Microbial based composition of methylobacterium species for enhancement of plant health and productivity

Abstract

The present invention relates to microbial based compositions comprising Methylobacterium species for improving plant growth and productivity.

Description

[0001] “MICROBIAL BASED COMPOSITION OF METHYLOBACTERIUM SPECIES FOR ENHANCEMENT OF PLANT HEALTH AND PRODUCTIVITY”

[0002] This is a Cognate Application of the originally filed Indian Patent Application no. 202441048500 dated 25-07-2024 and Indian Patent Application no. 202541006271 dated 25-01-2025.

[0003] TECHNICAL FIELD OF INVENTION:

[0004] The present invention relates to microbial based compositions comprising Methylobacterium species for improving plant growth and productivity.

[0005] BACKGROUND OF THE INVENTION:

[0006] Among the vast list of plant growth promoting microbes, Methylobacterium species are well known plant phyllospheric microbes capable of producing growth hormone, mitogen and secondary metabolite release required for plant growth, thereby promoting the growth of crops such as field crops, cash crops, vegetables and not limited to Paddy, Maize, soybean, peanut, lettuce, tomato, melon and the like.

[0007] The genus Methylobacterium comprises of several pink pigmented facultatively methylotrophic - PPFM species, which can utilize one-carbon compounds such as formate, formaldehyde and methanol as the sole source of carbon and energy as well as on a wide range of multi carbon growth substrates. Methylobacteria are widely distributed in a variety of habitats including leaf surfaces - phyllosphere, soil, fresh water, lake sediments, nodules, rice grains, atmospheric air and hospital environments. There are prior art documents available pertaining to microbial compositions and Methylobacterium spp., for plant applications related to biostimulants / biofertilizers.

[0008] CN117511833A discloses the application capability of the Methylobacterium as plant probiotics and phyllosphaeria bacterial manure. The invention details methods of improving photosynthesis and promoting the growth capacity of the Methylobacterium on plant phyllosphere colonization, and application potential for promoting the growth and production of crops.

[0009] CA2874835A1 provides methods for the cultivation of the Methylobacterium genus of bacteria. In particular, the invention provides methods for the efficient and inexpensive cultivation of these bacteria. Additionally, the invention provides methods for the utilization of these bacterial cultures to improve plant agriculture.

[0010] CN114423291 A relates to a new strain of Methylobacterium sp. nov, deposited under accession number CECT9580 (denoted as Methylobacterium symbioticum) compositions comprising the same, and its use as a biostimulant and as an endogenous nitrogen-fixing bacterium in plants.

[0011] EP3550955Bldescribes application of a microbial composition comprising Methylobacterium species which inhibits fungal disease in a plant or on an isolated plant part, from plant group including rice, wheat, com, barley, millet, sorghum, and the like, wherein the Methylobacterium composition is applied as a seed treatment and further comprises an agriculturally acceptable excipient selected from the group consisting of wood flour, clay, activated carbon, diatomaceous earth, inorganic solids and calcium carbonate.

[0012] There is a need in the art to provide a novel composition of Methylobacterium strains in suitable ratio to get synergistic effect on improvement of plant growth, plant health, crop yield, etc. Hence, the present inventors have developed synergistic combinatorial composition of Methylobacterium species with or without other microbes and / or adjuvants, etc., that increases yield, emulates biotic and / or abiotic stress, enhances Nitrogen fixation, nutrient uptake efficiency, increases chlorophyll synthesis and photosynthesis efficiency thereby improves plant growth, crop yield, etc.

[0013] The present inventors observed that the in-house trials of the present novel Formulations showed better results when compared to the prior art formulations, with respect to crop yield and productivity.

[0014] SUMMARY OF THE INVENTION:

[0015] Accordingly, the present invention discloses novel microbial based compositions of Methylobacterium species for the application in improvement of plant health and productivity.

[0016] In a preferred aspect, the present invention relates to a microbial based composition comprising at least two Methylobacterium species with or without other microbes selected from endophytes, ectophytes ephiphytes, Rhizospheric and / or free living; other materials selected from macro nutrients and micronutrients, simple and complex carbohydrates including and not limited to monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides; nitrogen containing compounds including enzymes / proteins / peptides / amino acids, vitamins, organic acids; minerals secondary metabolites including flavonoids, aromatic, phenolics compounds; alcohols; lipids; growth hormones; polyphenolic compounds; pesticides, adjuvants, preservatives, for agricultural applications as bio stimulant, biofertilizer, plant growth promotion, nitrogen fixation, increased nutrient use efficiency, plant protection improved plant health, abiotic and biotic stress tolerances, increased crop productivity and yields, increased produce quality, increased produce nutrient density, increased shelf life of produce and reduced greenhouse gas emissions. In another aspect, the composition of the present invention comprises at least two Methylobacterium species selected from Methylobacterium symbioticum, Methylobacterium extorquens, Methylobacterium mesophilicum, Methylobacterium aquaticum. Methylobacterium fujisawaense, Methylobacterium gnaphalii. Methylobacterium hispanicum. Methylobacterium oryzae. Methylobacterium phyllo sphaerae. Methylobacterium phyllostachyos. Methylobacterium radiotolerans. Methylobacterium Populi, Methylobacterium adhaesivam, Methylobacterium longum, Methylobacterium brachythecii, Methylobacterium aerolatum, Methylobacterium bullatum, Methylobacterium gossipiicola, Methylobacterium phyllostachyos, Methylobacterium planium.

[0017] In yet another aspect, the other microbes are selected from aerobic / anaerobic bacteria, fungi, algae, archaea, epiphytes, endophytes, ectophytes, endosymbiont, rhizosphere, free-living, diazotrophic, non-diazotrophic microbe endo, ecto mycorrhiza, microbes including Rhizobium leguminosorum, Azospirillum, Azotobacter, Pseudomonas, Bacillus, Streptomyces, Bradyrhizobium japonicum, Nitrospira inopinata, Rhodopseudomonas palustris, Sphingomonas, Nitrosopumilus maritimus, Methylobacterium, Rhodobacter sphaeroides, Reyranella massiliensis, Alcaligene, Saccharomyces cerevisiae, Saccharomyces lactis, Trichoderma, Brevibacterium, Kluyveromyces lactis, Epichloe typhina, Enterococcus, Corynebacterium, Arthobacter, Pichia, Zymomonas, Saccharomyces carlsbergensis, Salmonella, Zymomonas, Rhodacoccus, Escherichia (e.g., E. Coli), Hansenula, Firmicutes, Rubrivivax, Dinoroseobacter shibae, Beijerinckiaindica, Ralstonia eutropha / Cupriavidus necator, Methyloversatilis, Nigrospora oryzae, Candida, Reyranella massiliensis, Novosphingobium aromaticivorans, Microbacterium, Acidovorax, Bordetella, Phomopsis liquidambaris, Nitrosopumilus maritimus, Ralstonia eutrophus / Cupriavidus necator, Nitrospira inopinata, Nigrospora oryzae, Rhodopseudomonas palustris, Periconia spp, Paenibacillus beijingensis, Thiobacillus sp, Sinorhizobium meliloti, Methyloversatilissp, Reyranella massiliensis Cyanobacteria, Rhodobacter capsulatus, Epichloetyphina, Metallosphaera, Sulfolobus, Archaeoglobus, Cenarchaeum spp, Clostridium autoethanogenum, Xanthobacter flavus, Oligotropha carboxidovorans, Acidithiobacillus thiooxidans, Desulfobacter hydrogenophilus, Thiomicrospira denitrificans.

[0018] In a further aspect, the nitrogen containing compounds in the composition of the present invention are selected from proteins including but not limited to protein hydrolysate, protein isolates and / or amino acids and or enzymes including but not limited to phenyl alanine, amino lyase; coenzymes including but not limited to NAD+, NADP+ and adenosine triphosphate (ATP); coenzymes, such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamine pyrophosphate(TPP), and tetrahydrofolate (THF), derived from vitamins; Vitamins including but not only limited to a tocopherol and / or vitamin-D and / or vitamin K and / or and / or vitamin B and or Ascorbic acid and the like in an amount of 0.001 % to 5 % by weight of the composition.

[0019] In another aspect, the other materials in the composition of the present invention are selected from but not limited macro nutrients and micro nutrients such as primary, secondary, micro and trace elements including but not limited to nitrogen, phosphate, potash, poly phosphates, sulphates and their salts and / or pesticides and / or antioxidant pathway inducers (like coumaric acid and / or cinnamic acid and / or salicylic acid etc.) in an amount of 0.01 to 50 %.

[0020] In yet another aspect, the minerals in the composition of the present invention are selected from but not limited to Potassium, Calcium, Magnesium, Scandium, Titanium, Vanadium, Molybdenum, Iron, Cobalt, Nickel, Copper, Zinc, Iodine selenium, , and other trace elements etc. in an amount of 0.01 % to 50 % by weight of the composition. In another aspect, the carbohydrates in the composition of the present invention are selected from but not limited to monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, Carboxy methyl cellulose, sugars and / or sugar alcohols including but not limited to mannitol, sorbitol, erythritol, laminarin fucoidan, chitin, chitosan, alginic acid etc and / or carbohydrate containing substances etc., in an amount of 0.01 % to 50 %.

[0021] In yet another aspect, the alcohols in the composition of the present invention are selected from but not limited to monohydric alcohols including but not limited to ethanol and / or methanol in an amount of 0.01 to 30 %.

[0022] In another aspect, the lipids in the composition of the present invention are selected from but not limited to monoglycerides and / or diglycerides and / or triglycerides and / or phospholipids in an amount of 0.01 % to 25 %.

[0023] In yet another aspect, the hormone and or hormone mimic substances in the composition of the present invention are selected from but not limited to pesticides such as Glyphosate, azoxystrobin, fludioxonil etc present in non-inhibitory lower concentration.

[0024] In another aspect, the polyphenolic compounds in the composition of the present invention are selected from but not limited to Tannins in an amount of 0.01 to 50 0 / / o, .

[0025] In another aspect, the composition of the present invention may also include a surfactant selected from, but not limited to, anionic and non-ionic and cationic surfactants, tween-20, tween -80 etc. to ensure uniform and wide spreading of the composition on the plant when applied to give complete application of foliage by maintaining surface tension of leaf and molecule contact to the plant. In an aspect, the present invention provides the synergistic effect of combinatorial application of more than one Methylobacterium species on improvement in plant health, crop productivity, produce quality, nutrient content, shelf life, etc.

[0026] In another aspect, the effect of each strain of Methylobacterium species individually and in combination was evaluated on plant growth and productivity, and it was observed that the combinatorial, synergistic effect of the strains together showed enhanced plant growth and productivity, in comparison to individual microbe application.

[0027] In another aspect, the present invention provides the synergistic effect of combinatorial application of more than one Methylobacterium species on improvement in plant health, crop productivity, produce quality, nutrient content, shelflife, etc.

[0028] In another aspect, the present invention provides microbial compositions comprising of Methylobacterium sp, for the application as biostimulant, biofertilizer, plant growth promotion, plant protection, etc.

[0029] In another aspect, the present microbial based composition further comprises nutrients such as not limited to NPK, boron, calcium, silicon, magnesium, nickel, cobalt, manganese, iron, zinc, copper, molybdenum, etc either alone or combinations thereof.

[0030] In another aspect, the present microbial based composition further comprises compounds including but not limited to pesticides, organic acids, aromatic compounds, phenolics, proteins, flavonoids, peptides, amino acids, growth hormones, adjuvants, preservatives, etc either alone or combinations thereof.

[0031] In one aspect, the compositions of the present invention may be formulated optionally in combination with proteins (including but not limited to hydrolysate proteins, protein isolates) and / or amino acids and or enzymes (including but not limited to phenyl alanine, amino lyase) and or coenzymes (including but not limited to NAD+, NADP+ and adenosine triphosphate (ATP); certain coenzymes, such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamine pyrophosphate(TPP), and tetrahydrofolate (THF), derived from vitamins), wherein, the optional ingredients can be used in an amount of 0.01 % to 0.5 %; in combination with Vitamins including but not only limited to a tocopherol and / or vitamin-D and / or vitamin K and / or and / or vitamin B and or Ascorbic acid and the like in an amount of 0.001 % to 0.5 % by weight of the composition.

[0032] In another aspect, the compositions of the present invention may be formulated in combination with other materials including but not limited to materials containing fertilizers such as primary, secondary, micro, micro and trace elements including but not limited to nitrogen, phosphate, phosphite, poly phosphates, sulphates, sulphites and their salts and / or pesticides and / or antioxidant pathway inducers (coumaric acid and / or cinnamic acid and / or salicylic acid etc.) in an amount of 0.01 to 50 %.

[0033] In another aspect, the compositions of the present invention may be formulated in combination with minerals including but not limited to Potassium, Calcium, Magnesium, Scandium, Titanium, Vanadium, Molybdenum, Iron, Cobalt, Nickel, Copper, Zinc, Iodine selenium, and other trace elements etc. in an amount of 0.01 % to 50 % by weight of the composition.

[0034] In yet another aspect, the compositions of the present invention may be formulated in combination with carbohydrates including but not limited to mono, di, oligo, poly saccharides, Carboxymethyl cellulose, sugars and / or sugar alcohols including but not limited to mannitol, sorbitol, erythritol, laminarin fucoidan, chitin, chitosan, alginic acid etc. and / or carbohydrate containing substances etc., in an amount of 0.01 % to 50 %. In another aspect of the invention, the compositions of the present invention may be formulated in combination with alcohols including but not limited to monohydric alcohols including but not limited to ethanol and / or methanol in an amount of 0.01 to 30 %.

[0035] In certain aspects of the invention, the compositions of the present invention may be formulated in combination containing lipids including but not limited to monoglycerides and / or diglycerides and / or triglycerides and / or phospholipids in an amount of 0.01 % to 25 %.

[0036] The compositions of the present invention may be formulated in combination with hormone and or hormone mimic substances including but not limited to pesticides at non-inhibitory concentrations.

[0037] The compositions of the present invention may be formulated in combination with tannins in an amount of 0.01 to 50 %;

[0038] In another aspect, the compositions of the present invention may comprise a surfactant selected from anionic and non-ionic and cationic surfactants such as lignosulphonates, tween-20, tween -80 etc. to ensure uniform and wide spreading of the composition on the plant when applied to give complete application of foliage by maintaining surface tension of leaf and molecule contact to the plant.

[0039] In another aspect, the present invention provides application of the present microbial based composition on improvement of (a) nitrogen fixation, (b) phosphate accumulation and Phosphate solubilization, (c) potash, silica, calcium, magnesium, sulphur and zinc solubilization, (d) Providing abiotic stress tolerance and not restricted to temperature, salt and drought tolerance (e) Induce seed germination of the crop (f) plant growth hormone production, (g) siderophore production (h) reduction in inorganic fertilizer application (i) lesser irrigation (j) increase in chlorophyll content and increase in photosynthesis efficiency, etc. DETAILS OF THE MICROORGANISMS USED IN THE INVENTION:

[0040] DETAILED DESCRIPTION OF THE INVENTION:

[0041] The present invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects therefore maybe fully understood and appreciated. The term "composition" or "formulation" used herein interchangeably relates to and means the microbial based compositions of the present invention.

[0042] The present invention discloses novel microbial based composition of Methylobacteria, for the application on improvement of plant health, productivity, etc.

[0043] In a preferred embodiment, the present invention relates to a microbial based composition comprising at least two Methylobacterium species with or without other microbes selected from endophytes, ectophytes, ephiphytes, Rhizospheric and / or free living; other materials selected from macronutrients and micronutrients; simple and complex carbohydrates including and not limited to monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides; nitrogen containing compounds including enzymes / proteins / peptides / amino acids, vitamins, organic acids; minerals; secondary metabolites including flavonoids, aromatic, phenolics compounds; alcohols; lipids; growth hormones; polyphenolic compounds; pesticides, adjuvants, preservatives, for agricultural applications as bio stimulant, biofertilizer, plant growth promotion, nitrogen fixation, increased nutrient use efficiency, plant protection improved plant health, abiotic and biotic stress tolerances, increased crop productivity and yields, increased produce quality, increased produce nutrient density, increased shelf life of produce and reduced greenhouse gas emissions.

[0044] In another embodiment, the composition of the present invention comprises at least two Methylobacterium species selected from Methylobacterium symbioticum, Methylobacterium extorquens, Methylobacterium mesophilicum, Methylobacterium aquaticum. Methylobacterium fujisawaense. Methylobacterium gnaphalii. Methylobacterium hispanicum, Methylobacterium ory ae. Methylobacterium phyllo sphaerae. Methylobacterium phyllostachyos. Methylobacterium radiotolerans, Methylobacterium Populi, Methylobacterium adhaesivam, Methylobacterium longum, Methylobacterium brachythecii, Methylobacterium aerolatum, Methylobacterium bullatum, Methylobacterium gossipiicola, Methylobacterium phyllostachyos, Methylobacterium planium.

[0045] In yet another embodiment, the other microbes in the composition of the present invention are selected from aerobic / anaerobic bacteria, fungi, algae, archaea, epiphytes, endophytes, ectophytes, endosymbiont, rhizosphere, free-living, diazotrophic, non-diazotrophic microbe, endo, ecto, mycorrhiza, microbes including Rhizobium leguminosorum, Azospirillum, Azotobacter, Pseudomonas, Bacillus, Streptomyces, Bradyrhizobium japonicum, Nitrospira inopinata, Rhodopseudomonas palustris, Sphingomonas, Nitrosopumilus maritimus, Methylobacterium, Rhodobacter sphaeroides, Reyranella massiliensis, Alcaligene, Saccharomyces cerevisiae, Saccharomyces lactis, Trichoderma, Brevibacterium, Kluyveromyces lactis, Epichloe typhina, Enterococcus, Corynebacterium, Arthobacter, Pichia, Zymomonas, Saccharomyces carlsbergensis, Salmonella, Zymomonas, Rhodacoccus, Escherichia (e.g., E. Coli), Hansenula, Firmicutes, Rubrivivax, Dinoroseobacter shibae, Beijerinckiaindica. Ralstonia eutropha / Cupriavidus necator, Methyloversatilis, Nigrospora oryzae, Candida, Reyranella massiliensis, Novosphingobium aromaticivorans, Microbacterium, Acidovorax, Bordetella, Phomopsis liquidambaris, Nitrosopumilus maritimus, Ralstonia eutrophus / Cupriavidus necator, Nitrospira inopinata, Nigrospora oryzae, Rhodopseudomonas palustris, Periconia spp, Paenibacillus beijingensis, Thiobacillus sp, Sinorhizobium meliloti, Methyloversatilissp, Reyranella massiliensis, Cyanobacteria, Rhodobacter capsulatus,

[0046] Epichloetyphina, Metallosphaera, Sulfolobus, Archaeoglobus, Cenarchaeum spp, Clostridium autoethanogenum, Xanthobacter flavus, Oligotropha carboxidovorans, Acidithiobacillus thiooxidans, Desulfobacter hydrogenophilus, Thiomicro spir a denitrifican. In a further embodiment, the nitrogen containing compounds in the composition of the present invention are selected from proteins including but not limited to protein hydrolysate, protein isolates and / or amino acids and or enzymes including but not limited to phenyl alanine, amino lyase; coenzymes including but not limited to NAD+, NADP+ and adenosine triphosphate (ATP); coenzymes, such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamine pyrophosphate(TPP), and tetrahydrofolate (THF), derived from vitamins; Vitamins including but not only limited to a tocopherol and / or vitamin-D and / or vitamin K and / or and / or vitamin B and or Ascorbic acid and the like in an amount of 0.001 % to 5 % by weight of the composition.

[0047] In another embodiment, the other materials in the composition of the present invention are selected from but not limited to macro nutrients and micro nutrients such as primary, secondary, micro and trace elements including but not limited to nitrogen, phosphate, potash, poly phosphates, sulphates and their salts and / or pesticides and / or antioxidant pathway inducers (like coumaric acid and / or cinnamic acid and / or salicylic acid etc.) in an amount of 0.01 to 50 %.

[0048] In yet another embodiment, the minerals in the composition of the present invention are selected from but not limited to Potassium, Calcium, Magnesium, Scandium, Titanium, Vanadium, Molybdenum, Iron, Cobalt, Nickel, Copper, Zinc, Iodine selenium, , and other trace elements etc. in an amount of 0.01 % to 50 % by weight of the composition.

[0049] In another embodiment, the carbohydrates in the composition of the present invention are selected from but not limited to monosaccharides, disaccharides, trisaccharides, oligosaccharides, poly saccharides, Carboxy methyl cellulose, sugars and / or sugar alcohols including but not limited to mannitol, sorbitol, erythritol, laminarin fucoidan, chitin, chitosan, alginic acid etc. and / or carbohydrate containing substances etc., in an amount of 0.01 % to 50 %. In yet another embodiment, the alcohols in the composition of the present invention are selected from but not limited to monohydric alcohols including but not limited to ethanol and / or methanol in an amount of 0.01 to 30 %.

[0050] In another embodiment, the lipids in the composition of the present invention are selected from but not limited to monoglycerides and / or diglycerides and / or triglycerides and / or phospholipids in an amount of 0.01 % to 25 %.

[0051] In yet another embodiment, the hormone and or hormone mimic substances in the composition of the present invention are selected from but not limited to pesticides such as Glyphosate, azoxystrobin, fludioxonil etc present in non-inhibitory lower concentration.

[0052] In another embodiment, the polyphenolic compounds in the composition of the present invention are selected from but not limited to Tannins in an amount of 0.01 to 50 %;

[0053] In another embodiment, the composition of the present invention may also include a surfactant selected from, but not limited to, anionic and non-ionic and cationic surfactants, tween-20, tween -80 etc. to ensure uniform and wide spreading of the composition on the plant when applied to give complete application of foliage by maintaining surface tension of leaf and molecule contact to the plant.

[0054] In another embodiment, the composition of the present invention may also include binder and trace metals such as Talc, Bentonite, peat, lignite, vermiculite, charcoal, and press mud etc.

[0055] In another embodiment, the present invention provides the use of the present composition for enhancing Nitrogen fixation, phosphate accumulation and Phosphate solubilization, potash, silica, calcium, magnesium, sulphur and zinc solubilization, and increased production of siderophore. In another embodiment, the present invention provides compositions comprising of Methylobacterium species, for the application as biostimulant, biofertilizer, plant growth promotion, plant protection impacting on plant improvement in plant health, crop productivity, produce quality, nutrient content, shelflife etc.

[0056] In an embodiment, the present invention provides a microbial based composition for improving nitrogen fixation and providing Nitrogen supply to plants, wherein the combined effect of the microbial strains results in better yield even in terms of significant reduction in Nitrogen fertilization.

[0057] In another embodiment, the present invention provides synergistic microbial based composition for improving plant growth hormone production and thereby enhancing plant health and productivity.

[0058] In yet another embodiment, the present invention provides synergistic microbial based composition for improving plant immunity by providing biotic and abiotic stress tolerance and not restricted to temperature, salt and drought tolerance, biotic and abiotic stress tolerance, and protection against disease and insect attack, etc.

[0059] In yet another embodiment, the present invention provides synergistic microbial based composition for enhancing plant chlorophyll content and increasing photosynthesis efficiency, due to the increased nitrogen supply and uptake.

[0060] In yet another embodiment, the present invention provides the use of the present synergistic microbial based compositions for enhancement of phosphate accumulation and phosphate solubilization, potash, silica, calcium, magnesium, sulphur and zinc solubilization, and increased production of siderophore, etc. In another embodiment, the present invention provides synergistic microbial based compositions for the application as biostimulant, biofertilizer, plant growth promotion, plant protection, etc.

[0061] In another embodiment, the microbial based composition of the present invention reduces the requirement of chemical based Nitrogen fertilizer such as Urea in plants / crops. The Methylobacterium species present in the microbial based composition of the present invention fixes the atmospheric nitrogen which is utilised by the plants / crops for improving plant growth and crop yield. Thereby, reducing the need for chemical based fertilizers.

[0062] In yet another embodiment, the present invention provides biostimulant, biofertilizer compositions comprising of Methylobacterium species along with or without other microbes belonging to classes such as aerobic / anaerobic bacteria, fungi, algae, archaea, epiphytes, endophytes, endosymbiont, rhizosphere, free- living, diazotrophic, non-diazotrophic microbe endo, ecto mycorrhiza, etc.

[0063] In another embodiment, the present invention provides synergistic microbial based composition comprising Methylobacterium species along with any one or combinations of nutrients and / or micronutrients such as and not limited to NPK, boron, calcium, silicon, magnesium, nickel, cobalt, manganese, iron, zinc, copper, molybdenum, etc.

[0064] In another embodiment, the present invention provides synergistic microbial based compositions comprising Methylobacterium species along with any one or combination of compounds including but not limited to pesticides, organic acids, aromatic compounds, phenolics, proteins, flavonoids, peptides, amino acids, growth hormones, adjuvants, preservatives, etc.

[0065] In another embodiment, the compositions of the present invention may be formulated optionally in combination with proteins (including but not limited to hydrolysate proteins, protein isolates) and / or amino acids and or enzymes (including but not limited to phenyl alanine, amino lyase) and or coenzymes (including but not limited to NAD+, NADP+ and adenosine triphosphate (ATP). Certain coenzymes, such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamine pyrophosphate(TPP), and tetrahydrofolate (THF), derived from vitamins), wherein, the optional ingredients can be used in an amount of 0.01 % to 0.5 % ; in combination with Vitamins including but not only limited to a tocopherol and / or vitamin-D and / or vitamin K and / or and / or vitamin B and or Ascorbic acid and the like in an amount of 0.001 % to 0.5 % by weight of the composition.

[0066] In another embodiment, the compositions of the present invention may be formulated in combination with other materials including but not limited to materials containing fertilizers such as primary, secondary, micro, micro and trace elements including but not limited to nitrogen, phosphate, phosphite, poly phosphates, sulphates, sulphites and their salts and / or pesticides and / or antioxidant pathway inducers (coumaric acid and / or cinnamic acid and / or salicylic acid etc.) in an amount of 0.01 to 50 %.

[0067] In yet another embodiment, the compositions of the present invention may be formulated in combination with minerals including but not limited to Potassium, Calcium, Magnesium, Scandium, Titanium, Vanadium, Molybdenum, Iron, Cobalt, Nickel, Copper, Zinc, Iodine selenium, and other trace elements etc. in an amount of 0.01 % to 50 % by weight of the composition.

[0068] In certain embodiment, the compositions of the present invention may be formulated in combination with carbohydrates including but not limited to monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, Carboxy methyl cellulose, sugars and / or sugar alcohols including but not limited to mannitol, sorbitol, erythritol, laminarin fucoidan, chitin, chitosan, alginic acid etc., and / or carbohydrate containing substances etc., in an amount of 0.01 % to 50 0 / / o.

[0069] In one embodiment, the compositions of the present invention may be formulated in combination with alcohols including but not limited to monohydric alcohols including but not limited to ethanol and / or methanol in an amount of 0.01 to 30 %.

[0070] In another embodiment, the compositions of the present invention may be formulated in combination with lipids including but not limited to monoglycerides and / or diglycerides and / or triglycerides and / or phospholipids in an amount of 0.01 % to 25 %.

[0071] In yet another embodiment, the compositions of the present invention may be formulated in combination with hormone and or hormone mimic substances including but not limited to pesticides such as Glyphosate, azoxystrobin, fludioxonil etc present in non inhibitory lower concentration.

[0072] In another embodiment, the compositions of the present invention may be formulated in combination with tannins in an amount of 0.01 to 50 %.

[0073] In another embodiment, the composition of the present invention may comprise a surfactant selected from anionic and non-ionic and cationic surfactants such as CMC, lignosulphonates, tween-20, tween -80 etc. to ensure uniform and wide spreading of the composition on the plant when applied to give complete application of foliage by maintaining surface tension of leaf and molecule contact to the plant.

[0074] In another embodiment, the present invention provides a process for preparation of the synergistic microbial composition wherein the said process comprising the steps of i. Growing of individual microorganism separately ii. Checking the compatibility of the different microorganisms; and iii. Mixing the microorganisms after adding binder and tracemetals in the formulated materials such as Talc, Bentonite, peat, lignite, vermiculite, charcoal, and press mud etc

[0075] In another embodiment, the effect of each strain of Methylobacterium species individually and in combination on plant growth and productivity was evaluated, and it was observed that the combinatorial, synergistic effect of strains showed significant enhancement in plant growth, reduction in Nitrogen fertilizer requirement and improved crop productivity in comparison to individual microbe application as depicted in Examples 1 to 12.

[0076] In another embodiment, the present invention provides the synergistic effect of combinatorial application of more than one Methylobacterium species on improvement in plant health, crop productivity, produce quality, nutrient content, shelflife etc.

[0077] In another embodiment, the present invention provides application of the present microbial based composition on improvement of (a) nitrogen fixation, (b) phosphate accumulation and Phosphate solubilization, (c) potash, silica, calcium, magnesium, sulphur and zinc solubilization, (d) Providing abiotic stress tolerance and not restricted to temperature, salt and drought tolerance (e) Induce seed germination of the crop (f) plant growth hormone production, (g) siderophore production (h) reduction in inorganic fertilizer application (i) lesser irrigation (j) increase in chlorophyll content and increase in photosynthesis efficiency, etc.

[0078] In another embodiment, the present invention details the mutual relationship and impact of each microbe on the other microbe and inter-microbial symbiosis effects, wherein the inter-microbial symbiosis is supposed to impact on further enhancement of plant productivity, growth, and overall plant health. In another embodiment, the microbial based composition of the present invention are useful for improving plant immunity by providing biotic and abiotic stress tolerance and protection against disease and insect attack.

[0079] In another embodiment, the microbial based composition of the present invention are useful for enhancing plant chlorophyll content and increasing photosynthesis efficiency, due to the increased nitrogen supply and uptake.

[0080] In yet another embodiment, the microbial based composition of the present invention are useful for application for crops such as and not limiting to field crops, cash crops cereals, pulses, fruits, vegetables, flowers, leafy vegetables, aromatic crops, biomass crops, and the like.

[0081] In another embodiment, the microbial based composition of the present invention may be prepared in various forms such as solid powder, granule, paste, liquid, gel, pellet, pills, capsule or tablet.

[0082] In another embodiment, the microbial based composition of the present invention is effective on crop varieties such as tomato, okra, brinjal, soybean, cotton, onion, potatoes, cereals com, wheat, rice, sorghum, bajra, grains, pulses crops, fruits, vegetables, plantation crops, vegetables, sugarcane, fodder grass, oilseed crops, tea and the like, that can lead to increased photosynthesis and improved crop yield.

[0083] In another embodiment, the synergistic microbial composition of the present invention can be applied on leaves by sprinkler application, sprayer application or drip application, more preferably by sprayer application such as foliar sprays, sprays to be applied to plants leaves, shoots and the like. The composition can also be mixed with soil or added during irrigation; mixed with fertilizers or impregnated or coated on fertilizers; or mixed with pesticides, weedicides and plant growth regulators. In another embodiment, the synergistic microbial composition of the present invention is applied at the dosage of 0.1 kg to 5 kg per acre per application depending upon the crop requirement.

[0084] Further details of the composition of the present invention will be apparent from the examples presented below. Examples presented are purely illustrative and should not be construed as limiting the scope of the invention in any manner. Numerous modifications or alterations may be made therein without departing from the spirit and the scope of the present invention.

[0085] EXAMPLES

[0086] In the following examples 1 to 12, the terms 100%N, 75%N reduced and 50% N reduced relates to the following:

[0087] 100%N means the nitrogen is present in 100% concentration in the nitrogen containing fertilizer.

[0088] 75 %N reduced means the nitrogen is present in 25% concentration in the nitrogen containing fertilizer.

[0089] 50%N reduced means the nitrogen is present in 50% concentration in the nitrogen containing fertilizer.

[0090] Example 1:

[0091] Pot-trial analysis of combinatorial synergistic effect of Methylobacterium extorquens ATCC14718 and Methylobacterium symbioticum PYCC8351 on enhancement of Green gram productivity:

[0092] The following example details the impact of Methylobacterium formulations on green gram yield and productivity in terms of enhancement in Nitrogen content, chlorophyll content, pod productivity, etc. In order to assess the synergistic impact of formulation comprising Methylobacterium extorquens and Methylobacterium symbioticum on crop yield and productivity, green gram was used as the crop in pot-trial analysis.

[0093] The novel formulations of Methylobacterium strains comprising Methylobacterium extorquens and Methylobacterium symbioticum were applied on green gram plantlets for pot-trial analysis, in following compositions.

[0094] Controls:

[0095] • Control 100%N

[0096] • Control 50%N Reduced • Control 75%N Reduced

[0097] Combination used for application:

[0098] • M.Symbioticum Wild type Foliar application

[0099] • M.Symbioticum Wild type + Methylobacterium extorquens Foliar application • Methylobacterium extorquens foliar application

[0100] Pot-trial analysis showed significant enhancement in productivity, in terms of increased number of pods, enhanced chlorophyll content, enhanced Nitrogen content, etc (as depicted in Table 1).

[0101] Table 1

[0102] Results:

[0103] 1. M. Symbioticum, showed 80% increase in yield compared to control 100% Nitrogen added (Farmer regular practice).

[0104] 2. M. extorquens showed 12% increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0105] 3. M.Symbioticum and M.extorquens showed 100% increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0106] 4. M.Symbioticum, Methylobacterium extorquens individually and M.Symbioticum+ Methylobacterium extorquens combination along with 50% N reduction showedl00% increase in yield compared to control 50% Nitrogen.

[0107] Example 2:

[0108] Pot-trial analysis of combinatorial synergistic effect of Methylobacterium extorquens and Methylobacterium symbioticum on enhancement of chlorophyll content, productivity of Maize:

[0109] In order to assess the synergistic impact of Methylobacterium extorquens and Methylobacterium symbioticum formulation on crop yield and productivity, Green gram was used as the crop in pot-trial analysis in terms of enhancement in Nitrogen content, chlorophyll content, etc. The novel formulations of Methylobacterium strains were applied on Green gram under pot-trial analysis, in following compositions

[0110] Controls:

[0111] • Control 100%N • Control 50%N Reduced

[0112] • Control 75%N Reduced

[0113] Combination used for application:

[0114] • M.Symbioticum Wild type Foliar application

[0115] • M.Symbioticum Wild type + Methylobacterium extorquens Foliar application in 50:50 ratio

[0116] • Methylobacterium extorquens foliar application

[0117] Pot-trial analysis showed significant enhancement in productivity, in terms of enhanced chlorophyll content, enhanced Nitrogen content, etc (as depicted in Table 2)

[0118] Table 2

[0119]

[0120] Results: 1. M.Symbioticum and M. Extorquens 50% N (Average wt- 18.5 gm) foliar application showed50% Highest yield compared to control 100% N (Average wt-9.5) (100% Nitrogen added Farmer practice).

[0121] 2. M. Symbioticum and M. Extorquens combination increased 30% of the Chlorophyll and Nitrogen content compared to control. (100% Nitrogen added Farmer practice).

[0122] Example 3:

[0123] Pot-trial analysis of combinatorial synergistic effect of Methylobacterium extorquens and Methylobacterium symbioticum on enhancement of chlorophyll content, productivity of Tomato, in comparison to Nano Urea application:

[0124] In order to assess the synergistic impact of Methylobacterium extorquens and Methylobacterium symbioticum formulation on crop yield and productivity, Tomato was used as the crop in pot-trial analysis, and compared with Nano Urea application, in terms of enhancement in Nitrogen content, chlorophyll content, etc.

[0125] The novel formulations of Methylobacterium strains were applied on Tomato under pot-trial analysis, in following compositions

[0126] Controls:

[0127] • Control 100%N

[0128] • Control 50%N Reduced

[0129] • Control 75%N Reduced

[0130] Combination used for application:

[0131] • M.Symbioticum Wild type Foliar application

[0132] • M.Symbioticum Wild type + Methylobacterium extorquens Foliar application in 50:50 ratio

[0133] • Methylobacterium extorquens foliar application • Nano Urea 100% N

[0134] • Nano Urea 50% N

[0135] • Nano Urea 25% N

[0136] Pot-trial analysis showed significant enhancement in productivity, in terms of enhanced chlorophyll content, enhanced Nitrogen content, etc (as observed from

[0137] Table 3)

[0138] Table 3

[0139] Results:

[0140] 1. M.Symbioticum + M.extorquens 50% N combination showed 15% higher yield than control 100% N and 35% higher yield compared to 50% control. 2. M.Symbioticum + M.extorquens 50% N combination also showed around 14%

[0141] Higher yield compared to Nano Urea application (100% N).

[0142] Example 4:

[0143] Pot-trial analysis of combinatorial synergistic effect of Methylobacterium symbioticum, M. extorquens, Methylobacterium mesophilicum and essential trace elements on productivity of Green gram:

[0144] A bioinoculant composition comprising Methylobacterium symbioticum PYCC8351, M. extorquens ATCC14718, Methylobacterium mesophilicum DSM 1708 and essential trace elements was formulated to significantly boosts Green gram yield through enhanced nutrient assimilation, root growth, and hormonal balance. This synergistic approach was ideal for organic and sustainable for agriculture.

[0145] Trace metals solution - Calcium chloride - 0.65 mg / kg, Magnesium sulphate -0.5 mg / kg, Cobalt chloride - 0.18 mg / kg, sodium molybdate - 0.3 mg / kg, Copper chloride - 0.16 mg / kg, Zinc sulphate-0.18 mg / kg, K2HPO4 - 0.7 mg / kg, KH2PO4 -

[0146] 0.6 mg / kg, Amm.citrate - 0.025 mg / kg, Manganese sulphate - 0.5 mg / kg, boric acid

[0147] - 0.5 mg / kg (1 liter ).

[0148] The results are depicted in Table 4.

[0149] Table 4

[0150] Results:

[0151] 1. Methylobacterium mesophilicum with TE solution showed60% increase in yield compared to control 100% Nitrogen added (Farmer regular practice). 2. M. Symbioticum Wild type and Methylobacterium extorquens with TE solution showed 96% increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0152] 3. M. Symbioticum, M. extorquens Methylobacterium mesophilicum with TE solution showed 100% increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0153] Example 5:

[0154] Pot-trial analysis to study effect of Methylobacterium symbioticum and M. extorquens. when used alone or in a microbial consortium with other PGPR (e.g., Bacillus, Pseudomonas, Azospirillum) on productivity of Tomato:

[0155] Methylobacterium symbioticum and M. extorquens, when used alone or in a microbial consortium with other PGPR (e.g., Bacillus, Pseudomonas, Azospirillum) significantly enhanced tomato growth and yield in pot trials (depicted in Table 5). This was largely due to phytohormone production, improved nutrient uptake, and stress tolerance.

[0156] These microbes promoted plant growth through multiple synergistic pathways, primarily: 1. Phytohormone Production: Produce cytokinins, auxins (IAA), and gibberellins, which:

[0157] ■ Stimulate root elongation and lateral root formation

[0158] ■ Promote cell division and expansion

[0159] ■ Delay leaf senescence, improving photosynthesis duration 2. Biological Nitrogen Fixation (BNF):Fix atmospheric nitrogen into plant- available forms, reducing fertilizer requirement

[0160] 3. Siderophore Production: Solubilize iron, enhancing nutrient uptake

[0161] 4. ACC Deaminase Activity: Lowers plant ethylene levels under stress, improving growth in sub-optimal conditions 5. Methanol Utilization: Methylobacterium species utilize methanol emitted by plant stomata, forming a beneficial phyllosphere symbiosis

[0162] Table 5

[0163] Results:

[0164] 1. Methylobacterium symbioticum and Methylobacterium extorquens showed 69% highest yield compared to control.

[0165] 2. M.Symbioticum and M.extorquens with other PGPR microbes combination Azospirillum brasilense, Bacillus licheniformis, P. fluorescens showed 100% Highest yield compared to control. Example 6:

[0166] Pot-trial analysis of combinatorial synergistic effect of

[0167] Methylobacterium aquaticum. Methylobacterium hispanicum and

[0168] Methylobacterium gnaphalii in Paddy crop: Methylobacterium aquaticum DSM16371, Methylobacterium hispanicum DSM 16372 and Methylobacterium gnaphalii DSM 24027 were screened under both normal and saline stress conditions. Many improved seedling vigor, shoot / root length and biomass. M. aquaticum, M. hispanicum and M. gnaphalii showed consistent ability to increase individual grain weight as depicted in Table 6.

[0169] Table 6

[0170] Results: 1. M. aquatic um. M. Gnaphalii and M. hispanicum combination showed 13% increase in yield compared to control (100% Nitrogen Farmers practice).

[0171] Example 7: Pot-trial analysis of combinatorial synergistic effect of Methylobacterium oryzae and Methylobacterium radiotolerans in Chilli:

[0172] For evaluating the effect of Methylobacterium oryzae DSM18207 and Methylobacterium radiotolerans DSM1819 inoculation on the growth of Chilli plants at different fertilizer levels, a pot trial experiment was conducted in a greenhouse.

[0173] The treatments consisted for organic fertilizer application, i.e. 100 and 50%. Each fertilizer treatment set was further divided into those with or without M. oryzae inoculation.

[0174] The results are depicted in Table 7

[0175] Table 7

[0176] Results:

[0177] 1. Methylobacterium oryzae with 100% nitrogen showed 50% increase in yield compared to control 100% Nitrogen added (Farmer regular practice).

[0178] 2. Methylobacterium oryzae with 50% Nitrogen showed 31% increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0179] 3. Methylobacterium radiotolerans with 100% nitrogen showed 56% increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0180] 4. Methylobacterium radiotolerans with 50% nitrogen showed 50% increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0181] Example 8:

[0182] Pot-trial analysis of combinatorial synergistic effect of Methylobacterium oryzae, Methylobacterium Nodulans, Methylobacterium extorquens and A. Brasilense

[0183] In this study, the effect of rhizosphere inoculation with A. brasilense and M. oryzae DSM18207, M.extorquens ATCC14718, M.nodulans ORS2060 was investigated. The results displayed growth promoting effects in pot experiments, on the growth and fruit yield of Chilli plant under a reduce fertilization level in the field (as depicted in Table 8). Table 8

[0184] Results:

[0185] 1. A.brasilense and Methylobacterium Extorquens with 50% nitrogen showed 22% increase in yield compared to control 100% Nitrogen added (Farmer regular practice).

[0186] 2. A. brasilense + Methylobacterium oryzae with 50% Nitrogen showed 14% increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0187] 3. A. brasilense + Methylobacterium nodulans with 50% nitrogen showed 28% increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0188] Example 9:

[0189] Pot-trial analysis of combinatorial synergistic effect of Methylobacterium phyllo sphaerae and / W. adhaesivum as plant- growth-promoting rhizobacteria

[0190] The composition comprising Methylobacterium phyllosphaerae DSM19779 and Methylobacterium adhaesivum DSM17169 was investigated and results showed increased plant growth and yield as depicted in Table 9.

[0191] Methylobacterium was known to contribute to plant growth via:

[0192] Production of Phytohormones: Indole-3-acetic acid (IAA): Promotes root elongation and branching.

[0193] Cytokinins: Stimulate cell division and shoot formation. ACC Deaminase Activity: Breaks down 1 -aminocyclopropane- 1 -carboxylate (ACC), the precursor of ethylene, reducing plant stress and improving root growth.

[0194] Biological Nitrogen Fixation: fix atmospheric nitrogen, though this trait varies by species and strain.

[0195] Methanol Utilization: Utilizes methanol emitted from plant stomata as a carbon source, forming a mutualistic relationship.

[0196] Induction of Systemic Resistance (ISR): Enhances plant defense against pathogens.

[0197] Phosphate Solubilization and Siderophore Production: Makes nutrients more bioavailable to plants.

[0198] Treatments:

[0199] 1. Control (no bacteria)

[0200] 2. Control + 50% Nitrogen

[0201] 3. M. adhaesivum + 50% Nitrogen

[0202] 4. M. adhaesivum +Azospirillum + 50% Nitrogen

[0203] 5. M. adhaesivum + Rhizobium + 50% Nitrogen

[0204] 6. M. adhaesivum + Bacillus sp + 50% Nitrogen

[0205] 7. Methylobacterium phyllo sphaerae + 50% Nitrogen

[0206] 8. Methylobacterium phyllosphaerae + Azospirillum + 50% Nitrogen

[0207] 9. Methylobacterium phyllosphaerae + Rhizobium+ 50% Nitrogen

[0208] 10. Methylobacterium phyllosphaerae + Bacillus sp+ 50% Nitrogen

[0209] 11. M. adhaesivum+ Methylobacterium phyllosphaerae

[0210] 12. M. adhaesivum+ Methylobacterium phyllosphaerae + 50% Nitrogen Using a consortium approach or comparing it with other well-known PGPRs like Azospirillum, Rhizobium, Bacillus helped benchmark effectiveness.

[0211] Table 9

[0212] Results:

[0213] 1. M. adhaesivum and Azospirillum with 50% nitrogen showed 56% increase in yield compared to control 100% Nitrogen added (Farmer regular practice). 2. M. phyllosphaerae and Bacillus sp with 50% Nitrogen showed 10% increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0214] 3. M. adhaesivum and M. phyllosphaerae with 50% Nitrogen showed 59 % increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0215] 4. M. adhaesivum and M. phyllosphaerae with 0% Nitrogen showed 45 % increase in yield when compared to control 100% Nitrogen added (Farmer regular practice). Example 10:

[0216] Pot-trial analysis to evaluate growth promotion effects of composition comprising of Methylobacterium longum DSM 23933, M. Brachythecii DSM 24105 and M. aerolatum DSM 19013 under controlled conditions:

[0217] Methanol metabolism: Efficiently consumes plant-emitted methanol, key for phyllosphere colonization

[0218] Phytohormone production produces IAA, cytokinins, and possibly gibberellins, which drive root / shoot growth

[0219] ACC deaminase activity: Methylobacterium spp helping reduce plant stress ethylene

[0220] Phosphate solubilization & siderophore production

[0221] Nitrogen fixation: PPFMs, boosting nutrient availability

[0222] Induced systemic resistance (ISR): to activate plant defense mechanisms

[0223] Treatments:

[0224] 1. Control

[0225] 2. Control 50%

[0226] 3. M. longum

[0227] 4. M. longum + 50% Nitrogen

[0228] 5. M. Aerolatum

[0229] 6. M. Aerolatum + 50% Nitrogen

[0230] 7. M. Brachythecii

[0231] 8. M. Brachythecii + 50% Nitrogen

[0232] 9. M. longum + M. Aerolatum + M. Brachythecii + 50% Nitrogen

[0233] Table 10

[0234] Results:

[0235] 1. Methylobacterium longum with 50% nitrogen showed 17.5% increase in yield compared to control 100% Nitrogen added (Farmer regular practice).

[0236] 2. Methylobacterium Aerolatum with 50% Nitrogen showed 58% increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0237] 3. Methylobacterium Brachythecii with 50% Nitrogen showed 48 % increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0238] 4. Methylobacterium longum + Methylobacterium Aerolatum + Methylobacterium Brachythecii with 50% N reduced resulted in 100 % increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0239] 5. Methylobacterium longum + Methylobacterium Aerolatum + Methylobacterium Brachythecii with 75% N reduced showed 86 % increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0240] Example 11:

[0241] Pot-trial analysis of combinatorial synergistic effect of Methylobacterium bullatum, Mb. Fujisawaense and Methylobacterium gossipiicola in Green gram:

[0242] Pot-trial analysis of combinatorial synergistic effect of Methylobacterium bullatum DSM 21893, Mb. Fujisawaense DSM 5686, Methylobacterium gossipiicola CCM 7572 as plant-growth-promoting rhizobacteria was investigated and results showed increased plant growth and yield. Treatments:

[0243] • Control

[0244] • Methylobacterium bullatum

[0245] • Methylobacterium bullatum +50% Nitrogen • Mb. Fujisawaense

[0246] • Mb. Fujisawaense +50% Nitrogen

[0247] • Methylobacterium gossipiicola

[0248] • Methylobacterium gossipiicola +50% Nitrogen

[0249] • Methylobacterium bullatum + Mb. Fujisawaense +Methylobacterium gossipiicola

[0250] • Methylobacterium bullatum + Mb. Fujisawaense +Methylobacterium gossipiicola +50% Nitrogen

[0251] The results are depicted in Table 11.

[0252] Table 11

[0253]

[0254] Results: 1. Methylobacterium bullatum with 50% nitrogen showed 25% increase in yield compared to control 100% Nitrogen added (Farmer regular practice).

[0255] 2. Mb. Fujisawaense with 50% Nitrogen showed 30% increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0256] 3. Methylobacterium gossipiicola with 50% Nitrogen showed 23 % increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0257] 4. Methylobacterium bullatum + Mb. Fujisawaense + Methylobacterium gossipiicola with 0% N reduced showed 87 % increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0258] 5. Methylobacterium bullatum + Mb. Fujisawaense + Methylobacterium gossipiicola with 50% N reduced showed 100 % increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0259] Example 12:

[0260] Pot-trial analysis of combinatorial synergistic effect of Methylobacterium phyllostachyos NBRC 105206, Methylobacterium planium, Methylobacterium populi ATCC BAA-705:

[0261] Treatments:

[0262] • Control

[0263] • Methylobacterium phyllostachyos

[0264] • Methylobacterium phyllostachyos + 50% nitrogen

[0265] • Methylobacterium planium

[0266] • Methylobacterium planium + 50% Nitrogen

[0267] • Methylobacterium populi

[0268] • Methylobacterium populi + 50% Nitrogen

[0269] • Methylobacterium phyllostachyos + Methylobacterium planium + Methylobacterium populi • Methylobacterium phyllostachyos + Methylobacterium planium +

[0270] Methylobacterium populi

[0271] Table 12

[0272] Results:

[0273] 1. Methylobacterium phyllostachyos with 50% nitrogen showed 61% increase in yield compared to control 100% Nitrogen added (Farmer regular practice). 2. Methylobacterium planium with 50% Nitrogen showed 64% increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0274] 3. Methylobacterium populi with 50% Nitrogen showed 68 % increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0275] 4. Methylobacterium phyllostachyos + Methylobacterium planium+ Methylobacterium populi with 0% N reduced showed 84.5 % increase in yield when compared to control 100% Nitrogen added (Farmer regular practice). 5. Methylobacterium phyllostachyos + Methylobacterium planium+ Methylobacterium populi with 50% N reduced showed 100 % increase in yield when compared to control 100% Nitrogen added (Farmer regular practice).

[0276] Although the invention has been described in detail in the foregoing for the purpose of illustration, it is to be understood that such detail is solely for that purpose and that variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention except as it may be limited by the claims.

Claims

We claim,1. A microbial based composition comprising at least two Methylobacterium species with or without other microbes selected from endophytes, ectophytes, ephiphytes, Rhizospheric and / or free living; other materials selected from macro nutrients and micronutrients; simple and complex carbohydrates including and not limited to monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides; nitrogen containing compounds including enzymes / proteins / peptides / amino acids, vitamins, organic acids; minerals; secondary metabolites including flavonoids, aromatic, phenolics compounds; alcohols; lipids; growth hormones; polyphenolic compounds; pesticides, adjuvants, preservatives, for agricultural applications as bio stimulant, biofertilizer, plant growth promotion, nitrogen fixation, increased nutrient use efficiency, plant protection improved plant health, abiotic and biotic stress tolerances, increased crop productivity and yields, increased produce quality, increased produce nutrient density, increased shelf life of produce and reduced greenhouse gas emissions.

2. The composition as claimed in claim 1, wherein the composition comprises at least two Methylobacterium species selected from Methylobacterium symbioticum, Methylobacterium extorquens, Methylobacterium mesophilicum, Methylobacterium aquaticum. Methylobacterium fujisawaense, Methylobacterium gnaphalii. Methylobacterium hispanicum,Methylobacterium oiyzae. Methylobacterium phyllosphaerae,Methylobacterium phyllostachyos, Methylobacterium radiotolerans,Methylobacterium Populi, Methylobacterium adhaesivam,Methylobacterium longum, Methylobacterium brachythecii,Methylobacterium aerolatum, Methylobacterium bullatum,Methylobacterium gossipiicola, Methylobacterium phyllostachyos,Methylobacterium planium.

3. The composition as claimed in claim 1, wherein the other microbes are selected from aerobic / anaerobic bacteria, fungi, algae, archaea, epiphytes, endophytes, ectophytes, endosymbiont, rhizosphere, free-living, diazotrophic, non-diazotrophic microbe endo, ecto mycorrhiza, microbes including Rhizobium leguminosorum, Azospirillum, Azotobacter, Pseudomonas, Bacillus, Streptomyces, Bradyrhizobium japonicum, Nitrospira inopinata, Rhodopseudomonas palustris, Sphingomonas, Nitrosopumilus maritimus, Methylobacterium, Rhodobacter sphaeroides, Reyranella massiliensis, Alcaligene, Saccharomyces cerevisiae, Saccharomyces lactis, Trichoderma, Brevibacterium, Kluyveromyces lactis, Epichloe typhina, Enterococcus, Corynebacterium, Arthobacter, Pichia, Zymomonas, Saccharomyces carlsbergensis, Salmonella, Zymomonas, Rhodacoccus, Escherichia (e.g., E. Coli), Hansenula, Firmicutes, Rubrivivax, Dinoroseobacter shibae, Beijerinckiaindica. Ralstonia eutropha / Cupriavidus necator, Methyloversatilis, Nigrospora oryzae, Candida, Reyranella massiliensis, Novosphingobium aromaticivorans, Microbacterium, Acidovorax, Bordetella, Phomopsis liquidambaris, Nitrosopumilus maritimus, Ralstonia eutrophus / Cupriavidus necator, Nitrospira inopinata, Nigrospora oryzae, Rhodopseudomonas palustris, Periconia spp, Paenibacillus beijingensis, Thiobacillus sp, Sinorhizobium meliloti, Methyloversatilissp, Reyranella massiliensis, Cyanobacteria, Rhodobacter capsulatus, Epichloetyphina, Metallosphaera, Sulfolobus, Archaeoglobus, Cenarchaeum spp, Clostridium autoethanogenum, Xanthobacter flavus, Oligotropha carboxidovorans, Acidithiobacillus thiooxidans, Desulfobacter hydrogenophilus, Thiomicrospira denitrificans.

4. The composition as claimed in claim 1, wherein the nitrogen containing compounds are selected from proteins including but not limited to protein hydrolysate, protein isolates and / or amino acids and or enzymes including but not limited to phenyl alanine, amino lyase; coenzymes including but not limited to NAD+, NADP+ and adenosine triphosphate (ATP); coenzymes, such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD),thiamine pyrophosphate(TPP), and tetrahydrofolate (THF), derived from vitamins; Vitamins including but not only limited to a tocopherol and / or vitamin-D and / or vitamin K and / or and / or vitamin B and or Ascorbic acid and the like in an amount of 0.001 % to 5 % by weight of the composition.

5. The composition as claimed in claim 1, wherein the macro nutrients and micro nutrients are selected from primary, secondary, micro and trace elements including but not limited to nitrogen, phosphate, potash, poly phosphates, sulphates and their salts and / or pesticides and / or antioxidant pathway inducers (like coumaric acid and / or cinnamic acid and / or salicylic acid etc.) in an amount of 0.01 to 50 %.

6. The composition as claimed in claim 1, wherein the minerals are selected from but not limited to Potassium, Calcium, Magnesium, Scandium, Titanium, Vanadium, Molybdenum, Iron, Cobalt, Nickel, Copper, Zinc, Iodine selenium, and other trace elements etc. in an amount of 0.01 % to 50 % by weight of the composition.

7. The composition as claimed in claim 1, wherein the carbohydrates are selected from monosaccharides, disaccharides, trisaccharides, oligosaccharides, poly saccharides, Carboxy methyl cellulose, sugars and / or sugar alcohols including but not limited to mannitol, sorbitol, erythritol, laminarin fucoidan, chitin, chitosan, alginic acid etc. and / or carbohydrate containing substances etc., in an amount of 0.01 % to 50 %.

8. The composition as claimed in claim 1, wherein the alcohols are selected from but not limited to monohydric alcohols including but not limited to ethanol and / or methanol in an amount of 0.01 to 30 %.

9. The composition as claimed in claim 1, wherein the lipids are selected from but not limited to monoglycerides and / or diglycerides and / or triglycerides and / or phospholipids in an amount of 0.01 % to 25 %.

10. The composition as claimed in claim 1, wherein the hormone and / or hormone mimic substances are selected from but not limited to pesticides selected from but not limited to Glyphosate, azoxystrobin, fludioxonil present in non inhibitory lower concentration.

11. The composition as claimed in claim 1 , wherein the polyphenolic compounds are selected from but not limited to Tannins in an amount of 0.01 to 50 %.

12. The composition as claimed in claim 1, wherein the composition may comprise a surfactant selected from, but not limited to, anionic and nonionic and cationic surfactants, tween-20, tween -80 and the like.

13. The composition as claimed in claim 1, wherein the composition may comprise binder and trace metals selected from Talc, Bentonite, peat, lignite, vermiculite, charcoal, and press mud and the like.

14. The composition as claimed in claim 1, wherein composition further comprises other microbes selected from aerobic / anaerobic bacteria, fungi, algae, archaea, epiphytes, endophytes, endosymbiont, rhizosphere, free- living, diazotrophic, non-diazotrophic microbe endo, ecto mycorrhiza, etc as biostimulant, biofertilizer for improving plant growth and productivity.

15. The composition as claimed in claim 1, wherein the composition further comprises of nutrients selected from NPK, boron, calcium, silicon, magnesium, nickel, cobalt, manganese, iron, zinc, copper, molybdenum, and the like.

16. The composition as claimed in claim 1, wherein the composition further comprises compounds selected from pesticides, organic acids, aromatic compounds, phenolics, proteins, flavonoids, peptides, amino acids, growth hormones, adjuvants, preservatives and the like.

17. A process for preparation of the microbial based composition as claimed in claim 1, wherein the said process comprises the steps of; i. Growing the microorganisms separately; ii. Checking the compatibility of the different microorganisms; and iii. Mixing the microorganisms after adding binder and trace metals in the formulated materials such as Talc, Bentonite, peat, lignite, vermiculite, charcoal, and press mud.

18. The composition as claimed in any one of the preceding claims, wherein the said compositions are useful for enhancing Nitrogen fixation, phosphateaccumulation and Phosphate solubilization, potash, silica, calcium, magnesium, sulphur and zinc solubilization, and increased production of siderophore.

19. The composition as claimed in any one of the preceding claims, wherein said compositions are useful for improving plant immunity by providing biotic and abiotic stress tolerance and protection against disease and insect attack.

20. The composition as claimed in any one of the preceding claims, wherein said compositions are useful for enhancing plant chlorophyll content and increasing photosynthesis efficiency, due to the increased nitrogen supply and uptake.

21. The composition as claimed in any one of the preceding claims, wherein said compositions are useful for application for crops such as and not limiting to field crops, cash crops cereals, pulses, fruits, vegetables, flowers, leafy vegetables, aromatic crops, biomass crops, and the like.

22. The composition as claimed in any one of the preceding claims, wherein the composition may be in the form of solid powder, granule, paste, liquid, gel, pellet, pills, capsule or tablet and the like.

23. The composition as claimed in any one of the preceding claims, wherein said composition may be applied by sprinkler application, sprayer application or drip application, more preferably by sprayer application such as foliar sprays, sprays to be applied to plants shoots and the like.

24. A method for improving plant growth and productivity comprising applying the present microbial based composition to the plant in an effective amount.

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