Biofertilizer composition for plant growth promotion

Abstract

A novel strain of bacteria of the species Pseudomonas azotoformans, designated as strain AL-336, is useful for enhancing plant growth and development. Methods of promoting the growth of plants, including agriculturally relevant crops, using biofertilizer compositions comprising Pseudomonas azotoformans strain AL-336 are provided.

Description

BIOFERTILIZER COMPOSITION FOR PLANT GROWTH PROMOTIONField

[0001] The present application is directed to a composition for use in promoting plant growth. More specifically, the present application is directed to a new strain of Pseudomonas azotoformans, designated as strain AL-336, and a biofertilizer composition comprising the strain, for use in promoting plant growth.Background

[0002] Microorganisms such as bacteria which colonize root surfaces (the rhizosphere) or internal plant tissues (the endosphere) can exert beneficial effects on plant growth. These beneficial effects may be realized by several mechanisms. For example, microbes can increase the availability to the plant of micro- or macro-nutrients such as nitrogen (N), phosphorus (P), especially in the form of phosphate, potassium (K), manganese (Mn), calcium (Ca), zinc (Zn), copper (Cu), iron (Fe) and silicon (Si) through biofertilization. Microbes can improve root health by stimulating production by plants of plant hormones which encourage root growth, by producing such plant hormones themselves, or by metabolizing and eliminating toxic compounds in the soil such as pesticides or heavy metals. In addition, microbes can contribute to reducing stresses on the plant caused by unfavourable conditions such as drought, high temperature or high salt concentration in soil or by the presence of pathogens. For example, beneficial bacteria can produce and release secondary metabolites that are antagonistic to plant pathogens (antibiosis), can compete with pathogenic species for nutrients and niches, and can induce systemic resistance in the plant to pathogen invasion.

[0003] Species of Pseudomonas bacteria, including Pseudomonas azotoformans and other species within the Pseudomonas fluorescens subgroup have been shown to be beneficial to plants and show biofertilizer / biostimulant properties. Such properties have been described for strains of P. azotoformans, alone and in combination with other agents, in US Patent No. 8,796,179 and by Phour, M. and Sindhu, S.S., (2020) “Amelioration of salinity stress and growth stimulation of mustard (Brassica juncea L.) by salt-tolerant Pseudomonas species”. Applied Soil Ecology, 149, p.103518; Bahena, M.H.R., Salazar, S., Velazquez, E., Laguerre, G. and Peix, A., (2015) “Characterization of phosphate solubilizing rhizobacteria associated with pea (Pisum sativum L.) isolated from two agricultural soils”. Symbiosis, 67(1-3), pp.33- 41 ; Sarin, P. and Riddech, N., (2018) “Effects of Agricultural Residues as Carriers for Biofertilizer Production to Promote Tomato Growth in Saline Soil”. CHIANG MAI JOURNAL OF SCIENCE, 45(4), pp.1699-1712; Saha, M., Maurya, B.R., Meena, V.S., Bahadur, I. and Kumar, A., (2016) “Identification and characterization of potassium solubilizing bacteria(KSB) from Indo-Gangetic Plains of India”. Biocatalysis and Agricultural Biotechnology, 7, pp.202-209; Ma, Y., Rajkumar, M., Moreno, A., Zhang, C. and Freitas, H., (2017) “Serpentine endophytic bacterium Pseudomonas azotoformans ASS1 accelerates phytoremediation of soil metals under drought stress”. Chemosphere, 185, pp.75-85; Nie, Z.J., Hang, B.J., Cai, S., Xie, X.T., He, J. and Li, S.P., (2011) “Degradation of cyhalofop- butyl (CyB) by Pseudomonas azotoformans strain QDZ-1 and cloning of a novel gene encoding CyB-hydrolyzing esterase”. Journal of Agricultural and Food Chemistry, 59(11), pp.6040-6046; Lee, Chan-Jung, Ji-Won Moon, Young-Mi Yoo, Ju-Yeon Han, Jong-Chun Cheong, and Won-Sik Kong. (2015) "Optimum cultivation conditions for mass production of antagonistic bacterium Pseudomonas azotoformans HC5 effective in antagonistic of brown blotch disease caused by Pseudomonas tolaasii." Journal of Mushrooms 13, no. 2: 97-102; Sang, M.K., Kim, E.N., Han, G.D., Kwack, M.S., Jeun, Y.C. and Kim, K.D., (2014) “Priming- mediated systemic resistance in cucumber induced by Pseudomonas azotoformans GC-B19 and Paenibacillus elgii MMB22 against Colletotrichum orbiculare”. Phytopathology, 104(8), pp.834-842; Banerjee, S., Singh, S., Pandey, S., Bhandari, M.S., Pandey, A. and Giri, K., (2020) “Biocontrol potential of Pseudomonas azotoformans, Serratia marcescens and Trichoderma virens against Fusarium wilt of Dalbergia sissoo". Forest Pathology, 50(2), p.e12581 ; Fang, Y., Wu, L., Chen, G. and Feng, G., (2016) “Complete genome sequence of Pseudomonas azotoformans S4, a potential biocontrol bacterium”. Journal of Biotechnology, 227, pp.25-26; and Mustafa, S., Kabir, S., Shabbir, U. and Batool, R., (2019) “Plant growth promoting rhizobacteria in sustainable agriculture: from theoretical to pragmatic approach”. Symbiosis, 78(2), pp.115-123.

[0004] However, the discovery of new strains of bacteria showing beneficial properties for promoting the growth of plants, including but not limited to agriculturally relevant crop plants, and methods of using such bacterial strains, are desirable.Summary

[0005] In one aspect, the present application provides an isolated novel strain of bacteria of the species Pseudomonas azotoformans, herein designated as strain AL-336, deposited on April 26, 2023 with the International Depository Authority of Canada (IDAC), National Microbiology Laboratory of Canada, 1015 Arlington Street, Winnipeg, Manitoba R3E 3R2, under accession number 260423-01 .

[0006] In another aspect, the present application provides a biofertilizer composition comprising Pseudomonas azotoformans strain AL-336 and an agriculturally acceptable carrier. Another aspect of the present application provides a kit for preparation of a biofertilizer composition as described herein, the kit comprising a culture of Pseudomonasazotoformans strain AL-336 and instructions for preparation of a biofertilizer composition as described herein.

[0007] In a further aspect, the present application provides the use of Pseudomonas azotoformans strain AL-336 or of a biofertilizer composition as described herein to promote plant growth, enhance early germination and plant emergence, reduce environmental stress, including but not limited to reducing transplant shock and protecting plants against salt stress, or increase crop yield under greenhouse and field conditions. Yet another aspect of the present application provides a method of promoting plant growth, enhancing early germination and plant emergence, reducing environmental stress, including but not limited to reducing transplant shock and protecting plants against salt stress, or increasing crop yield under greenhouse and field conditions, comprising applying Pseudomonas azotoformans strain AL-336 or a biofertilizer composition as described herein to a plant or a portion thereof or to a seed thereof, or to a substrate in or on which the plant or seed is planted or transplanted.Brief Description of the Drawings

[0008] Further features of the present invention will become apparent from the following written description and the accompanying figures, in which:

[0009] Figure 1 is a diagram illustrating the evolutionary relationship between Pseudomonas azotoformans strain AL-336 and other Pseudomonas species;

[0010] Figure 2A is a series of photographs (cropped for clarity) illustrating detection of AL-336 DNA (indicated by boxes) in DNA extracted from surface sterilized (SS) corn leaves (panel A) and from non-surface sterilized (NS) corn leaves (panel B) seven days after application of a biofertilizer composition as described herein to corn plants. In each case, DNA was extracted from five corn plants (R1-R5). C+: positive control. C-: negative control;

[0011] Figure 2B is a series of photographs (cropped for clarity) illustrating detection of AL-336 DNA (indicated by a box) in DNA extracted from surface sterilized tomato roots (R1- R5) and shoots (S1-S5) five weeks after drench application of a biofertilizer composition as described herein to tomato plants at planting. In each case, DNA was extracted from five tomato plants. C+: positive control. C-: negative control;

[0012] Figure 3A is a photograph of untreated wheat plants (control) or wheat plants treated with an embodiment of a biofertilizer composition as described herein (Biofertilizer Composition A) taken 7 days after germination and growth in paper towel pouches;

[0013] Figure 3B is a chart comparing the length of untreated and treated wheat plants grown as described for Figure 3A;

[0014] Figure 3C is a chart comparing the dry biomass of untreated and treated wheat plants grown as described for Figure 3A;

[0015] Figure 4 is a photograph of untreated wheat plants (control) or of wheat plants treated with an embodiment of a biofertilizer composition as described herein (Biofertilizer Composition B) taken 21 days after planting;

[0016] Figure 5A is a photograph taken 40 days after planting of untreated (control) tomato plants grown in vermiculite;

[0017] Figure 5B is a photograph taken 40 days after planting of tomato plants grown as described for Figure 5A from seeds treated with an embodiment of a biofertilizer composition as described herein (Biofertilizer Composition A);

[0018] Figure 5C is a photograph of the tomato plants of Figures 5A (control) and 5B (Biofertilizer Composition A) taken after harvest 40 days after planting;

[0019] Figure 6A is a chart comparing the shoot length of untreated and treated tomato plants grown and harvested as described for Figures 5A-C;

[0020] Figure 6B is a chart comparing the dry biomass of untreated and treated tomato plants grown and harvested as described for Figures 5A-C;

[0021] Figure 7 is a photograph taken 14 days after planting of mini-green (mixed lettuce) plants grown in vermiculite from untreated (control) seeds or from seeds treated with an embodiment of a biofertilizer composition as described herein (Biofertilizer Composition A);

[0022] Figure 8A is a chart comparing the plant emergence over time of untreated and treated mini-green (mixed lettuce) plants grown as described for Figure 7;

[0023] Figure 8B is a chart comparing the root length of untreated and treated mini-green (mixed lettuce) plants grown as described for Figure 7 and harvested 21 days after planting;

[0024] Figure 8C is a chart comparing the shoot length of untreated and treated mini-green (mixed lettuce) plants grown as described for Figure 7 and harvested 21 days after planting;

[0025] Figure 8D is a chart comparing the dry biomass of untreated and treated mini-green (mixed lettuce) plants grown as described for Figure 7 and harvested 21 days after planting;

[0026] Figure 9A is a photograph taken at 21 days after planting of lettuce plants grown in untreated (control) commercial potting mix, and treated with a foliar application of water at 10 days after planting;

[0027] Figure 9B is a photograph taken at 21 days after planting of lettuce plants grown in commercial potting mix treated with an embodiment of a biofertilizer composition as described herein (Biofertilizer Composition C), and treated with a foliar application of Biofertilizer Composition C at 10 days after planting;

[0028] Figure 9C is a photograph taken at 45 days after initial planting (three weeks after transplantation) of the lettuce plants of Figure 9A transplanted at 21 days after planting and grown for three weeks with weekly fertilization;

[0029] Figure 9D is a photograph taken at 45 days after initial planting (three weeks after transplantation) of the lettuce plants of Figure 9B transplanted at 21 days after planting and grown for three weeks with weekly fertilization;

[0030] Figure 10A is a chart comparing the number of leaves of untreated and treated lettuce plants grown as described for Figures 9A-D and harvested 45 days after initial planting;

[0031] Figure 10B is a chart comparing the fresh biomass of untreated and treated lettuce plants grown as described for Figures 9A-D and harvested 45 days after initial planting;

[0032] Figure 10C is a chart comparing the dry biomass of untreated and treated lettuce plants grown as described for Figures 9A-D and harvested 45 days after initial planting;

[0033] Figure 11A is a photograph taken at 22 days after planting of tomato plants grown in untreated (control) soil or in soil treated with an embodiment of a biofertilizer composition as described herein (Biofertilizer Composition A);

[0034] Figure 11 B is a photograph taken at 57 days after initial planting (35 days after transplantation) of the tomato plants of Figure 11 A transplanted at 22 days after planting;

[0035] Figure 12A is a chart comparing the chlorophyll content of untreated and treated tomato plants grown as described for Figures 11A-B and harvested 57 days after initial planting;

[0036] Figure 12B is a chart comparing the shoot length of untreated and treated tomato plants grown as described for Figures 11 A-B and harvested 57 days after initial planting;

[0037] Figure 12C is a chart comparing the dry biomass of untreated and treated tomato plants grown as described for Figures 11 A-B and harvested 57 days after initial planting;

[0038] Figure 13A is a photograph taken at 16 days after planting of cucumber plants grown in untreated (control) commercial potting mix;

[0039] Figure 13B is a photograph taken at 16 days after planting of cucumber plants grown in commercial potting mix treated with an embodiment of a biofertilizer composition as described herein (Biofertilizer Composition A);

[0040] Figure 13C is a photograph taken at 28 days after transplantation of the cucumber plants of Figures 13A and 13B transplanted at 17 days after initial planting;

[0041] Figure 13D is a photograph taken at 39 days after transplantation of the cucumber plants of Figure 13C;

[0042] Figure 14A is a chart comparing the number of flowers of untreated and treated cucumber plants grown as described for Figures 13A-D and harvested 43 days after transplantation;

[0043] Figure 14B is a chart comparing the shoot length of untreated and treated cucumber plants grown as described for Figures 13A-D and harvested 43 days after transplantation;

[0044] Figure 14C is a chart comparing the dry biomass of untreated and treated cucumber plants grown as described for Figures 13A-D and harvested 43 days after transplantation;

[0045] Figure 15A is a photograph taken 30 days after planting of wheat plants growing in potting mix in the absence of excess salt (negative control), in the presence of excess salt (positive control) or in the presence of excess salt and treated with an embodiment of a biofertilizer composition as described herein (Biofertilizer Composition A);

[0046] Figure 15B is a chart comparing the dry biomass of wheat plants grown as described for Figure 15A and harvested 36 days after planting; and

[0047] Figure 16 is a chart comparing the dry biomass of cucumber plants grown as described for Figure 15A and harvested 36 days after planting.Detailed Description

[0048] In one aspect, the present application provides an isolated novel strain of bacteria of the species Pseudomonas azotoformans, herein designated as strain AL-336, deposited on April 26, 2023 with the International Depository Authority of Canada (IDAC), National Microbiology Laboratory of Canada, 1015 Arlington Street, Winnipeg, Manitoba R3E 3R2, under accession number 260423-01 .

[0049] Pseudomonas azotoformans strain AL-336 is a plant endophyte Gram-negative bacterium and a member of the Pseudomonas fluorescens family. In at least one embodiment, strain AL-336 has been found to enhance one or more of early seed germination, early plant emergence, plant growth, environmental stress resistance, including but not limited to transplant stress resistance and saline stress resistance, and yield under field and greenhouse conditions of one or more agricultural crops belonging to plant families including but not limited to Poaceae (Gramineae), Solanaceae, Amaryllidaceae, Caryophyllaceae, Rosaceae, Compositae (Asteraceae), Cucurbitaceae, Brassicaceae, Apiaceae (Umbelliferae) and Fabaceae (Leguminosae). In addition, in at least one embodiment, strain AL-336 has been found to enhance root formation and growth of tree plantlets in plant nurseries. Furthermore, in at least one embodiment, strain AL-336 has been found to colonize plant tissues following application of the strain itself, or of a biofertilizer composition comprising the strain, to a seed, a plant, or a portion thereof.

[0050] In another aspect, the present application provides a biofertilizer composition comprising Pseudomonas azotoformans strain AL-336 and an agriculturally acceptable carrier. As used herein, the term "agriculturally acceptable" is intended to refer to carriers or components that are tolerable or beneficial to P. azotoformans strain AL-336 so as to maintain the viability of and / or cause no deleterious effects to P. azotoformans strain AL-336, and which do not typically cause untoward reactions or deleterious effects to a plant or crop being treated with such a carrier or components, or to a worker applying a composition including such a carrier or components to a plant or crop under normal agricultural conditions. Preferably, as used herein, the term "agriculturally acceptable" means approved by a regulatory agency of the federal or a state government for use in agricultural applications. Such agriculturally acceptable carriers and components are well known in the art.

[0051] In at least one embodiment, the biofertilizer composition contains about 1% to about 20% by weight of P. azotoformans strain AL-336. In at least one embodiment, the biofertilizer composition contains about 5% by weight of P. azotoformans strain AL-336. In at least one embodiment, the biofertilizer composition contains about 0.0025% to about 1% by weight of P. azotoformans strain AL-336. In at least one embodiment, the biofertilizer composition contains about 0.0025% to about 0.25% by weight of P. azotoformans strain AL-336. In at least one embodiment, the biofertilizer composition contains about 0.003% to about 0.3% by weight of P. azotoformans strain AL-336. In at least one embodiment, the biofertilizer composition contains about 0.0025% by weight of P. azotoformans strain AL- 336. In at least one embodiment, the biofertilizer composition contains about 1 x 108colony forming units (CFU)ZmL of P. azotoformans strain AL-336. In at least one embodiment, the biofertilizer composition contains about 1 x 107CFU / mL of P. azotoformans strain AL-336. In at least one embodiment, the biofertilizer composition contains about 1 x 106CFU / mL of P. azotoformans strain AL-336. In at least one embodiment, the biofertilizer composition contains about 1 x 105CFU / mL of P. azotoformans strain AL-336. In at least one embodiment, the biofertilizer composition contains about 1 x 108CFU / g of P. azotoformans strain AL-336. In at least one embodiment, the biofertilizer composition contains about 1 x 105CFU / g of P. azotoformans strain AL-336.

[0052] In at least one embodiment, the agriculturally acceptable carrier is a liquid carrier. In at least one embodiment, the liquid carrier comprises water. In at least one embodiment, the liquid carrier is an aqueous solution comprising agriculturally acceptable components as described herein. In at least one such embodiment, the liquid carrier can be a growth medium in which the culture of Pseudomonas azotoformans AL-336 can viably grow. In at least one embodiment, the agriculturally acceptable carrier is a solid carrier. In at least oneembodiment, the solid carrier is a powder. In at least one embodiment, the solid carrier includes granules comprising organic and / or inorganic agriculturally acceptable material.

[0053] In at least one embodiment, the biofertilizer composition contains other agriculturally acceptable components. In at least one embodiment, the biofertilizer composition comprises nutrients to support growth of the AL-336 strain. In at least one embodiment, the nutrients include but are not limited to sugars, lipids, amino acids, vitamins, minerals and combinations thereof. In at least one embodiment, the nutrients include but are not limited to yeast extract, enzymatic digest of casein, enzymatic digest of soybean, Lab-Lemco powder and peptone. In at least one embodiment, the biofertilizer composition contains about 0.002% to about 0.2% by weight of yeast extract. In at least one embodiment, the biofertilizer composition contains about 1% by weight of yeast extract. In at least one embodiment, the biofertilizer composition contains up to about 0.75% by weight of enzymatic digest of casein. In at least one embodiment, the biofertilizer composition contains about 0.017% to about 0.17% by weight of enzymatic digest of casein. In at least one embodiment, the biofertilizer composition contains about 0.085% by weight of enzymatic digest of casein. In at least one embodiment, the biofertilizer composition contains up to about 0.25% by weight of enzymatic digest of soybean. In at least one embodiment, the biofertilizer composition contains about 0.0003% to about 0.03% by weight of enzymatic digest of soybean. In at least one embodiment, the biofertilizer composition contains about 0.15% by weight of enzymatic digest of soybean. In at least one embodiment, the biofertilizer composition contains up to about 0.1% by weight of Lab-Lemco powder. In at least one embodiment, the biofertilizer composition contains up to about 0.5% by weight of peptone.

[0054] In at least one embodiment, the sugars include but are not limited to glucose and dextrose. In at least one embodiment, the biofertilizer composition contains about 0.025% to about 2.5% by weight of dextrose. In at least one embodiment, the biofertilizer composition contains about 7.5% by weight of dextrose. In at least one embodiment, the biofertilizer composition contains about 0.00025% to about 0.025% by weight of glucose. In at least one embodiment, the biofertilizer composition contains about 0.125% by weight of glucose.

[0055] In at least one embodiment, the amino acids include but are not limited to glutamic acid and glutamate salts. In at least one embodiment, the amino acids include monosodium glutamate. In at least one embodiment, the biofertilizer composition contains about 0.0904% to about 0.904% by weight of monosodium glutamate. In at least one embodiment, the biofertilizer composition contains about 1.55% by weight of monosodium glutamate.

[0056] In at least one embodiment, the minerals can be provided in the form of salts which can be readily ingested and utilized by bacteria without causing toxicity to the bacteria. In at least one embodiment, the minerals include but are not limited to salts containing cationsincluding but not limited to sodium, potassium, ammonium, magnesium, zinc, and iron, and containing anions including but not limited to phosphate, chloride and sulfate. In at least one embodiment, the minerals include but are not limited to sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), sodium chloride (NaCI), ammonium chloride (NH4CI), iron(ll) sulfate (FeSO4), magnesium sulfate (MgSO4) and zinc sulfate (ZnSO4).

[0057] In at least one embodiment, the biofertilizer composition contains about 0.0113% to about 1.13% by weight of disodium hydrogen phosphate (Na2HPO4). In at least one embodiment, the biofertilizer composition contains about 1.13% by weight of disodium hydrogen phosphate (Na2HPO4). In at least one embodiment, the biofertilizer composition contains about 0.003% to about 0.3% by weight of potassium dihydrogen phosphate (KH2PO4). In at least one embodiment, the biofertilizer composition contains about 0.3% by weight of potassium dihydrogen phosphate (KH2PO4). In at least one embodiment, the biofertilizer composition contains about 0.00025% to about 0.125% by weight of dipotassium hydrogen phosphate (K2HPO4).

[0058] In at least one embodiment, the biofertilizer composition contains about 0.00005% to about 0.025% by weight of sodium chloride (NaCI). In at least one embodiment, the biofertilizer composition contains about 0.05% to about 0.5% by weight of sodium chloride (NaCI). In at least one embodiment, the biofertilizer composition contains about 0.001 % to about 0.1% by weight of ammonium chloride (NH4CI). In at least one embodiment, the biofertilizer composition contains about 0.15% by weight of ammonium chloride (NH4CI). In at least one embodiment, the biofertilizer composition contains about 0.000004% to about 0.0004% by weight of iron(ll) sulfate heptahydrate (FeSO4-7H2O). In at least one embodiment, the biofertilizer composition contains about 0.0004% by weight of iron(ll) sulfate heptahydrate (FeSO4-7H2O). In at least one embodiment, the biofertilizer composition contains about 0.00049% to about 0.049% by weight of magnesium sulfate heptahydrate (MgSO4-7H2O). In at least one embodiment, the biofertilizer composition contains about 0.049% by weight of magnesium sulfate heptahydrate (MgSO4-7H2O). In at least one embodiment, the biofertilizer composition contains about 0.00005% to about 0.005% by weight of zinc sulfate heptahydrate (ZnSO4-7H2O). In at least one embodiment, the biofertilizer composition contains about 0.005% by weight of zinc sulfate heptahydrate (ZnSO4-7H2O).

[0059] In at least one embodiment, the biofertilizer composition contains an antifoam agent. In at least one embodiment, the antifoam agent is MCA 222 (Hydrite Chemical Co.). In at least one embodiment, the biofertilizer composition contains about 0.001% to about 0.1% byweight of MCA 222. In at least one embodiment, the biofertilizer composition contains about 0.1% by weight of MCA 222.

[0060] In at least one embodiment, the biofertilizer composition contains a preservative. In at least one embodiment, the preservative comprises one or more of ammonium dihydrogen phosphate (NH4H2PO4) and sodium nitrate (NaNO3). In at least one embodiment, the biofertilizer composition contains about 0.000002% to about 0.0002% by weight of ammonium dihydrogen phosphate (NH4H2PO4) and about 0.000013% to about 0.0013% by weight of sodium nitrate (NaNO3). In at least one embodiment, the biofertilizer composition contains about 0.0002% by weight of ammonium dihydrogen phosphate (NH4H2PO4) and about 0.0013% by weight of sodium nitrate (NaNO3).

[0061] In at least one embodiment, the biofertilizer composition contains about 0.01% to about 0.1% by weight of seaweed (Ascophyllum nodosum) extract. In at least one embodiment, the biofertilizer composition contains about 0.1% to about 0.2% by volume of seaweed (Ascophyllum nodosum) extract. In at least one embodiment, the biofertilizer composition contains about 0.05% (weight / volume) of seaweed (Ascophyllum nodosum) extract.

[0062] Various formulations of the biofertilizer composition have compositions as indicated in Table 1 below:Table 1 : Formulations of the present biofertilizer composition

[0063] In at least one embodiment, the biofertilizer composition can be prepared by mixing a culture of Pseudomonas azotoformans strain AL-336 with an agriculturally acceptable carrier. In at least one embodiment, the culture of Pseudomonas azotoformans strain AL-336 is a liquid culture. In at least one embodiment, the culture of Pseudomonas azotoformans strain AL-336 is a solid culture. In at least one embodiment, the solid culture is a powder culture. In at least one embodiment, the powder culture is a lyophilized culture. In at least one embodiment, the powder culture is a spray dried culture.

[0064] In at least one embodiment, the agriculturally acceptable carrier is a liquid carrier as described herein and a liquid or solid culture of Pseudomonas azotoformans strain AL-336 is mixed with the liquid carrier to form the biofertilizer composition as a liquid composition. In at least one embodiment, the liquid biofertilizer composition is suitable for application as described herein. In at least one embodiment, the liquid biofertilizer composition is a concentrated composition configured for further dilution with an agriculturally acceptable liquid carrier to form a diluted biofertilizer composition which is suitable for application as described herein. In at least one such embodiment, about 1 part of the concentrated composition can be mixed with about 9 to about 999 parts by volume of the liquid carrier to form the diluted biofertilizer composition. In at least one such embodiment, about 1 part of the concentrated composition can be mixed with about 49 to about 199 parts by volume of the liquid carrier to form the diluted biofertilizer composition. In at least one such embodiment, about 1 part of the concentrated composition can be mixed with about 99 parts by volume of the liquid carrier to form the diluted biofertilizer composition.

[0065] In at least one embodiment, the agriculturally acceptable carrier is a solid carrier as described herein and a liquid or solid culture of Pseudomonas azotoformans strain AL-336 is mixed with the solid carrier to form the biofertilizer composition as a solid composition. In at least one embodiment, the solid formulation is a lyophilized powder formulation. In at least one embodiment, the solid formulation is a spray-dried powder formulation. In at least one embodiment, the solid formulation is a granular formulation. Granular biofertilizer compositions containing Pseudomonas azotoformans strain AL-336 which are suitable foruse in agricultural applications can be prepared by mixing about 2% to about 16% (w / w) of a powder culture of Pseudomonas azotoformans strain AL-336 (lyophilized culture or spray dried culture), or about 1% to about 5% by weight of a biofertilizer composition as described herein with about 84% to about 99% by weight of one or more organic and / or inorganic agriculturally acceptable materials, including but not limited to one or more carriers, one or more binders and one or more detergents. Suitable carriers include but are not limited to up to about 60% by weight compost (containing up to about 60% moisture), up to about 25% by weight zeolite, up to about 95% by weight bentonite, up to about 3% by weight rice flour, up to about 15% by weight corn syrup solids, up to about 59% by weight mannitol, up to about 6% by weight sodium alginate and up to about 10% by weight starch. Suitable binders include but are not limited to up to about 10% by weight polyvinylpyrrolidone (PVP) or about 10% to about 15% by weight ammonium lignosulfonate. Suitable detergents include but are not limited to up to about 0.4% by weight Tween™ 80.

[0066] In light of the teaching herein, the person of skill in the art would be readily capable of identifying suitable formulations for the present biofertilizer composition and would readily be capable of identifying and using methods by which such solid formulations could be prepared. It will be clear to the skilled person that caution should be taken during the preparation of the present biofertilizer composition to avoid prolonged heating of the bioformulation or of the liquid or solid culture containing Pseudomonas azotoformans strain AL-336 at temperatures at or above 40°C.

[0067] In another aspect, the present application provides a kit for preparation of a biofertilizer composition as described herein, the kit comprising a culture of Pseudomonas azotoformans strain AL-336 and instructions for preparation of a biofertilizer composition as described herein. In at least one embodiment, the culture of Pseudomonas azotoformans strain AL-336 is a liquid culture. In at least one embodiment, the culture of Pseudomonas azotoformans strain AL-336 is a solid culture. In at least one embodiment, the solid culture is a powder culture. In at least one embodiment, the powder culture is a lyophilized powder. In at least one embodiment, the powder culture is a spray dried powder.

[0068] In at least one embodiment, the instructions for preparation of a biofertilizer composition include instructions for mixing the culture of Pseudomonas azotoformans strain AL-336 with an agriculturally acceptable carrier as described herein. In at least one embodiment, the agriculturally acceptable carrier is a liquid carrier. In at least one embodiment, the liquid carrier comprises water. In at least one embodiment, the liquid carrier is an aqueous solution comprising agriculturally acceptable components as described herein. In at least one embodiment, the kit further comprises the agriculturally acceptable carrier.

[0069] In at least one embodiment, the kit comprises a liquid culture of Pseudomonas azotoformans AL-336 and instructions for mixing the liquid culture with an agriculturally acceptable carrier prior to use to form a biofertilizer composition as described herein. In at least one embodiment, the liquid culture of Pseudomonas azotoformans AL-336 is a concentrated composition as described herein and the biofertilizer composition is a diluted biofertilizer composition as described herein. In at least one such embodiment, the carrier is water. In at least one such embodiment, the instructions include instructions to mix about 1 part by volume of the concentrated composition with about 9 to about 999 parts by volume of the carrier such that the concentration of Pseudomonas azotoformans strain AL-336 in the biofertilizer composition or diluted biofertilizer composition is about 1 / 10 to about 1 / 1000 of the concentration of Pseudomonas azotoformans strain AL-336 in the liquid culture or concentrated composition. In at least one such embodiment, the instructions include instructions to mix about 1 part by volume of the liquid culture or concentrated composition with about 49 to 199 parts by volume of the carrier such that the concentration of Pseudomonas azotoformans strain AL-336 in the biofertilizer composition or diluted biofertilizer composition is about 1 / 50 to about 1 / 200 of the concentration of Pseudomonas azotoformans strain AL-336 in the liquid culture or concentrated composition. In at least one such embodiment, the instructions include instructions to mix about 1 part by volume of the liquid culture or concentrated composition with about 99 parts by volume of the carrier such that the concentration of Pseudomonas azotoformans strain AL-336 in the biofertilizer composition or diluted biofertilizer composition is about 1 / 100 of the concentration of Pseudomonas azotoformans strain AL-336 in the liquid culture or concentrated composition.

[0070] Another aspect of the present application provides the use of Pseudomonas azotoformans strain AL-336 or of a biofertilizer composition comprising P. azotoformans strain AL-336 to enhance early germination and plant emergence. Another aspect of the present application provides the use of Pseudomonas azotoformans strain AL-336 or of a biofertilizer composition comprising P. azotoformans strain AL-336 to promote plant growth. Another aspect of the present application provides the use of Pseudomonas azotoformans strain AL-336 or of a biofertilizer composition comprising P. azotoformans strain AL-336 to reduce environmental stress, including but not limited to reducing transplant shock and protecting plants against salt stress. Another aspect of the present application provides the use of Pseudomonas azotoformans strain AL-336 or of a biofertilizer composition comprising P. azotoformans strain AL-336 to increase crop yield under greenhouse or field conditions.

[0071] Without being bound by theory, it has been found that Pseudomonas azotoformans strain AL-336 can act as a biofertilizer and can enhance the bioavailability of macro and micronutrients to a plant to which strain AL-336 has been applied. Strain AL-336 was foundto be capable of nitrogen fixation, phosphate solubilization, zinc solubilization, iron sequestration, so as to make nitrogen, phosphate, zinc and iron available to plants. Strain AL-336 was also found to produce the plant growth hormone indole-3-acetic acid via tryptophan-dependent and tryptophan-independent pathways, thereby acting to promote and regulate plant growth and development. In addition, strain AL-336 was found to be capable of protease activity, amylase activity and cellulase activity, producing simple amino acids and sugars from more complex proteins, starch and cellulose, so as to recycle plant debris in the soil and make nutrients available to plants and microbes.

[0072] In addition, Pseudomonas azotoformans strain AL-336 has been shown to exhibit 1 -aminocyclopropane-1 -carboxylic acid (ACC) deaminase activity. ACC deaminase catalyzes the degradation of ACC, a precursor of the plant stress hormone ethylene, into ammonia and a- keto butyrate, thus preventing formation of ethylene. Intracellular levels of ethylene change according to the growth stage of the plant and, more importantly, as a consequence of various environmental stresses. Ethylene is generally required in very low concentrations for normal plant growth and development (except during fruit ripening where ethylene levels are very high). However, the high levels of ethylene that plants produce when they are subject to either abiotic or biotic stresses are deleterious to plant growth and development and many of the plant senescence physiological responses are triggered by elevated levels of ethylene. A microbe showing ACC deaminase activity can therefore help host plants to reduce ethylene levels and grow under conditions of stress.

[0073] Furthermore, Pseudomonas azotoformans strain AL-336 has been shown to systemically colonize plants after application to seeds or plant tissues, including but not limited to roots, leaves and stems, under both laboratory and field conditions. Thus, strain AL-336 is believed to be an endophyte, which can survive on the surface of plants and systemically colonize internal plant tissues upon application to the plant. Bacterial endophytes are better protected from environmental conditions than non-endophytic bacteria or fungi which instead colonize plant rhizosphere, soil, or root surfaces. Thus, endophytes have been proposed to provide more persistent benefits to the host plant than non- endophytic microbes, formulations of which have been demonstrated to show inconsistent field performance.

[0074] Thus, a further aspect of the present application provides a method of enhancing early germination, promoting plant growth, reducing environmental stress, including but not limited to reducing transplant shock and protecting plants against salt stress, or increasing crop yield under greenhouse or field conditions, comprising applying Pseudomonas azotoformans strain AL-336 or of a biofertilizer composition comprising P. azotoformans strain AL-336 to a plant or a portion thereof.

[0075] The biofertilizer composition can be applied by any known method and at any stage in the plant lifecycle at which application of the biofertilizer composition will be effective to enhance early germination and plant emergence, promote plant growth, reduce environmental stress, including but not limited to reducing transplant shock and protecting plants against salt stress, or increase crop yield under greenhouse or field conditions as will be understood by one skilled in the art. In at least one embodiment, the biofertilizer composition can be applied to one or more seeds prior to, concurrently with or after germination. In at least one embodiment, the biofertilizer composition can be applied to the plant or to one or more portions thereof, including but not limited to roots, leaves, stems, flowers, buds, fruits, or any other portion of the plant. In at least one embodiment, the biofertilizer composition can be applied to a substrate prior to, concurrently with, or after planting one or more seeds or transplanting one or more plants in the substrate. As used herein, the term “substrate” is intended to mean a medium or base in or on which seeds can be germinated or plants can be planted or grown, and includes but is not limited to soil, potting mix, compost, vermiculite and other substrates known in the art. In at least one embodiment, the biofertilizer composition can be applied by coating, spraying, sprenching, soaking, drenching, or immersing the one or more seeds, plants or portions thereof. In at least one embodiment, the biofertilizer composition can be applied by broadcasting, spraying, drenching, or soaking a substrate prior to planting seeds or plants in or on the substrate. In at least one embodiment, the biofertilizer composition can be applied to seeds or plants in the field. In at least one embodiment, the biofertilizer composition can be applied to seeds or plants under cultivation, including but not limited to seeds or plants in cultivation in greenhouses or glasshouses.

[0076] As used herein, the terms “about” or “approximately” as applied to a numerical value or range of values are intended to mean that the recited values can vary within an acceptable degree of error for the quantity measured given the nature or precision of the measurements, such that the variation is considered in the art as equivalent to the recited values and provides the same function or result. For example, the degree of error can be indicated by the number of significant figures provided for the measurement, as is understood in the art, and includes but is not limited to a variation of ±1 in the most precise significant figure reported for the measurement. Typical exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" can mean values that are within an order of magnitude, preferably within 5- fold and more preferably within 2-fold of a given value. Numerical quantities given herein areapproximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

[0077] As used herein, the term “substantially” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is “substantially” in a given position including but not limited to vertical, horizontal, or adjacent to or aligned with another object, would mean that the object is either completely in that position or nearly completely in that position. The exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking, the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained.

[0078] The use of “substantially” is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result. For example, a composition that is “substantially free of’ an ingredient or element would either completely lack that ingredient or element, or so nearly completely lack that ingredient or element that the effect would be the same as if it completely lacked that ingredient or element. In other words, a composition that is “substantially free of’ an ingredient or element may still actually contain such item as long as there is no measurable or significant effect thereof.

[0079] As used herein, terms indicating relative direction or orientation, including but not limited to “upper”, “lower”, “top”, “bottom”, “vertical”, “horizontal”, “outer”, “inner”, “front”, “back”, and the like, are intended to facilitate description of the present invention by indicating relative orientation or direction in usual use, and are not intended to limit the scope of the present invention in any way to such orientations or directions.

[0080] As used herein and unless otherwise indicated, the terms “a” and “an” are intended to include both plural and singular forms and can be interpreted to mean “one or more”.EXAMPLES

[0081] Other features of the present invention will become apparent from the following nonlimiting examples which illustrate, by way of example, the principles of the invention.Example 1 : Isolation and identification of Pseudomonas azotoformans strain AL-336Corn sampling and sap extraction

[0082] Pseudomonas azotoformans strain AL-336 was isolated from corn sap as described by Ali et al, Environmental Sustainability (2018), 1 : 341-355. A total of thirty-four corn farms from Canada and United States were selected to collect samples. All corn plants were collected at the rapid growth phase (i.e. V10 stage) from each of the participating farms. A 10-cm long segment of corn stem was cut approximately 30 cm above the ground andstored at 4°C for 24 h. Ten plants (10 stem segments) were sampled from each farm and each stem segment was processed separately. The stem segments were surface sterilized as follows: washed for 3 min with tap water, then for 3 min with 70% ethanol, followed by a 3-min immersion in 1% commercial bleach, and finally rinsed three times with sterile water for 1 min each. To ensure that the surface sterilization process was successful, a 100 pL aliquot of the final rinse water was plated onto nutrient agar (NA) and incubated at 30°C for 3 days. If no bacterial growth was found, the sterile corn stem segment was transferred to a sterile plastic bag, and the sap was collected by crushing the stems using a mechanical device, (Engenho Para Cana B60, Botini®, Industria Brasileira, Brazil), designed to extract sap from sugar cane stalks.Corn sap plating and enumerating bacterial endophytes

[0083] All ten corn sap replicates from each farm were pooled and serially diluted (10°— 105) in sterile water, and 100 pl of each dilution was plated in duplicate onto Pseudomonas isolation agar (PIA Remel R454392; 1% pancreatic digest of gelatin, 1% peptide digest of animal tissue, 1.14% magnesium chloride, 1% potassium sulfate, 0.025% Irgasan™ (triclosan), 1.36% agar). Plates were incubated at 30°C for 72 h. After incubation, morphologically different colonies (based on size, shape, and color) were selected. Individual colonies were sub-cultured on PIA growth medium for further screening.

[0084] Strain AL-336 was found to produce characteristic pink colonies when plated on PIA or on nutrient agar (NA, 0.1% Lab-Lemco powder, 0.2% yeast extract, 0.5% peptone, 0.5% sodium chloride, 1.5% agar (Oxoid CM0003)), tryptic soy agar (TSA, 1.5% pancreatic digest of casein, 0.5% papaic digest of soybean, 0.5% sodium chloride, 1 .5% Agar (Difco 236920)), or Pseudomonas F agar (PF, 1% pancreatic digest of casein, 1% proteose peptone No. 3, 0.15% dipotassium phosphate, 0.15% magnesium sulfate, 1.5% Agar).Identification and taxonomic classification

[0085] Bacterial genomic DNA was extracted from the isolated strain AL-336 and sequenced. The sequences were annotated and assembled, and alignment analysis was performed using the BLAST (Basic Local Alignment Search Tool) algorithm against the National Center for Biotechnology Information (NCBI) data base, the European Molecular Biology Laboratory-European Bioinformatic Institute (EMBL-EBI) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Comparison of the contiguous sequence containing the gyrB and rrs (16S rRNA) concatenated gene sequences (SEQ ID NO:1 , Table 2) to the corresponding portion of reference genomes allowed identification of strain AL-336 as a strain of the species Pseudomonas azotoformans. The partial sequence of the 16S rRNA gene for strain AL-336 is identified in GenBank under accession number MG819457.Phylogenetic relationship to known Pseudomonas species

[0086] A phylogenetic tree was generated comparing the gyrB and rrs (16S rRNA) concatenated sequences from strain AL-336 to corresponding sequences from the reference strains listed in Table 2. Table 2: gyrB and rrs (16S rRNA) concatenated sequences from strain AL-336 and reference Pseudomonas strains

[0087] The evolutionary history was inferred using the neighbor-joining method, as described by Saitou et al., Molecular Biology and Evolution (1987), 4: 406-425. The optimal tree with the sum of branch length = 0.12197875 is shown in Figure 1. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test determined according to Felsenstein, Evolution (1985), 39: 783-791 (500 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method as described by Tamura et al., Proceedings of the National Academy of Sciences (USA) (2004), 101 : 11030-11035, and are in the units of the number of base substitutions per site. This analysis involved 15 nucleotide sequences. All positions with less than 85% site coverage were eliminated, i.e., fewer than 15% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There were a total of 1425 positions in the final dataset. Evolutionary analyses were conducted using the MEGA X software as described by Kumar et al., Molecular Biology and Evolution (2018). 35: 1547-1549.

[0088] Pathogenic species that are most closely related to Pseudomonas azotoformans are Pseudomonas aeruginosa, a known opportunistic human and animal pathogen, and Pseudomonas syringae, a plant pathogen. However, similarly to the results shown in Figure 1 , phylogenetic trees based on 16S rRNA, gyrB, rpoB and rpoD sequences of Pseudomonas species show that the P. fluorescens group, which includes P. azotoformans, is not closely clustered with the P. aeruginosa group or the P. syringae group. Notably, Pseudomonas azotoformans is classified in Risk Group 1 (no or low risk to individuals or the public community) by Health Canada and to the best knowledge of the present applicant, there are no reports in the literature of this bacterium acting as a human or animal pathogen.Example 2: Biochemical characterization of Pseudomonas azotoformans strain AL-336Nitrogen fixation

[0089] Pseudomonas azotoformans strain AL-336 showed growth in a nitrogen-free medium containing 20 g / l of malate as the carbon source; 0.2 g / L of K2HPO4; 0.2 g / L of NaCI; 0.2 g / L of MgSO4.7H2O; 0.1 g / L of K2SO4; 5.0 g / L of CaCO3; and 20 g / L of agar, adapted from Dbbereiner, J. (1988). Isolation and identification of root associated diazotrophs. Plant Soil 110, 207-212. In a separate experiment, wheat seeds were inoculated with a media- free suspension of strain AL-336 cells having a value of OD6oo of 0.5. The media-free suspension was formed by washing the AL-336 cells three times, each wash including centrifuging a culture of strain AL-336 cells, removing the supernatant and re-suspending the pellet in sterile water. The inoculated seeds were germinated and grown on a nitrogen-freesubstrate (sand washed with 2 M HCI and supplemented with nitrogen-free macro- and micronutrients) and the resulting plants grew better and for a longer time than plants from control seeds treated with water. These results indicate that strain AL-336 is capable of fixing atmospheric nitrogen.Solubilization of phosphorus and zinc

[0090] Pseudomonas azotoformans strain AL-336 was inoculated on agar plates supplemented with either insoluble phosphorous (Cas(PO4)2) or insoluble zinc (ZnO) and incubated as described by Ali et al, Environmental Sustainability (2018), 1 : 341-355. Clearing zones were observed around colonies of the bacterium, indicating that strain AL-336 can solubilize phosphorus and zinc, making those nutrients more available to plants.Siderophore production and sequestration of iron

[0091] Five microliters of a culture of Pseudomonas azotoformans strain AL-336 growing in King’s B (KB) medium overnight was spotted onto a chrome azurol S (CAS) agar plate and incubated at 30°C for 4-5 days, as described in Ali et al, Environmental Sustainability (2018), 1 : 341-355. CAS is an indicator dye that forms a blue-colored ferric complex containing iron. A positive reaction, signaled by a change in colour of the medium from blue to orange, was observed, indicating that iron was removed from the CAS dye, and that siderophores capable of sequestering iron were produced by strain AL-336.Indoleacetic acid (IAA) production

[0092] Indole acetic acid (IAA) production by Pseudomonas azotoformans strain AL-336 was measured as described in Ali et al, Environmental Sustainability (2018), 1 : 341-355. Pseudomonas azotoformans strain AL-336 produces IAA as confirmed by LC-MS analysis.1 -aminocyclopropane-1 -carboxylic acid (ACC) deaminase activity

[0093] ACC deaminase activity is tested according to the protocol described by Penrose and Glick (Physiologia Plantarum (2003), 118(1): 10-15) with a standard curve of 23 oc- ketobutyrate between 0.05 and 0.5 pM. Using this method, Pseudomonas azotoformans strain AL-336 was determined to show ACC deaminase activity.Carbon source usage

[0094] Strain AL-336 was tested for use of various carbon sources using the Biolog Gramnegative (GN) identification test panel. The results are shown in Table 3.Table 3: Carbon source utilization profile of AL-336Example 3: Preparation of biofertilizer compositions containing Pseudomonas azotoformans strain AL-336Bio fertilizer composition A

[0095] Sterile seed medium containing ammonium chloride (NH4CI, 1 g / L), potassium phosphate monobasic (KH2PO4, 3 g / L), sodium phosphate dibasic (Na2HPO4, 11.3 g / L), monosodium glutamate (9.04 g / L), dextrose monohydrate (30 g / L), iron sulfate heptahydrate (FeSO4-7H2O, 0.004 g / L), magnesium sulfate heptahydrate (MgSO4-7H2O, 0.49 g / L) and zinc sulfate heptahydrate (ZnSO4-7H2O, 0.05 g / L) in water is inoculated with Pseudomonas azotoformans strain AL-336 from an agar plate containing colonies of Pseudomonas azotoformans strain AL-336 grown from a frozen stock culture. The inoculated medium is incubated for 24 hours at 28°C with horizontal shaking at 250 rpm to provide a first seed culture.

[0096] Seed medium containing 0.1% v / v of an antifoam component (MCA 222, Hydrite Chemical Co.) is inoculated with 0.67%-1% v / v of the first seed culture. Fermentation is carried out for 12 to 14 hours at 28°C and 50% optical density (OD), with aeration at 1.2 L / min and horizontal shaking at 300 rpm, until the absorbance of the culture at 600 nm is at least 6, to provide a second seed culture. The pH is maintained at 6.8 during fermentation by addition of 2N phosphoric acid (H2PO4) and / or 4N potassium hydroxide (KOH) as required.

[0097] Fermentation medium containing yeast extract (10 g / L), monosodium glutamate (15.5 g / L), sodium phosphate dibasic (Na2HPO4, 11.3 g / L), ammonium chloride (NH4CI, 1.5 g / L), potassium phosphate monobasic (KH2PO4, 3 g / L), dextrose monohydrate (75 g / L), ironsulfate heptahydrate (FeSO4-7H2O, 0.004 g / L), magnesium sulfate heptahydrate (MgSO4-7H2O, 0.49 g / L) and zinc sulfate heptahydrate (ZnSO4-7H2O, 0.05 g / L) in water is mixed with 0.1% v / v of MCA 222, aseptically inoculated with 2% to 5% of the second seed culture and fermented for 72 h at 28°C, with aeration at 1 .2 L / min and horizontal shaking at 300 rpm. The pH is maintained at 6.8 during fermentation by addition of 2N phosphoric acid (H3PO4) and / or 4N potassium hydroxide (KOH) as required. Once fermentation is complete, up to 5% (v / v) of a preservative solution containing 0.04 g / L of ammonium phosphate monobasic (NH4H2PO4) and 0.26 g / L of sodium nitrate (NaNO3) in water is added to provide an embodiment of a biofertilizer composition according to the present application (herein referred to as Biofertilizer Composition A).Bio fertilizer composition B

[0098] Sterile seed medium containing 0.1 % Lab-Lemco powder, 0.2% yeast extract, 0.5% peptone, and 0.5% sodium chloride in water is inoculated with Pseudomonas azotoformans strain AL-336 from an agar plate containing colonies of Pseudomonas azotoformans strain AL-336 grown from a frozen stock culture. The inoculated medium is incubated for 24 hours at 30°C with horizontal shaking at 250 rpm to provide a first seed culture.

[0099] Seed medium is inoculated with 0.67%- 1% v / v of the first seed culture. The inoculated medium is incubated for 48 - 72 hours at 30°C with horizontal shaking at 250 rpm to provide an embodiment of a biofertilizer composition according to the present application (herein referred to as Biofertilizer Composition B).Bio fertilizer composition C

[0100] Biofertilizer composition B described above is mixed with 0.1% - 0.2% (V / V) or 0.05% - 0.1% (W / V) seaweed (Ascophyllum nodosum) extract to provide an embodiment of a biofertilizer composition according to the present application (herein referred to as Biofertilizer Composition C).Bio fertilizer composition D

[0101] Sterile seed medium containing 0.1 % Lab-Lemco powder, 0.2% yeast extract, 0.5% peptone, and 0.5% sodium chloride in water is inoculated with Pseudomonas azotoformans strain AL-336 from an agar plate containing colonies of Pseudomonas azotoformans strain AL-336 grown from a frozen stock culture. The inoculated medium is incubated for 48-72 hours at 30°C with horizontal shaking at 250 rpm to provide a second seed culture.

[0102] The second seed culture is freeze dried, and the powder is used to inoculate growing media (0.15% pancreatic digest of casein, 0.05% papaic digest of soybean, 0.05% sodium chloride). The inoculated medium is incubated for 48 - 72 hours at room temperature toprovide an embodiment of a biofertilizer composition according to the present application (herein referred to as Biofertilizer Composition D).Bio fertilizer composition E

[0103] The second seed culture used in the preparation of Biofertilizer Composition D is freeze dried, and the powder is used to inoculate growing media (0.75% pancreatic digest of casein, 0.25% papaic digest of soybean, 0.25% sodium chloride). The inoculated medium is incubated for 48 - 72 hours at room temperature to provide an embodiment of a biofertilizer composition according to the present application (herein referred to as Biofertilizer Composition E).Example 4: Plant tissue colonization by a biofertilizer composition containing Pseudomonas azotoformans strain AL-336

[0104] Two seeds of either corn (Dekalb 45-65) or tomato plants (Heinz 1439) were planted per pot (18D x 14.5W x 14.5H centimeters) prefilled with potting mix (Agro Mix™). Plants were watered and fertilized following standard practices. Treatments (Biofertilizer Composition B and water as a control) were applied as a drench on top of the seed at planting or as a foliar application sprayed onto the 3rdfully grown set of leaves to complete coverage at 4 weeks after planting for corn and 5 weeks after planting for tomatoes. Control plants were sprayed with water. Each treatment (biofertilizer and water control) was tested with 15 replications.

[0105] Plants were kept under controlled conditions of temperature and light (24°C - 14 h light, 21 °C - 10 h dark cycle). Plant foliage was sampled at 7, 10, and 14 days after treatment. Five pots (indicated as R1 to R5 in Figure 2A, for example) were sampled at each timepoint, and samples from each plant were divided into two groups: one group of plant foliage was surface sterilized as described in Example 1 ; the other group was kept as is. Each group was then sub-divided into three technical replications. Plant material was homogenized under sterile conditions using mortar and pestle, transferred to sterile microcentrifuge tubes, and stored at -20°C until further use. DNA was extracted from the homogenized plant material using a DNA extraction kit (Norgen, Biotek Corp.) as per manufacturer instructions. Detection of strain AL-336 was performed by polymerase chain reaction (PCR) using primers specific to strain AL-336 DNA. DNA extracted from a pure culture of strain AL-336 was used as positive control. Samples were run simultaneously.Representative results from treatment of corn and tomato plants are presented in Figures 2A and 2B. As seen from these results, strain AL-336 DNA was found in and on both surface sterilized and non-surface sterilized tissues from corn plants seven days after foliar application of the biofertilizer composition. In addition, strain AL-336 DNA was found in surface sterilized roots and shoots of tomato plants five weeks after application of thebiofertilizer composition as a drench at planting. Similar experiments carried out under field conditions confirmed colonization of plant tissues by strain AL-336 after application of a similar biofertilizer composition to seeds or plant tissues.Example 5: Plant growth promotion by biofertilizer compositions containing Pseudomonas azotoformans strain AL-336Statistical analysis

[0106] All statistical analysis was performed using the GraphPad Prism software package 6.04 (GraphPad Software, Inc., San Diego, CA, USA). The data for seedling root growth were analyzed through analysis of variance (ANO A). To identify which treatments were significantly different (P < 0.05), Tukey’s post hoc tests were performed.Wheat gnotobiotic assay

[0107] The ability of Biofertilizer Composition A to promote plant growth was tested on wheat under gnotobiotic conditions, using paper towels as a support material. Briefly, eighteen wheat (Triticum aestivunr, Pioneer, 2547) seeds were treated with Biofertilizer Composition A for one hour at room temperature. Control seeds were treated with water. Each paper towel pouch (7 x 5 in2) received 6 treated or control seeds with a total of three pouches per treatment. The paper towel pouches were then rolled separately, kept in a plastic container in an upright position, and incubated under controlled conditions of light and temperature for seven days. Root growth of the resulting plants was observed and the vigor indices of length and dry biomass were measured. The vigour index of length corresponds to the average root length of all plants plus the average shoot length of 18 plants, multiplied by the percentage of germinated seeds. The vigour index of dry biomass corresponds to the total dry weight of each treatment multiplied by the percentage of germinated seeds. As seen from the results shown in Figures 3A-C, treatment with Biofertilizer Composition A stimulates root growth and results in increased stem length and dry biomass in wheat.Wheat growth assay

[0108] The ability of Biofertilizer Composition B to promote plant growth was tested on wheat under controlled conditions, using potting mix (Miracle Grow™) as a growing material. Briefly, twenty wheat seeds (Triticum aestivum, Pioneer, 2547) were drenched with Biofertilizer Composition B at planting. Control seeds were treated with water. Seeds were planted in coffee cups containing 200 g of potting mix. Four seeds were planted per cup and each treatment had five replications. Cups were incubated under controlled conditions of light and temperature (16 hr light at 23°C / 8hr dark at 21°C) for 21 days. Root growth of the resulting plants was observed, and the dry biomass of the plants was measured. As seenfrom the results shown in Figure 4, treatment with Biofertilizer Composition B stimulates root growth in wheat.Tomato growth assay

[0109] Tomato seeds were germinated in the presence of Biofertilizer Composition A, and plants were grown using vermiculite as a substrate and were harvested 40 days after planting. At harvest, root length, plant height and dry biomass were measured. As seen from the results shown in Figures 5A to 5C, 6A and 6B, treatment with Biofertilizer Composition A increased root length, plant height and biomass in a statistically significant manner, as compared to control plants.Mini-greens growth assay

[0110] Mini greens seeds (lettuce Salad Bowl and Salad Bowl Trio, William Dam Seeds) were germinated in the presence of Biofertilizer Composition A, and plants were grown using vermiculite as a substrate and were harvested 21 days after planting. At harvest, root length, shoot length and dry biomass were measured. As seen in Figures 7 and 8A to 8D, treatment with Biofertilizer Composition A increased early plant emergence, root length, plant height or shoot length, and biomass in a statistically significant manner, as compared to control plants.Lettuce transplant assay

[0111] Lettuce (Buttercrunch lettuce, William Dam Seeds Ltd.) seeds were planted in commercial potting mix (MiracleGro™ Moisture Control) treated with freshly prepared Biofertilizer Composition C (100 mL of Biofertilizer Composition C per 200 g of potting mix) or in untreated potting mix (control). Ten days after planting, plants were treated with a foliar application of Biofertilizer Composition C or water (control). Treated and control plants were transplanted in soil collected from a research farm located in London, ON at 21 days after planting. Photographs of control (Figure 9A) and treated (Figure 9B) plants were taken immediately prior to transplantation. Plants were grown for three more weeks with weekly fertilization. At harvest (45 days after planting), photographs were again taken of the control (Figure 9C) and treated (Figure 9D) plants, and their number of leaves (Figure 10A), fresh biomass (Figure 10B) and dry biomass (Figure 10C) were recorded. As can be seen from Figures 9A to 9D and 10A to 10C, treatment with Biofertilizer Composition C resulted in increased lettuce growth and plants looked greener and healthier and had more leaves and higher fresh and dry weight as compared to the untreated control plants. No signs of disease were observed on any of the treated or control plants.Tomato transplant assay

[0112] Tomato seeds were planted in potting mix treated with Biofertilizer Composition A as described above for the lettuce transplant assay or in untreated potting mix (control). At 22days after planting, plants were transplanted into soil collected from a research farm located in London, ON. A photograph, shown in Figure 11 A, of control and treated plants was taken immediately prior to transplantation. Control and treated plants were photographed again immediately before harvesting at 57 days after planting, and shoot length, chlorophyll content, and dry shoot biomass were recorded. Chlorophyll content was measured with a handheld meter (SPAD-502). As seen in Figure 11 B, immediately before harvest, control plants, though tall, appeared sparse and light in color while, in contrast, the plants treated with Biofertilizer Composition A appeared denser with more leaves and were a darker shade of green. As seen from the results shown in Figure 12A, statistical analysis of the data showed that plants treated with Biofertilizer Composition A had significantly more chlorophyll content than control plants. However, there was no statistically significant difference in shoot length or biomass between treated plants and control plants, as seen from the results shown in Figures 12B and 12C.Cucumber transplant assay

[0113] Cucumber seeds (Straight Eight, William Dam Seeds) were planted in potting mix treated with Biofertilizer Composition A as described above for the lettuce transplant assay or in untreated potting mix (control). At 17 days after planting, five plants per treatment were transplanted into 4-inch pots containing a sandy loam soil collected from a farm near London, Ontario. Photographs of untreated (Figure 13A) and treated (Figure 13B) plants were taken before transplant. As seen in Figure 13C, the plants treated with Biofertilizer Composition A were larger than the control plants even at 28 days after transplant.

[0114] At 39 days after transplantation, control and treated plants were photographed and harvested, and shoot length, dry shoot biomass, and number of flowers were recorded. As seen in Figure 13D and in the graphs shown in Figures 14A to 14C, control plants treated with water were significantly lighter, smaller and showed a smaller number of flowers that the plants treated with Biofertilizer Composition A. No signs of disease were detected on any of the control or treated plants.Example 6: Plant growth promotion by biofertilizer compositions containing Pseudomonas azotoformans strain AL-336 under saline stressWheat salinity trial

[0115] Wheat seeds (Triticum aestivum, Pioneer, 2547; three seeds per pot) were planted in potting mix (Agromix™; Fafard) previously mixed with a 100 mM NaCI solution. Biofertilizer composition A (300 pl) was drenched on top of the seeds at planting. Seeds drenched with water and planted in potting mix with or without salt were used as positive and negative controls, respectively. Each treatment was replicated 5 times. Pots were incubated undercontrolled conditions of light and temperature (16 h light at 23°C / 8 h dark at 21 °C) for 36 days. Plants were watered with 60 ml / pot of a 100 mM NaCI solution twice a week, except for the negative controls (untreated potting mix) where water was used. As seen in Figure 15A, 30 days after planting wheat plants treated with biofertilizer composition A appear taller and greener than those plants growing without bacterial treatment in potting mix with (positive control) or without (negative control) salt. As seen from the results shown in Figure 15B, statistical analysis of the data showed that plants treated with Biofertilizer Composition A had a larger biomass than control plants grown in a salty environment. Similarly, plants grown without excess of salt (negative control) had statistically significantly larger biomass than those plants grown in an excess of salt, indicating that the experimental set up for the salinity trial is correct.Cucumber salinity trial

[0116] Two cucumber seeds (Straight Eight, William Dam Seeds) were planted in potting mix (Agromix; Fafard) previously mixed with a 100 mM NaCI solution. Biofertilizer composition A (300 pl) was drenched on top of the seeds at planting. Seeds drenched with water and planted in potting mix with or without salt were used as positive and negative controls, respectively. Each treatment was replicated 5 times. Pots were incubated under controlled conditions of light and temperature (16 h light at 23°C / 8 h dark at 21 °C) for 36 days. Plants were thinned out to 1 per pot after full emergence was achieved. Pots were watered with 60 ml / pot of a 100 mM NaCI solution every two days for 3 weeks, starting 1 week after planting. Negative controls (untreated potting mix) where watered with water. As seen from the results shown in Figure 16, statistical analysis of the data showed that plants treated with Biofertilizer Composition A had a statistically significantly larger biomass than control plants grown on a salty environment. Similarly, plants grown without excess of salt (negative control) had a statistically significantly larger biomass than those plants grown in an excess of salt.Example 7: Crop yield increase by biofertilizer compositions containing Pseudomonas azotoformans strain AL-336Tomato Field Trial

[0117] A tomato field trial was conducted on a farm in llderton, Ontario, Canada (43° 0.4.47’ North 181 ° 20.24’ West) in 2022, to determine the effect of biofertilizer compositions containing Pseudomonas azotoformans strain AL-336 on tomato yield.

[0118] The soil is a Bryanston loam, silt-loam, pH 7.0 - 7.7, and the field was previously cultivated with soybeans & fall rye cover crop in 2021 . The field was fertilized withmonoammonium phosphate (MAP) 100 lbs, K-Mag™ 100 lbs, muriate of potash (MOP) 150 lbs, 6-24-24 50 lbs, and Urea 200 lbs before planting.

[0119] A total of 120 tomato seedlings were utilized per treatment. On April 19, 2022, 128- cell seedling germination trays were filled with premoistened potting mix (AgroMix™) and one tomato seed (Roma processing tomato variety TSH18) was planted per cell. Trays were covered and incubated in a climate-controlled growth chamber. Plants were fertilized with half strength MiracleGro™ Ultrabloom fertilizer 20:20:20 (NPK) once a week, starting 15 days after germination. Two weeks prior to transplant into the field, the tomatoes were fertilized daily and exposed to the outdoors to harden at increasing periods of time over the 2 weeks. Tomato seedlings were transplanted into the field five weeks after planting (DAP).

[0120] Six treatments were applied as root dip at transplant: Water Control, Biofertilizer composition A, Biofertilizer composition B, Biofertilizer composition D, Biofertilizer composition E, and an unrelated treatment. Each treatment was replicated six times and the plots were distributed in the field using a Latin Square Design (6x6). Each plot had two rows of 10 tomatoes each, planted 40 cm apart. Rows were planted 1 .2 m apart. Plots were 4 m long. There was total 6 blocks with 6 plots on each with a 2 m alley between blocks. There was one guard row of tomato planted on each side of the outer block lengthwise.Fertilizers / pesticides were applied throughout the season to all plots following standard agronomic practices.

[0121] Harvest was carried out on August 29, 2022. To obtain yield data, 8 plants per row were individually harvested for a total of 16 plants per plot. The number of tomatoes per plant, weight of tomato per plant, number of marketable tomatoes per plant, and weight of marketable tomatoes per plant were recorded in the field. Marketable tomatoes were defined as those with a diameter of 40 mm or bigger. For field tomatoes, all Canadian grades require that tomatoes should be sized between 38.1 to 51 mm. Total yield was determined by combining the yield of all plants in each plot and expressed in tonnes per hectare (t / h). Brix index of tomatoes was measured on a subsample for each plot.

[0122] Statistical analysis of the data showed that all 4 yield categories (Total number of tomatoes, total weight, marketable number of tomatoes, marketable weight) were nonparametric (non-normal) and skewed to the left and were analyzed accordingly. Outliers were identified and eliminated from the analysis.

[0123] As shown by the data provided in Tables 4 and 5, application of biofertilizer compositions containing Pseudomonas azotoformans AL-336 resulted in the production of more tomatoes than the control treatment, and all treatments produced more marketable tomatoes than the control treatment. The data presented was normalized to 100 plants for easy comparison.Table 4: Effect of various treatments on tomato yieldsTable 5: Effect of various treatments on tomato yieldsCorn field trial

[0124] A corn field trial was conducted on a farm in llderton, Ontario, Canada (43° 0.4.47’ North 181 ° 20.24’ West) in 2021 , to determine the effect of Biofertilizer composition A on corn yield.

[0125] The soil is a Bryanston loam, silt-loam, pH 7.0 - 7.7, and the field was previously cultivated with tomatoes in 2020. The field was fertilized with urea 175 lbs, monoammonium phosphate (MAP) 150 lbs, muriate of potash (MOP) 125 lbs, and K-Mag™ 100 lbs, before planting.

[0126] Corn seeds (DKC48-56RIB Hybrid Seed Corn Blend Lot# H18YFV4JX Bayer) were used in this experiment. Biofertilizer composition A was applied in furrow at planting at a rate of 2L / ac. Control was treated with water.

[0127] Plots were planted in 4 rows on 75 cm row spacing to a length of 10M with a 2 row John Deere plot planter fitted with 10 CO2pressurized bottles to apply various in furrow treatments. Plots measured 3m x 10m. The treatments were applied to a length of 10M, and lines were flushed with CO2and water between treatments.

[0128] At the end of the season, 25 corn plants from each row were harvested by hand, excluding the two outer plants on either end. All plants from each row were fed through themanual corn thresher and the total weight of corn kernels was obtained for each row. Yield in bushels / acre were calculated.

[0129] As seen from the data presented in Table 6, application of Biofertilizer composition A in furrow at planting, increased corn yield by 7.9%.Table 6. Effect of Biofertilizer Composition A on corn yieldConclusion

[0130] The results of these trials clearly indicate that the present biofertilizer compositions enhance plant growth and development via biostimulation in the absence or under reduced pressure of plant pathogens. Without being bound by theory, it is contemplated that the mechanisms by which the present biofertilizer compositions stimulate plant growth include, but are not limited to, acquiring and providing a constant supply of nutrients (N, P, Fe, K) to the plants. The bacterial strain Pseudomonas azotoformans strain AL-336 in the present biofertilizer compositions also produces an adequate amount of indoleacetic acid (IAA) and can regulate deleterious levels of plant stress hormone ethylene by producing the enzyme 1 -aminocyclopropane-1 -carboxylate (ACC) deaminase. The results presented here indicate that using the present biofertilizer compositions to treat agriculturally relevant crops would yield added benefits to growers and help achieve sustainability in agricultural production.

[0131] The embodiments described herein are intended to be illustrative of the present compositions and methods and are not intended to limit the scope of the present invention. Various modifications and changes consistent with the description as a whole and which are readily apparent to the person of skill in the art are intended to be included. The appended claims should not be limited by the specific embodiments set forth in the examples but should be given the broadest interpretation consistent with the description as a whole.

Claims

CLAIMS1 . An isolated bacterial strain of Pseudomonas azotoformans designated as strain AL-336, deposited with the International Depository Authority of Canada (IDAC) under accession number 260423-01.

2. A biofertilizer composition comprising the isolated bacterial strain of Pseudomonas azotoformans according to claim 1 and at least one agriculturally acceptable carrier.

3. The biofertilizer composition according to claim 2 wherein the biofertilizer composition is a liquid formulation.

4. The biofertilizer composition according to claim 2 wherein the biofertilizer composition is a solid formulation.

5. The biofertilizer composition according to claim 4 wherein the solid formulation is a granular formulation.

6. A kit for preparing the liquid formulation according to claim 3, the kit comprising a culture of Pseudomonas azotoformans strain AL-336 and instructions for mixing the culture of Pseudomonas azotoformans strain AL-336 with an agriculturally acceptable liquid carrier.

7. The kit according to claim 6 wherein the agriculturally acceptable liquid carrier is water.

8. The kit according to claim 6 wherein the agriculturally acceptable carrier is an aqueous solution comprising one or more agriculturally acceptable components.

9. The kit according to any one of claims 6 to 8 further comprising the agriculturally acceptable carrier.

10. The kit according to any one of claims 6 to 9 wherein the culture of Pseudomonas azotoformans strain AL-336 is a solid culture.11 . The kit according to claim 10 wherein the solid culture is a lyophilized culture or a spray-dried culture.

12. The kit according to any one of claims 6 to 9 wherein the culture of Pseudomonas azotoformans strain AL-336 is a liquid culture.

13. The kit according to claim 12 wherein the instructions for mixing the liquid culture of Pseudomonas azotoformans strain AL-336 with the agriculturally acceptable carrier include instructions for mixing about 1 part by volume of the liquid culture of Pseudomonas azotoformans strain AL-336 with about 9 to about 999 parts by volume of the agriculturally acceptable carrier.

14. The kit according to claim 12 wherein the instructions for mixing the liquid culture of Pseudomonas azotoformans strain AL-336 with the agriculturally acceptable carrier include instructions for mixing about 1 part by volume of the liquid culture of Pseudomonas azotoformans strain AL-336 with about 99 parts by volume of the agriculturally acceptable carrier.

15. A method of promoting growth of a plant, enhancing early germination and plant emergence, reducing environmental stress, or increasing crop yield, the method comprising applying the biofertilizer composition according to any one of claims 2 to 5 to the plant or to a part thereof or to a substrate for receiving the plant.

16. The method according to claim 15 wherein the biofertilizer composition is applied to the plant or to the part thereof or to the substrate by spraying, drenching, sprenching, coating or soaking.

17. A method of promoting growth of a plant, enhancing early germination and plant emergence, reducing environmental stress, or increasing crop yield, the method comprising applying the biofertilizer composition according to any one of claims 2 to 5 to a seed or to a substrate for receiving the seed.

18. The method according to claim 17 wherein the biofertilizer composition is applied to the seed prior to planting.

19. The method according to claim 17 or 18 wherein the biofertilizer composition is applied to the seed or to the substrate by spraying, drenching, sprenching, coating or soaking.

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