
The untreated bacterium showed the lowest nitrogenase activity (~9 nanomol/min). Both NAC and Vitamin C individually enhanced activity to ~12–16 nanomol/min. The combination of NAC and Vitamin C yielded the highest nitrogenase activity (~17–18 nanomol/min).
Conclusion:
Anti-stress agents significantly stimulate nitrogenase enzyme activity, a key factor in biological nitrogen fixation. The combination treatment was most effective, indicating a synergistic interaction between NAC and Vitamin C. These findings demonstrate that anti-stress compounds not only promote growth but also enhance ammonia production and nitrogenase function, thereby improving nitrogen metabolism and microbial efficiency. Among the agents tested, NAC consistently outperformed Vitamin C. Consequently, subsequent studies focused solely on NAC, which has shown promising results.

[0058] Example 4
Effect of carbohydrate-binding lectin and carrier on the growth kinetics of a free-living nitrogen fixing bacterium: aggregation and Biofilm Behavior of Azos Treated with WGA and Succinoglycan.
For this experiment, Azos from Example 1 was implemented. This bacterium (pre-treated with NAC) was then exposed to wheat germ agglutinin (WGA), succinoglycan, and their combination to assess cell aggregation and biofilm-like behavior. aggregation patterns were visualized under 100X magnification (Figs. 5A–5D).
Observations (see Table 1)
[Table 1]
Sample Observation
Fig. 5A (Azos + NAC) – Control: Cells appeared individually dispersed, rod-shaped, and Gram-negative.
No visible clumping or aggregation was observed, indicating the absence of cell-cell adhesion.
Fig. 5B (Azos + NAC + WGA): Presence of large, dense clusters due to aggregation.
Indicates that WGA (a lectin binding to N-acetyl-D-glucosamine residues on the bacterial surface) promotes strong cell-cell adhesion.
Fig. 5C (Azos + NAC + Succinoglycan): Moderate aggregation observed with cells embedded in an exopolysaccharide matrix.
Suggests early biofilm formation and enhanced structural stability via succinoglycan.
Fig. 5D (Azos + NAC + WGA + Succinoglycan): Dense, well-formed clumps of bacteria with visible matrix-like structure.
Indicates a synergistic interaction between WGA and succinoglycan, enhancing aggregation and possibly promoting biofilm maturation and stress resilience.

Overall Interpretation:
The control (Azos + NAC) displayed dispersed cells with no aggregation. Treatments with either WGA or succinoglycan individually promoted bacterial aggregation, with WGA inducing stronger clumping through lectin-mediated adhesion. The combination of WGA and succinoglycan resulted in maximal clustering, suggesting a synergistic enhancement of cell adhesion and biofilm development.
Biological relevance: WGA binds specifically to sugar moieties such as N-acetyl-D-glucosamine on Azos, causing aggregation. This interaction may not only facilitate colonization and biofilm formation but also stimulate nitrogen fixation and offer plant-protective effects. These findings highlight the potential of using lectins and exopolysaccharides to improve microbial performance and plant-microbe interactions.

Estimation of Ammoniacal Nitrogen and Nitrogenase Activity:
Ammoniacal nitrogen concentration and nitrogenase activity were quantified in the cultures subjected to various aggregation treatments. The data represents the average of triplicate measurements with a standard deviation ranging from ±1% to ±10% (see Figs. 6A & 6B).
Observations:
Nitrogenase activity (see Table 2)
[Table 2]
Sample Nitrogenase Activity (nmol/min)
Azos: ~9
Azos + NAC: ~15
Azos + NAC + WGA: ~20
Azos + NAC + Succinoglycan: ~25
Azos + NAC + WGA + Succinoglycan: ~30

Ammoniacal Nitrogen (mg/L):
Azos + NAC + WGA + Succinoglycan: 160 mg/L (highest recorded)

Interpretation:
A progressive increase in both ammoniacal nitrogen and nitrogenase activity was observed with successive treatments. The combination of WGA and succinoglycan with NAC significantly enhanced nitrogenase activity (~30 nmol/min) and ammoniacal nitrogen production (160 mg/L), suggesting a synergistic or additive effect. This combination proved to be the most effective in enhancing nitrogen fixation capacity, highlighting the role of cell aggregation and biofilm formation in boosting microbial nitrogen metabolism.

[0059] Example 5
Application of agrobiological formulation as a seed coating composition: effect of a seed coating composition on plant growth
Seed coating compositions comprising the agrobiological formulations incorporating Azos, NAC, WGA, and succinoglycan (as detailed in Examples 1–4) were applied to seeds (commercially sourced fenugreek seeds and mustard seeds) and the seeds were analyzed using scanning electron microscopy (SEM) to evaluate microbial colonization and surface modification (Figs. 7A–7E).
The application was carried out by coating about 1 g of seeds with about 5 mL of the agrobiological formulation.

Observations (see Table 3)
[Table 3]
Sample Observation
Fig. 7A (Seeds + NAC) – Control: Smooth seed surface with no visible microbial presence. Represents the untreated control, showing natural seed coat morphology.
Fig. 7B (Azos + NAC – without seeds) The image depicts a cluster of rod-shaped bacterial cells characterized by a rough surface texture and rounded termini. The cells appear aggregated, likely as a result of sample preparation procedures. Notably, only bacterial cells were observed in the sample; no plant seeds were present
Fig. 7C (Seeds + Azos + NAC): Visible microbial colonies indicate successful inoculation; and moderate surface colonization, with partial bacterial coverage.
Fig. 7D (Seeds + Azos + NAC + WGA): Denser bacterial colonization observed. Structured layering suggests enhanced adhesion facilitated by WGA’s lectin-sugar binding interactions.
Fig. 7E (Seeds + Azos + NAC + Succinoglycan): Extensive surface coverage with biofilm-like matrix. Irregular coating attributed to succinoglycan’s role in microbial encapsulation and adhesion.
Fig. 7F (Seeds + Azos + NAC + WGA + Succinoglycan): Most substantial colonization among all treatments. Compact, thick biofilm indicates a synergistic effect of WGA and succinoglycan. Maximal surface modification suggests improved microbial delivery and persistence on the seed coat.

Interpretation:
SEM analysis revealed a progressive increase in microbial colonization and biofilm formation with the addition of WGA and succinoglycan to the base formulation. The combination of all components (Fig. 7F) provided the most effective seed coating, enhancing microbial adherence and potential for sustained interaction with the seed, which may contribute to improved plant-microbe establishment post-sowing.
As is well understood by those skilled in the art, bacterial aggregation facilitates improved access of the aggregated microbial community to available nutrients, thereby promoting enhanced proliferation and increased adhesion to plant surfaces or soil particles.
The results demonstrated that the agrobiological formulation improved bacterial aggregation, and bacterial proliferation by about 70-90%, and about 70-90%, respectively. Furthermore, the agrobiological formulation also improved the seed surface colonization, and the seedling biomass by about 70%-90%, and 100%-120%, respectively.

Pot experimentation:
A pot experiment was conducted using seeds coated with various agrobiological formulations to assess plant growth performance. Treatments included Azos, NAC, WGA, succinoglycan, and their combinations. Plant vigor and density were visually assessed after germination (Figs. 8A–8E).
Observations: (see Table 4)
[Table 4]
Pot Observations
Fig. 8A (Azos + NAC): Displays robust, healthy seedlings with high plant density.
Represents the most favorable treatment, indicating optimal growth promotion.
Fig. 8B (Azos + NAC + WGA) Healthy seedling growth observed, though slightly less dense than 8A.
Suggests moderate effectiveness of WGA inclusion.
Fig. 8C (Azos + NAC + Succinoglycan): Dense and healthy seedling growth, comparable to 8A.
Indicates effective enhancement of plant vigor through succinoglycan treatment.
Fig. 8D (Azos + NAC + WGA + Succinoglycan): Healthy growth with visibly strong seedlings.
Suggests a beneficial synergistic effect of WGA and succinoglycan.
Fig. 8E (NAC only – No microbial inoculation): Sparse, wilted seedlings with visible signs of fungal infection.
Indicates poor plant performance due to absence of microbial protection and stimulation.

All treatments containing Azos with NAC—either alone or in combination with WGA and succinoglycan—supported healthy plant growth, with the Azos + NAC treatment (Fig. 8A) showing the most vigorous development. While combinations also performed well, the absence of microbial inoculant (Fig. 8E) led to weak growth and disease susceptibility, underscoring the critical role of beneficial microbes and bioactive additives in enhancing plant health and resilience.

Seedling growth and root development under varying conditions:
Seedlings (of the plants described previously) grown under five treatment conditions were compared for root and shoot development (see Fig. 9 – wherein 1- Control plant (without any treatment) Seed+NAC ; 2- Azos +NAC; 3- Azos+ NAC+ WGA; 4- Azos +NAC+ Succinoglycan; 5- Azos+ NAC+ WGA+ Succinoglycan).
Observations:
Fig. 9(5-) demonstrates the strongest root system with visibly longer, thicker roots and brownish pigmentation suggesting healthy vascular tissue and possible nodulation; upright, well-developed shoots; overall, the healthiest seedlings among all groups; and correlates with enhanced nitrogenase activity observed in earlier experiments.
Conclusion:
The visual data in Figure 9 clearly show that combination treatments—especially Azos + NAC + WGA + Succinoglycan—significantly improve seedling vigor, root system development, and overall growth, supporting their role in boosting nitrogen fixation and plant performance. The agrobiological formulation thereby demonstrated about 80%-90% improvement in root and shoot development.

Figures 10A–10C: Root Length, Shoot Length, and Plant Biomass
Plant growth parameters (root length, shoot length, and plant weight) were recorded on Days 5, 7, and 10 post-germination. Plant weight (biomass) was measured on Day 10.
Observations (Plant Weight – Day 10) (see Table 5)
[Table 5]
Group Plant weight (g)
Seed+ NAC 0.11±0.008
Azos+NAC 0.16±0.018
Azos +NAC+WGA 0.17±0.015
Azos+ NAC+Succino 0.19±0.02
Azos+NAC+WGA+Succino 0.22±0.01

Seed + NAC (control): Baseline growth.; Azos + NAC: Moderate increase in biomass; Azos + NAC + WGA: Further increase due to enhanced microbial adhesion; Azos + NAC + Succinoglycan: Greater growth, likely due to improved microbial colonization and biofilm formation; and Azos + NAC + WGA + Succinoglycan: Highest plant weight, showing a 125% increase over the control.
Conclusion:
The synergistic use of WGA and succinoglycan in the seed coating significantly boosted plant biomass, indicating improved microbial interactions and nutrient assimilation.

Table 6: Microbial Count Estimation (Spread Plate Method)
[Table 6]
Days 5th day 7th day 10th day
Treatments cfu/g cfu/g cfu/g
No treatment 3x10^6 9x10^6 1x10^7
Azos+NAC 3x10^5 8x10^6 1x10^9
Azos +NAC+ WGA 2x10^7 10x10^7 6x10^9
Azos+ NAC+ Succino 1x10^6 7x10^5 5x10^9
Azos+NAC+ WGA+Succino 5x10^7 1.7x10^7 1.4x10^12

Purpose: To quantify microbial population in soil under different treatments over time.
Results: The Azos + NAC + WGA + Succinoglycan group showed the highest microbial count, suggesting enhanced microbial survival and colonization in the rhizosphere; thereby demonstrating an increase in microbial count by 100000 times, and an improvement in the microbial count by at least 25%-30%, respectively by the agrobiological formulation.

Table 7: Ammoniacal Nitrogen in Soil
[Table 7] Observations:
Group Ammonical nitrogen (mg/kg) Percentage increase (%)
Control (Seed+NAC) 80 ± 1
Azos+NAC 116.7 ± 0.58 45.87
Azos + NAC+WGA 140.67 ± 0.47 75.8
Azos+ NAC+ Succino 145 ± 1 81.00
Azos+NAC+WGA+Succino 180 ± 0.76 125.00

Findings: Azos + NAC + WGA: 75.8% increase in soil ammoniacal nitrogen; Azos + NAC + Succinoglycan: 81% increase; Azos + NAC + WGA + Succinoglycan: 125% increase over control, indicating significant improvement in nitrogen availability by the agrobiological formulation.

Figure 11: Nitrogenase Activity
Key Result: The Azos + NAC + WGA + Succinoglycan treatment recorded the highest nitrogenase activity at 33.6 nanomoles/min, far surpassing the control and other groups; thereby demonstrating a 140%-150% improvement in the nitrogenase activity by the agrobiological formulation.

Overall Conclusion:
The integrated treatment using Azos, NAC, WGA, and succinoglycan proved to be the most effective strategy for promoting plant growth, enhancing root and shoot development, increasing microbial populations, and improving nitrogen availability in soil. The synergistic action of WGA and succinoglycan plays a crucial role in stimulating nitrogen fixation and ensuring microbial persistence, making this formulation highly promising for sustainable agricultural applications.

[0060] The agrobiological formulation described herein may be implemented with all the nitrogen-fixing bacterium, the at least one anti-stress agent, the carbohydrate-binding lectin, and/or the carrier(s) described above. Furthermore, the agrobiological formulation may be applied as an artificial nodule stimulant, as well as a root dipping composition for promoting plant growth.

[0061] Advantages of the Agrobiological Formulation and Its Applications
- Enhanced Soil Nitrogen Availability: Increases ammoniacal nitrogen content in soil by up to 125%, significantly improving nutrient availability for plants.
- Improved Nitrogen Fixation Efficiency: Boosts nitrogenase activity by 140–150%, leading to enhanced biological nitrogen fixation.
- Stimulation of Microbial Aggregation and Proliferation: Enhances bacterial aggregation and proliferation by 70–90%, promoting stable microbial colonization.
- Superior Seed Surface Colonization: Improves microbial adhesion and colonization on seed surfaces by 70–90%, enabling effective inoculum delivery.
- Increased Seedling Biomass: Enhances plant biomass by 100–120%, reflecting improved nutrient uptake and microbial support.
- Enhanced Root and Shoot Development: Promotes root development by 80–90% and shoot growth by 80–90%, supporting overall plant vigor.
- Boost in Microbial Count in Soil: Increases rhizospheric microbial population by up to 100,000 times, contributing to sustained microbial activity and soil health.
- Synergistic Biofilm Formation and Microbial Persistence: The combined effect of WGA (lectin) and succinoglycan (exopolysaccharide) results in robust biofilm formation, improving microbial survival and efficacy under field conditions.
- Versatile Application Modes The formulation is effective as a: Seed coating composition – for pre-sowing microbial delivery; root dipping solution – for transplant-stage colonization; and Artificial nodule stimulant – enhancing nodulation-like root zones and biological nitrogen activity
- Improved Plant Resistance and Growth under Stress

[0062] While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the scope of the present application as defined by the following claims, and equivalents thereof
,CLAIMS:WE CLAIM:
1. An agrobiological formulation for plant growth comprises:
a free-living nitrogen-fixing bacterium;
at least one anti-stress agent;
a carbohydrate-binding lectin; and
a carrier,
wherein the microbial formulation improves ammoniacal nitrogen in soil and nitrogenase activity.

2. The agrobiological formulation as claimed in claim 1, wherein the free-living nitrogen-fixing bacterium is selected from Azospirillum lipoferum, Azospirillum brasilense, Azotobacter chrrococcum, Azotobacter beijerinckii, Azotobacter insignis, Azolla, Anabaena, Herbaspirillum seropedicae, Gluconacetobacter diazotrophicus, and Pseudomonas sp.

3. The agrobiological formulation as claimed in claim 1, wherein the at least one anti-stress agent is a water-soluble component, an endogenous tripeptide, a lipid-soluble component, or an antioxidant.

4. The agrobiological formulation as claimed in claim 3, wherein the antioxidant is selected from N-acetyl cysteine (NAC), vitamin C (Ascorbic acid), glutathione (GSH), vitamin E (tocopherol), melatonin and/or polyphenols.

5. The agrobiological formulation as claimed in claim 1, wherein the carbohydrate-binding lectin is selected from Concanavalin A (Con A), Snodrop Lectin, Ricinus communis Agglutin, Peanut Agglutin (PNA), Wheat Germ Agglutin (WGA), Aleuria europaeus Agglutin, Soybean Agglutinin (SBA), Jacalin- related lectins, ZmLEC1 – like lectins, PSA (Pea seed agglutinin), Dolichos biflorus Agglutinin (DBA), Ulex europaeus Agglutinin I (UEA-I), MLL ( Morus alba lectin), CsinLec ( Citrus sinensis lectin), VvML (Vitis vinifera mannose lectin), MsLec (Medicago sativa lectin), CaLec (Coffea arabica lectin), OliLec (Olea europaea lectin), PsL (Prunus species lectin), and AlLec (Almond Lectin).

6. The agrobiological formulation as claimed in claim 1, wherein the carrier is selected from exopolysaccharides, lipopolysaccharides, mucopolysaccharides, polypeptides, lipids, modified polysaccharides, Glycoproteins, and derived polysaccharides.

7. The agrobiological formulation as claimed in claim 6, wherein the exopolysaccharide is selected from succinoglycan, succinylated alginate, xanthan gum, levan, alginate, and glucomannan.

8. The agrobiological formulation as claimed in claim 1, wherein concentration of a free-living nitrogen-fixing bacterium; at least one anti-stress agent; a carbohydrate-binding lectin; and a carrier is 0.01% to 10%, 0.01% to 0.03%, 0.004% to 0.02%, and 0.01% to 10% (w/v) respectively of total agrobiological formulation.

9. The agrobiological formulation as claimed in claim 1, wherein the agrobiological formulation improves the ammoniacal nitrogen in the soil by 125%.

10. The agrobiological formulation as claimed in claim 1, wherein the agrobiological formulation improves nitrogenase activity by 140-150%.

11. The agrobiological formulation as claimed in claim 1, wherein the agrobiological formulation improves at least one of bacterial aggregation, and bacterial proliferation by 70-90%, and 70-90%, respectively.

12. The agrobiological formulation as claimed in claim 1, wherein the agrobiological formulation improves at least one of seed surface colonization, seedling biomass, root and shoot development, and microbial count by 70-90%, 100-120%, 80-90%, and 25-30%, respectively.

13. The agrobiological formulation as claimed in claim 1, wherein the agrobiological formulation increases microbial count by 100000 times.

14. The agrobiological formulation as claimed in claim 1, wherein the plant is selected from legumes (Fabaceae), monocots, dicots, Actinorhizal plants, Parasponia (non-legume), aquatic legumes, graminaceous crops, vegetables, Native trees, and shrubs.

15. A seed coating composition for plant growth comprising the agrobiological formulation as claimed in claim 1.

16. An artificial nodule stimulant for plant growth comprising the agrobiological formulation as claimed in claim 1.

17. A root dipping composition for plant growth comprising the agrobiological formulation as claimed in claim 1.

Dated this 26th Day of May 2024


ABHIJEET GIDDE
IN/PA-4407
AGENT FOR THE APPLICANT